WO2023159316A1 - Formulation of anti-sars-cov-2 neutralizing antibodies and uses thereof - Google Patents

Formulation of anti-sars-cov-2 neutralizing antibodies and uses thereof Download PDF

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Publication number
WO2023159316A1
WO2023159316A1 PCT/CA2023/050235 CA2023050235W WO2023159316A1 WO 2023159316 A1 WO2023159316 A1 WO 2023159316A1 CA 2023050235 W CA2023050235 W CA 2023050235W WO 2023159316 A1 WO2023159316 A1 WO 2023159316A1
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Prior art keywords
composition
sequence
seq
antibody
antigen
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PCT/CA2023/050235
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French (fr)
Inventor
Alexandre FUGÈRE
Frédéric Leduc
Bruno Maranda
Simon GAUDREAU
Djordje GRBIC
Jean-François LARRIVÉE
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Immune Biosolutions Inc.
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Publication of WO2023159316A1 publication Critical patent/WO2023159316A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/544—Mucosal route to the airways
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present disclosure generally relates to viral infection, and more particularly to the prevention and/or treatment of coronavirus infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.
  • coronavirus infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • Coronaviruses are large, roughly spherical, RNA viruses with bulbous surface projections that cause diseases in mammals and birds. In humans, these viruses cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold (which is also caused by other viruses, predominantly rhinoviruses), while more lethal varieties can cause severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Coronavirus disease 2019 (COVID-19). Coronaviruses have four structural proteins, namely the Spike (S), Envelope (E), and Membrane (M) proteins, forming the viral envelope, as well as the Nucleocapsid (N) protein, holding the viral RNA genome.
  • S Spike
  • E Envelope
  • M Membrane
  • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the strain of coronavirus that causes COVID-19, the respiratory illness responsible for the COVID-19 pandemic.
  • the spike protein SARS-CoV-2 is the glycoprotein responsible for allowing the virus to attach to and fuse with the membrane of a host cell; specifically, its S1 subunit contains the receptor-binding domain (RBD) that interacts with the cellular receptor angiotensin-converting enzyme 2 (ACE2) and catalyzes virus attachment, and its S2 subunit possesses the fusion machinery, which can mediate host-viral membrane fusion after S1 shedding.
  • RBD receptor-binding domain
  • ACE2 angiotensin-converting enzyme 2
  • the main receptor involved in SARS- CoV-2 entry into human cells is the angiotensin converting enzyme 2 (ACE2).
  • TMPRSS2 protease transmembrane protease, serine 2
  • B.1.1.7 also known as VOC-202012/01
  • 501Y.V2 B.1.351
  • P.1 B.1.1.28.1 delta
  • B.1.617.2 Omicron
  • B.1.1.529 Omicron
  • the B.1.1.7 variant had been reported in 93 countries, the 501 Y.V2 variant in 45, and the P.1 variant in 21. All three variants have the N501Y mutation, which changes the amino acid asparagine (N) to tyrosine (Y) at position 501 in the receptor-binding domain of the spike protein.
  • the 501Y.V2 and P.1 variants both have two additional receptor-binding-domain mutations, K417N/T and E484K. These mutations increase the binding affinity of the receptorbinding domain to the angiotensin-converting enzyme 2 (ACE2) receptor.
  • ACE2 angiotensin-converting enzyme 2
  • the B.1.617.2 delta variant comprises the following substitutions in the Spike protein that are known to affect transmissibility of the virus: D614G, T478K, P681 R and L452R.
  • WHO designated the variant B.1 .1 .529 (Omicron) a variant of concern based on several mutations that may have an impact on how it behaves, for example, on how easily it spreads or the severity of illness it causes.
  • the present disclosure provides the following items:
  • composition comprising a combination of anti-SARS-CoV-2 antibodies or antigenbinding fragments thereof, said combination comprising:
  • a first antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs): a light chain CDR1 (CDR-L1) comprising the sequence RASQSVSSSYLA (SEQ ID NO:7); a light chain CDR2 (CDR-L2) comprising the sequence GASSRAT (SEQ ID NO:8); a light chain CDR3 (CDR-L3) comprising the sequence QQYGTSPWT (SEQ ID NO:9); a heavy chain CDR1 (CDR-H1) comprising the sequence GFTFTSS (SEQ ID NO: 10); a heavy chain CDR2 (CDR-H2) comprising the sequence WGSGN (SEQ ID NO:11); and a heavy chain CDR3 (CDR-H3) comprising the sequence PSCSGGRCYDGFDI (SEQ ID NO:12);
  • CDRs complementarity determining regions
  • a second antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising the sequence RASQGISSWLA (SEQ ID NO: 13); a CDR- L2 comprising the sequence AASSLQS (SEQ ID NO:14); a CDR-L3 comprising the sequence QQGNSFPYT (SEQ ID NO:15); a CDR-H1 comprising the sequence GYTFTRY (SEQ ID NO:16); a CDR-H2 comprising the sequence YPGDSD (SEQ ID NO:17); and a CDR-H3 comprising the sequence LPQYCSNGVCQRWFDP (SEQ ID NO:18); and
  • a third antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising the sequence RASQSVSSSYLA (SEQ ID NO:7); a CDR-L2 comprising the sequence GASSRAT (SEQ ID NO:8); a CDR-L3 comprising the sequence QQYGSSYT (SEQ ID NO:19); a CDR-H1 comprising the sequence GITVSSN (SEQ ID NO:20); a CDR-H2 comprising the sequence YSGGS (SEQ ID NO:21); and a CDR-H3 comprising the sequence DLEMAGAFDI (SEQ ID NO:22).
  • composition of item 1 wherein the first antibody or antigen-binding fragment thereof comprises a light chain FR1 comprising the sequence of the FR1 depicted in FIG. 1A (SEQ ID NO:27).
  • composition of item 1 or 2 wherein the first antibody or antigen-binding fragment thereof comprises a light chain FR2 comprising the sequence of the FR2 depicted in FIG. 1A (SEQ ID NO:28).
  • composition of any one of items 1 to 3, wherein the first antibody or antigen-binding fragment thereof comprises a light chain FR3 comprising the sequence of the FR3 depicted in FIG. 1A (SEQ ID NO:29).
  • composition of any one of items 1 to 6, wherein the third antibody or antigen-binding fragment thereof comprises a light chain FR2 comprising the sequence of the FR2 depicted in FIG. 1C (SEQ ID NO:44).
  • composition of any one of items 1 to 7, wherein the third antibody or antigen-binding fragment thereof comprises a light chain FR3 comprising the sequence of the FR3 depicted in FIG. 10 (SEQ ID NO:45).
  • composition of any one of items 1 to 8, wherein the third antibody or antigen-binding fragment thereof comprises a light chain FR4 comprising the sequence of the FR4 depicted in FIG. 1C (SEQ ID NO:46).
  • composition of any one of items 1 to 9, wherein the second antibody or antigenbinding fragment thereof comprises a light chain FR1 comprising the sequence of the FR1 depicted in FIG. 1 B (SEQ ID NO:35).
  • composition of any one of items 1 to 10, wherein the second antibody or antigenbinding fragment thereof comprises a light chain FR2 comprising the sequence of the FR2 depicted in FIG. 1 B (SEQ ID NO:36).
  • composition of any one of items 1 to 11 , wherein the second antibody or antigenbinding fragment thereof comprises a light chain FR3 comprising the sequence of the FR3 depicted in FIG. 1 B (SEQ ID NO:37).
  • composition of any one of items 1 to 12, wherein the second antibody or antigenbinding fragment thereof comprises a light chain FR4 comprising the sequence of the FR4 depicted in FIG. 1 B (SEQ ID NO:38).
  • composition of any one of items 1 to 13, wherein the first antibody or antigenbinding fragment thereof comprises a heavy chain comprising the heavy chain sequence depicted in FIG. 1A (SEQ ID NO:1), and a light chain comprising the light chain sequence depicted in FIG. 1A (SEQ ID NO:2).
  • composition of any one of items 1 to 14, wherein the third antibody or antigenbinding fragment thereof comprises a heavy chain comprising the heavy chain sequence depicted in FIG. 1C (SEQ ID NO:3), and a light chain comprising the light chain sequence depicted in FIG. 1C (SEQ ID NO:4).
  • composition of any one of items 1 to 15, wherein the second antibody or antigenbinding fragment thereof comprises a heavy chain comprising the heavy chain sequence depicted in FIG. 1 B (SEQ ID NO:5), and a light chain comprising the light chain sequence depicted in FIG. 1 B (SEQ ID NO:6).
  • composition of item 20 wherein the first, second and/or third antibodies are of the lgG1 subclass.
  • composition of any one of items 1 to 21 , wherein the combination comprises about 55% to about 65% of the first antibody or antigen-binding fragment thereof.
  • composition of item 22, wherein the combination comprises about 63% of the first antibody or antigen-binding fragment thereof.
  • composition of any one of items 1 to 23, wherein the combination comprises about 28% to about 37% of the third antibody or antigen-binding fragment thereof.
  • composition of item 24, wherein the combination comprises about 32% of the third antibody or antigen-binding fragment thereof.
  • composition of item 26, wherein the combination comprises about 5% of the second antibody or antigen-binding fragment thereof.
  • composition of item 28 wherein the pharmaceutically acceptable carrier or excipient is adapted for administration of the composition by the pulmonary route.
  • composition of item 28 or 29, wherein the composition is an aqueous solution, suspension or emulsion.
  • composition of any one of items 28 to 30, wherein the pharmaceutically acceptable carrier or excipient comprises a saline solution or buffer.
  • composition of any one of items 28 to 31 wherein the pharmaceutically acceptable carrier or excipient comprises a surfactant or emulsifier.
  • composition of item 32, wherein the surfactant or emulsifier is a polysorbate.
  • 36. A composition comprising one or more nucleic acids encoding the first, second and third antibodies or antigen-binding fragments thereof defined in any one of items 1 to 27.
  • composition of item 36 wherein the one or more nucleic acids comprise a first nucleic acid encoding the first antibody or antigen-binding fragment thereof defined in any one of items 1 to 27, a second nucleic acid encoding the second antibody or antigen-binding fragment thereof defined in any one of items 1 to 27, and a third nucleic acid encoding the third antibody or antigen-binding fragment thereof defined in any one of items 1 to 27.
  • composition of item 36 or 37, wherein the one or more nucleic acids are mRNA molecules.
  • composition of item 39, wherein the vesicles are lipid nanoparticles or liposomes.
  • An inhaler or nebulization device comprising the composition of any one of items 1 to 40.
  • a method for preventing or treating a betacoronavirus infection or a related disease in a subject in need thereof comprising administering to the subject an effective amount of the composition of any one of items 1 to 40.
  • a method for reducing the risk of developing a betacoronavirus-related disease or the severity of a betacoronavirus-related disease in a subject comprising administering to the subject an effective amount of the composition of any one of items 1 to 40.
  • a method for blocking the entry of a betacoronavirus in an ACE2-expressing cell comprising contacting the cell and/or the virus with an effective amount of the composition of any one of items 1 to 40.
  • SARS-CoV-2 strain such as an Omicron variant.
  • composition is administered with (i) at least one additional anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, or nucleic acid(s) encoding said at least one additional anti-SARS-CoV-2 antibody or antigenbinding fragment thereof; and/or (ii) at least one antiviral or anti-inflammatory drug.
  • composition of any one of items 1 to 40 for preventing or treating a betacoronavirus infection or a related disease in a subject.
  • composition of any one of items 1 to 40 for the manufacture of a medicament for preventing or treating a betacoronavirus infection or a related disease in a subject.
  • composition of any one of items 1 to 40 for reducing the risk of developing a betacoronavirus-related disease or the severity of a betacoronavirus-related disease in a subject.
  • composition of any one of items 1 to 40 for the manufacture of a medicament for reducing the risk of developing a betacoronavirus-related disease or the severity of a betacoronavirus-related disease in a subject.
  • composition of any one of items 1 to 40 for blocking the entry of a betacoronavirus in an ACE2-expressing cell.
  • composition of any one of items 1 to 40 for the manufacture of a medicament for blocking the entry of a betacoronavirus in an ACE2-expressing cell.
  • CoV-2 strain such as an Omicron variant.
  • composition is for administration with (i) at least one additional anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, or nucleic acid(s) encoding said at least one additional anti-SARS-CoV-2 antibody or antigenbinding fragment thereof; and/or (ii) at least one antiviral or anti-inflammatory drug.
  • composition of any one of items 1 to 40 for use in blocking the entry of a betacoronavirus in an ACE2-expressing cell is a composition of any one of items 1 to 40 for use in blocking the entry of a betacoronavirus in an ACE2-expressing cell.
  • composition for use of item 64, wherein the sarbecovirus is SARS-CoV-2.
  • FIG. 1A shows the amino acid sequence of the heavy and light chain variable regions of Antibody IBIO1 described herein.
  • the amino acid corresponding to the CDR1 , CDR2 and CDR3 according to the Clothia numbering scheme are underlined.
  • FIG. 1B shows the amino acid sequence of the heavy and light chain variable regions of Antibody IBIO2 described herein.
  • the amino acid corresponding to the CDR1 , CDR2 and CDR3 according to the Clothia numbering scheme are underlined.
  • FIG. 1C shows the amino acid sequence of the heavy and light chain variable regions of Antibody IBIO3 described herein.
  • the amino acid corresponding to the CDR1 , CDR2 and CDR3 according to the Clothia numbering scheme are underlined.
  • FIG. 2A shows the proportion of antibody I BI01 , antibody IBIO2 and antibody IBIO3 in the antibody cocktails A-J tested in the study reported in FIG. 2B.
  • FIG. 2B is a graph showing the IC50, IC 8 o and IC99 of the antibody cocktails A-J in a SARS- CoV-2 Spike Neutralization Assay.
  • FIG. 3 is a graph showing the results of a dose-response direct ELISA assay of antibody cocktail J (IBIO123) binding on trimeric SARS-CoV-2 Spike Protein (100 ng). EC50 and correlation determined by the 5-parameter logistic non-linear regression curve fit analysis.
  • FIG. 4 is a graph showing the results of a dose-response neutralization assay of SARS- CoV-2 viral particles using antibody cocktail J (IBIO123).
  • FIG. 5 is a graph showing the results of a dose-response neutralization assay of SARS- CoV-2 viral particles using antibody cocktail J (IBIO123) prior and after virus filtering of the composition, or a control SARS-CoV-2 Spike Neutralizing Antibody from Sino Biological (Cat. No. 40592-MM57).
  • FIG. 6A and 6B show the results of a dose-response neutralization assay of SARS-CoV-2 variants of concern (VOCs) using the cell-virus fusion assay. Relative viral infectivity is shown at increasing antibody concentrations. Inhibition dose-response nonlinear regression curve fit (three parameters) was used to analyze the results.
  • VOCs SARS-CoV-2 variants of concern
  • FIG. 6C shows the results of a dose-response neutralization assay of IBIO123 on complete SARS-CoV-2 virus variants (Delta, Omicron BA.1 , Omicron BA.2 and Omicron BA.5).
  • the cytopathic effect (CPE) score was set at 100 for the virus control and at 0.1 for the medium control.
  • FIG. 7A is a graph showing the results of a dose-response ELISA with initial (pre- nebulization, circles) and nebulized (squares) IBIO123 antibody cocktail on 100 ng on SARS- CoV-2 Wuhan Trimeric Spike glycoprotein (SMT1-1) (CNRC).
  • SMT1-1) SARS- CoV-2 Wuhan Trimeric Spike glycoprotein
  • FIG. 7B is a graph showing the results of a dose-response neutralization assay of SARS- CoV-2 Wuhan pseudovirus with initial (pre-nebulization, circles) and nebulized (post-nebulization, squares) IBIO123 antibody cocktail.
  • IC 5 o values were determined using the log(inhibitor) vs. response (three parameters) dose-response inhibition curve-fit model.
  • FIG. 8 is a graph depicting the mutations in the Spike protein from SARS-CoV-2 VOCs Omicron sublineages BA.1 , BA.2 and BA.3 (from William A. Haseltine, Birth of The Omicron Family: BA.1 , BA.2, BA.3. Each As Different As Alpha Is From Delta, Forbes, Jan 26, 2022).
  • the term “about” has its ordinary meaning.
  • the term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).
  • composition such as an aerosol composition, comprising a combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof, said combination comprising at least two of the following antibodies:
  • a first antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs): a light chain CDR1 (CDR-L1) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQSVSSSYLA (SEQ ID NO:7); a light chain CDR2 (CDR-L2) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GASSRAT (SEQ ID NO:8); a light chain CDR3 (CDR- L3) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQYGTSPWT (SEQ ID NO:9); a heavy chain CDR1 (CDR-H1) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100%
  • a second antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQGISSWLA (SEQ ID NO: 19); a CDR- L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence AASSLQS (SEQ ID NO:20); a CDR-L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQGNSFPYT (SEQ ID NO:21); a CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GYTFTRY (SEQ ID NO:22); a CDR-H2 comprising or consisting or consist
  • a third antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQSVSSSYLA (SEQ ID NO:13); a CDR- L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GASSRAT (SEQ ID NO:14); a CDR-L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQYGSSYT (SEQ ID NO:15); a CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GITVSSN (SEQ ID NO: 16); a CDR-H2 comprising or consisting of an amino acid
  • composition such as an aerosol composition, comprising a combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof, said combination comprising:
  • CDRs complementarity determining regions
  • a light chain CDR1 CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQSVSSSYLA (SEQ ID NO:7)
  • a light chain CDR2 CDR- L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GASSRAT (SEQ ID NO:8)
  • a light chain CDR3 CDR-L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQYGTSPWT (SEQ ID NO:9)
  • a heavy chain CDR1 CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%
  • a second antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQGISSWLA (SEQ ID NO: 19); a CDR-L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence AASSLQS (SEQ ID NQ:20); a CDR- L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQGNSFPYT (SEQ ID NO:21); a CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GYTFTRY (SEQ ID NO:22); a CDR-H
  • a third antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQSVSSSYLA (SEQ ID NO: 13); a CDR-L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GASSRAT (SEQ ID NO:14); a CDR- L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQYGSSYT (SEQ ID NO:15); a CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GITVSSN (SEQ ID NO:16); a CDR-H2 comprising
  • composition comprising a combination of anti- SARS-CoV-2 antibodies or antigen-binding fragments thereof, said combination comprising:
  • CDRs complementarity determining regions
  • a light chain CDR1 CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQSVSSSYLA (SEQ ID NO:7)
  • a light chain CDR2 CDR- L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GASSRAT (SEQ ID NO:8)
  • a light chain CDR3 CDR-L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQYGTSPWT (SEQ ID NO:9)
  • a heavy chain CDR1 CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%
  • a second antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQGISSWLA (SEQ ID NO: 19); a CDR-L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence AASSLQS (SEQ ID NQ:20); a CDR- L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQGNSFPYT (SEQ ID NO:21); a CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GYTFTRY (SEQ ID NO:22); a CDR-H
  • a third antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQSVSSSYLA (SEQ ID NO: 13); a CDR-L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GASSRAT (SEQ ID NO:14); a CDR- L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQYGSSYT (SEQ ID NO:15); a CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GITVSSN (SEQ ID NO:16); a CDR-H2 comprising or
  • antibody or antigen-binding fragment thereof refers to any type of antibody/antibody fragment including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, humanized antibodies, CDR-grafted antibodies, chimeric antibodies and antibody fragments so long as they exhibit the desired antigenic specificity/binding activity.
  • Antibody fragments comprise a portion of a full-length antibody, generally an antigen binding or variable region thereof.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain Fv, scFv), single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments.
  • Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, H regions ( H , V H -V H ), anticalins, PepBodies, antibody-T- cell epitope fusions (Troybodies) or Peptibodies.
  • monoclonal antibody refers to an antibody from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are substantially similar and bind the same epitope(s), except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts.
  • Such monoclonal antibody typically includes an antibody comprising a variable region that binds a target, wherein the antibody was obtained by a process that includes the selection of the antibody from a plurality of antibodies.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones.
  • the selected antibody can be further altered, for example, to improve affinity for the target, to humanize the antibody, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered variable region sequence is also a monoclonal antibody of this disclosure.
  • the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including the hybridoma method (e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 , (Elsevier, N.
  • Methods 284(1 -2): 119-132 (2004) and technologies for producing human or human-like antibodies from animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences see, e.g., WO98/24893, WO96/34096, WO96/33735, and WO91/10741 , Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immune, 7:33 (1993); U.S. Patent Nos.
  • the monoclonal antibodies herein specifically include "chimeric” or “recombinant” antibodies in which a portion of the light and/or heavy chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
  • Chimeric antibodies of interest herein include "humanized" antibodies.
  • the antibody is a monoclonal antibody, preferably a human antibody.
  • the first, second and/or third antibody of the present disclosure may be of any class or isotype, e.g., IgG, IgM, IgA, IgD or IgE. In an embodiment, the first, second and/or third antibody of the present disclosure is an IgA. In an embodiment, the first, second and/or third antibody of the present disclosure is an IgG.
  • the IgG may be of any subclass, e.g., lgG1 , lgG2, lgG3, or lgG4. In an embodiment, the first, second and/or third antibody of the present disclosure is an igGi .
  • the first, second and/or third antibody of the present disclosure is a multispecific antibody, such as a bispecific antibody.
  • multispecific (e.g., bispecific) antibodies at least one of the antigen-binding domains comprise one of the combinations of CDRs or variable regions described herein.
  • the multispecific (e.g., bispecific) antibody comprises two of the combinations of CDRs or variable regions described herein.
  • the multispecific (e.g., bispecific) antibody may also comprise a binding domain that binds to another antigen (i.e., other than the Spike protein from a betacoronavirus), for example a binding domain that binds to ACE2.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions (HVRs) both in the lightchain and heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework region (FR).
  • CDRs complementarity-determining regions
  • HVRs hypervariable regions
  • the more highly conserved portions of variable domains are called the framework region (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a p-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the p-sheet structure.
  • both light and heavy chain variable regions comprise alternating FRs and CDRs: FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each region may be made in accordance with the definitions of Kabat, Chothia (Al- Lazikani eta!., J Mol Biol.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and binding site.
  • this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.
  • one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species.
  • variable domain interacts to define an antigen-binding site on the surface of the V H -V L dimer.
  • the six CDRs are involved in conferring the antigen-binding specificity to the antibody.
  • a single variable domain or half of an Fv comprising only three CDRs specific for an antigen has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • HVR hypervariable region
  • the hypervariable region generally comprises amino acid residues from a "complementarity determining region” or “CDR” (Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (Al-Lazikani et al., supra).
  • CDR complementarity determining region
  • CDRs complementarity determining regions
  • the CDRs of immunological receptors are the most variable part of the receptor protein, giving receptors their diversity, and are carried on six loops at the distal end of the receptor's variable domains, three loops coming from each of the two variable domains of the receptor.
  • framework region refers to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved (i.e., other than the CDRs) among different immunoglobulins in a single species, as defined by Kabat et al. supra) or Chothia (Al- Lazikani et al., supra).
  • a "human framework region” is a framework region that is substantially identical to the framework region of a naturally occurring human antibody.
  • sequences of the CDR and FR as defined herein are defined according to the Clothia numbering scheme. However, the skilled person would understand that the amino acids forming the CDRs and FRs regions in the sequences of the antibodies defined herein may vary depending on the numbering scheme used. Other numbering schemes include the AbM, Kabat, Contact and IMGT schemes.
  • one or two residues in the above-noted CDRs sequences are substituted. In a further embodiment, one residue in the above-noted CDRs sequences is substituted. In another embodiment, the antibody or antigen-binding fragment thereof comprises the above-noted CDRs sequences.
  • the first antibody or antigen-binding fragment thereof comprises a light chain FR1 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR1 depicted in FIG. 1A.
  • the first antibody or antigen-binding fragment thereof comprises a light chain FR2 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR2 depicted in FIG. 1A.
  • the first antibody or antigen-binding fragment thereof comprises a light chain FR3 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR3 depicted in FIG. 1A.
  • the first antibody or antigen-binding fragment thereof comprises a light chain FR4 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR4 depicted in FIG. 1A.
  • the second antibody or antigen-binding fragment thereof comprises a light chain FR1 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR1 depicted in FIG. 1B.
  • the second antibody or antigen-binding fragment thereof comprises a light chain FR2 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR2 depicted in FIG. 1B.
  • the second antibody or antigen-binding fragment thereof comprises a light chain FR3 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR3 depicted in FIG. 1B.
  • the second antibody or antigen-binding fragment thereof comprises a light chain FR4 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR4 depicted in FIG. 1B.
  • the third antibody or antigen-binding fragment thereof comprises a light chain FR1 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR1 depicted in FIG. 1C.
  • the third antibody or antigen-binding fragment thereof comprises a light chain FR2 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR2 depicted in FIG. 1C.
  • the third antibody or antigen-binding fragment thereof comprises a light chain FR3 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR3 depicted in FIG. 1C.
  • the third antibody or antigen-binding fragment thereof comprises a light chain FR4 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR4 depicted in FIG. 1C.
  • the first antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the heavy chain sequence depicted in FIG. 1A (SEQ ID NO:1), and a light chain comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the light chain sequence depicted in FIG. 1A (SEQ ID NO:2).
  • the second antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the heavy chain sequence depicted in FIG. 1B (SEQ ID NO:5), and a light chain comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the light chain sequence depicted in FIG. 1B (SEQ ID NO:6).
  • the third antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the heavy chain sequence depicted in FIG. 1C (SEQ ID NO:3), and a light chain comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the light chain sequence depicted in FIG. 1C (SEQ ID NO:4).
  • Variations in the antibodies or antigen-binding fragments thereof described herein can be made, for example, using any of the techniques and guidelines for conservative and non- conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the antibody that results in a change in the amino acid sequence as compared with the native sequence antibody. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the antibody or antigen-binding fragment thereof.
  • Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the antibody or antigen-binding fragment thereof with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids.
  • the variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
  • the variant exhibits at least 50%, 55% or 60%, preferably at least 65, 70, 75, 80, 90, 95, 96, 97, 98 or 99% sequence identity with the sequence of the antibody or antigen-binding fragment thereof described herein and maintains the ability to specifically bind to SARS-CoV-2 Spike protein and/or to neutralize SARS-CoV-2 infection.
  • Identity refers to sequence identity between two polypeptides. Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B.
  • Covalent modifications of antibodies or antigen-binding fragments thereof are included within the scope of this disclosure.
  • Covalent modifications include reacting targeted amino acid residues of the antibody or antigen-binding fragment thereof with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the antibody or antigen-binding fragment thereof.
  • Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (T.E.
  • the first, second, and/or third antibody or antigen-binding fragment thereof of the present disclosure may further comprise one or more modifications that confer additional biological properties to the antigenic peptide such as protease resistance, plasma protein binding, increased plasma half-life, intracellular penetration, etc.
  • modifications include, for example, covalent attachment of molecules/moiety to the antibody or antigen-binding fragment thereof such as fatty acids (e.g., C 6 -Ci 8 ), attachment of proteins such as albumin (see, e.g., U.S. Patent No. 7,268,113); sugars/polysaccharides (glycosylation), biotinylation or PEGylation (see, e.g., U.S. Patent Nos.
  • the present disclosure provides a conjugate comprising the antibody or antigen-binding fragment thereof described herein and one or more additional molecules or agents (hereinafter secondary molecules or agents).
  • the antigenic peptide may be conjugated to any type of synthetic or natural secondary molecules or agents, such as peptides, proteins, saccharides/polysaccharides, lipids, naturally-occurring or synthetic polymers/co- polymers, etc. to modify one or more properties of the antibody or antigen-binding fragment thereof.
  • the conjugate comprises a covalent link or bond between the antibody or antigen-binding fragment thereof and the molecule conjugated thereto.
  • the molecule may be conjugated directly to the antibody or antigen-binding fragment thereof, or indirectly via a linker.
  • the linker may be a polypeptide linker comprising one or more amino acids or another type of chemical linker (e.g., a carbohydrate linker, a lipid linker, a fatty acid linker, a polyether linker, PEG, etc.
  • the molecule may be conjugated/attached to the side chain of one the amino acids of the antibody or antigen-binding fragment thereof.
  • Methods for conjugating moieties to side chains of amino acids are well known in the art.
  • chemical groups that react with primary amines (-NH 2 ) present in the side-chain of lysine residues such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters may be used to conjugate the molecule to the antibody or antigen-binding fragment thereof.
  • Cysteine residues present in the antibody or antigen-binding fragment thereof may also be used to attach the molecule.
  • the first, second, and/or third antibody or antigen-binding fragment thereof of the present disclosure is labelled or conjugated with one or more moieties.
  • the antibody or antigen-binding fragment thereof may be labeled with one or more labels such as a biotin label, a fluorescent label, an enzyme label, a coenzyme label, a chemiluminescent label, or a radioactive isotope label.
  • the antibody or antigen-binding fragment thereof is labelled with a detectable label, for example a fluorescent moiety (fluorophore).
  • Useful detectable labels include fluorescent compounds (e.g., fluorescein isothiocyanate, Texas red, rhodamine, fluorescein, Alexa Fluor® dyes, and the like), radiolabels, enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in a protein detection assays), streptavidin/biotin, and colorimetric labels such as colloidal gold, colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.). Chemiluminescent compounds may also be used.
  • Such labelled antibodies or antigen-binding fragments thereof may be useful, for example, for the detection of SARS-CoV-2 and/or SARS-CoV-2-infected cells in vivo or in vitro, e.g., by flow cytometry, immunohistochemistry, etc.
  • the antibody or antigen-binding fragment thereof can also be conjugated to detectable or affinity tags that facilitate detection and/or purification of the antibody or antigen-binding fragment thereof. Such tags are well known in the art.
  • detectable or affinity tags examples include polyhistidine tags (His-tags), polyarginine tags, polyaspartate tags, polycysteine tags, polyphenylalanine tags, glutathione S-transferase (GST) tags, Maltose binding protein (MBP) tags, calmodulin binding peptide (CBP) tags, Streptavidin/Biotin-based tags, HaloTag®, Profinity eXact® tags, epitope tags (such as FLAG, hemagglutinin (HA), HSV, S/S1 , c-myc, KT3, T7, V5, E2, and Glu-Glu epitope tags), reporter tags such as p-galactosidase (
  • the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 55% to about 65% of the first antibody or antigen-binding fragment thereof. In a further embodiment, the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 60% to about 65% or about 62 to about 64% of the first antibody or antigen-binding fragment thereof. In a further embodiment, the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 63% of the first antibody or antigen-binding fragment thereof.
  • the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 3% to 8% of the second antibody or antigen-binding fragment thereof. In a further embodiment, the combination of anti-SARS-CoV-2 antibodies or antigenbinding fragments thereof comprises about 3% to 7% or about 4% to 6% of the second antibody or antigen-binding fragment thereof. In a further embodiment, the combination of anti-SARS-CoV- 2 antibodies or antigen-binding fragments thereof comprises about 5% of the second antibody or antigen-binding fragment thereof.
  • the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 28% to about 37% of the third antibody or antigen-binding fragment thereof. In a further embodiment, the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 30% to about 35% or about 31% to about 33% of the third antibody or antigen-binding fragment thereof. In a further embodiment, the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 32% of the third antibody or antigen-binding fragment thereof.
  • the first, second, and/or third antibody or antigen-binding fragment thereof described herein binds to an epitope in the viral envelope spike protein (S) of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2.
  • the antibody or antigen-binding fragment thereof binds to an epitope in the receptor binding domain (RBD) of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 S protein.
  • the antibody or antigen-binding fragment thereof binds to an epitope that is outside the RBD of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 S protein.
  • a betacoronavirus such as a sarbecovirus, e.g., SARS-CoV-2 S protein.
  • the antibody or antigen-binding fragment thereof neutralizes a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2.
  • the antibody or antigen-binding fragment thereof inhibits viral and cell membrane fusion.
  • the antibody or antigen-binding fragment thereof binds to the S1 subunit of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 S protein. In some embodiments, the antibody or antigenbinding fragment thereof binds to the S2 subunit of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 S protein. In some embodiments, the antibody or antigen-binding fragment thereof also binds to the S protein of one at least two betacoronaviruses, such as SARS-CoV-1 and SARS-CoV-2.
  • Residues 1-12 correspond to the signal peptide
  • residues 13-685 correspond to the Spike protein subunit S1
  • residues 686-1273 correspond to the Spike protein subunit S2.
  • Residues 816-837 define the fusion peptide 1
  • residues 835-855 define the fusion peptide 2
  • residues 920-970 define the heptad repeat 1
  • residues 1163-1202 define the heptad repeat 2.
  • SARS-CoV2 variants comprise mutations in the Spike protein including L5F, S13I, L18F, T19R, T20N, P26S, A67V, del69-70, G75V, T76I, D80Y, D80A, T95I, S98F, R102I, D138Y, G142D, del142-144, del144, W152C, E154K, EFR156-158G, F157L, R190S, ins214EPE, D215G, A222V, del246-252, D253G, W258L, N354D, F342L, V367F, K417N, K417T, A435S, W436R, N439K, N440K, G446V, L452R, Y453F, K458R, G476S, S477N, S477G, T478K, V483A, E484K, E484Q, F490
  • the Delta variant comprises the following Spike protein mutations: T19R, (V70F*), T95I, G142D, E156-, F157-, R158G, (A222V*), (W258L*), (K417N*), L452R, T478K, D614G, P681 R, D950N.
  • the Omicron variant (sublineage BA.1) comprises the following Spike protein mutations: A67V, del69-70, T95I, del142-144, Y145D, del211 , L212I, ins214EPE, G339D, S371 L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681 H, N764K, D796Y, N856K, Q954H, N969K, L981 F.
  • FIG. 8 depicts the mutations in Omicron sublineages BA.1 , BA.2 and BA.3.
  • the combination of antibodies or antigen-binding fragments thereof described herein binds to and neutralizes the Spike protein from a SARS-CoV2 variant. In an embodiment, the combination of antibodies or antigen-binding fragments thereof described herein binds to and neutralizes the Spike protein from the SARS-CoV2 Delta variant. In an embodiment, the combination of antibodies or antigen-binding fragments thereof described herein binds to and neutralizes the Spike protein from the SARS-CoV2 Omicron variant.
  • a further aspect of the present disclosure provides nucleic acids encoding the first, second, and/or third antibody or antigen-binding fragment described herein.
  • the isolated nucleic acid may be a synthetic DNA, a non-naturally occurring mRNA, or a cDNA, for example.
  • the nucleic acid may be inserted within a plasmid, vector, or transcription or expression cassette.
  • the nucleic acids encoding the first, second, and/or third antibody or antigen-binding fragment described herein may be made and the expressed antibodies or antigen-binding fragments described may be tested using conventional techniques well known in the art.
  • the nucleic acid encoding the first, second, and/or third antibody or antigen-binding fragment described herein can be maintained in the vector in a host cell.
  • a nucleic acid comprising a sequence encoding the amino acid sequence of any one of SEQ ID NOs:1-6.
  • the nucleic acid is an expression vector.
  • the nucleic acid sequence encoding the antibody can be maintained in the vector in a host cell.
  • the nucleic acid(s) (DNA, mRNA) encoding the first, second and third antibodies or antigen-binding fragments described herein is/are comprised within vesicles such as lipid nanoparticles (e.g., liposomes) or any other suitable vehicle.
  • the nucleic acid(s) is/are mRNA and is/are encapsulated into nanoparticulate delivery vehicles (see, e.g., Van Hoecke and Roose, Journal of Translational Medicine, volume 17, Article number: 54 (2019); Sanz and Alvarez-Vallina, Antibodies (Basel). 2021 Sep 26;10(4):37).
  • the present disclosure provides a cell, for example a recombinant host cell, expressing the first, second, and/or third antibody or antigen-binding fragment described herein.
  • Methods of preparing antibodies or antigen-binding fragments comprise expressing the encoding nucleic acid(s) in a host cell under conditions to produce the antibodies or antigenbinding fragments, and recovering the antibodies or antigen-binding fragments.
  • the process of recovering the antibodies or antigen-binding fragments may comprise isolation and/or purification of the antibodies or antigen-binding fragments.
  • the method of production may comprise formulating the antibodies or antigen-binding fragments into a composition including at least one additional component, such as a pharmaceutically acceptable excipient.
  • a cell expressing one or more antibodies of the disclosure comprises one or more nucleic acid sequences encoding the amino acid sequence of any one of SEQ ID NOs: 1-6.
  • host cell is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein.
  • host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life.
  • the nucleic acid or nucleic acids encoding the light and heavy chains of the antibody or antigen-binding fragment thereof are introduced in a cell which is able to produce the recombinant antibody.
  • examples thereof include CHO-K1 (ATCC CCL-61), DUkXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies®, Cat #11619), rat myeloma cell YB2/3HL.P2.G11.16Ag.2O (also called YB2/0), mouse myeloma cell NSO, mouse myeloma cell SP2/0-Ag14 (ATCC No.
  • CRL1581 mouse P3- X63-Ag8653 cell (ATCC No. CRL1580), CHO cell in which a dihydrofolate reductase gene is defective, lectin resistance-acquired Led 3, CHO cell in which a1 ,6-fucosyltransaferse gene is defective, rat YB2/3HL.P2.G11.16Ag.2O cell (ATCC No. CRL1662), CHO-3E7 cells (expressing a truncated but functional form of EBNA1 , U.S. Patent No. 8,637,315) or the like.
  • transformants which stably express a recombinant antibody are selected by culturing them in a medium for animal cell culture containing an agent such as G418 sulfate or the like.
  • the medium for animal cell culture include RPMI1640 medium (manufactured by Invitrogen®), GIT medium (manufactured by Nihon Pharmaceutical®), EX- CELL301® medium (manufactured by JRH®), IMDM medium (manufactured by Invitrogen®), Hybridoma-SFM medium (manufactured by Invitrogen®), media obtained by adding various additives such as FBS to these media, or the like.
  • the recombinant antibody can be produced and accumulated in a culture supernatant by culturing the obtained transformants in a medium.
  • the expression level and antigen binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA or the like.
  • the expression level of the recombinant antibody can be increased by using DHFR amplification system or the like.
  • the recombinant antibody can be purified from the culture supernatant of the transformant by using a protein A column.
  • the recombinant antibody can be purified by combining the protein purification methods such as gel filtration, ion-exchange chromatography, ultrafiltration or the like.
  • the molecular weight of the H chain or the L chain of the purified recombinant antibody or the antibody molecule as a whole is determined by polyacrylamide gel electrophoresis, Western blotting, or the like.
  • Suitable vectors comprising nucleic acid(s) encoding the antibody or antigen-binding fragment described herein can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, phage, phagemids, adenoviral, AAV, lentiviral, for example.
  • Techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells, and gene expression, are well known in the art.
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector, wherein additional DNA segments may be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • nucleic acids into a host cell can be accomplished using techniques well known in the art.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, for example.
  • suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.
  • the nucleic acid of the invention is integrated into the genome, e.g., chromosome, of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.
  • the composition further comprises the above-mentioned first, second, and third antibodies or antigen-binding fragments thereof and a carrier or excipient, in a further embodiment a pharmaceutically acceptable carrier or excipient.
  • a carrier or excipient in a further embodiment a pharmaceutically acceptable carrier or excipient.
  • Such compositions may be prepared in a manner well known in the pharmaceutical art by mixing the antibodies or antigenbinding fragments thereof having a suitable degree of purity with one or more optional pharmaceutically acceptable carriers or excipients (see Remington: The Science and Practice of Pharmacy, by Loyd Allen, Jr, 2012, 22 nd edition, Pharmaceutical Press; Handbook of Pharmaceutical Excipients, by Rowe et al., 2012, 7 th edition, Pharmaceutical Press).
  • the carrier/excipient can be suitable for administration of the antibodies or antigen-binding fragments thereof by any conventional administration route, for example, for oral, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration.
  • the carrier/excipient is adapted for administration of the antibodies or antigen-binding fragments thereof by the intravenous or subcutaneous route.
  • the carriers/excipients are adapted for administration of the antibodies or antigen-binding fragments thereof by the intravenous route.
  • the carriers/excipients are adapted for administration of the antibodies or antigen-binding fragments thereof by the subcutaneous route. In an embodiment, the carriers/excipients are adapted for administration of the antibodies or antigen-binding fragments thereof by the pulmonary route.
  • excipient as used herein has its normal meaning in the art and is any ingredient that is not an active ingredient (drug) itself. Excipients include for example binders, lubricants, diluents, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizing agent, release-delaying agents and other components. "Pharmaceutically acceptable excipient” as used herein refers to any excipient that does not interfere with effectiveness of the biological activity of the active ingredients and that is not toxic to the subject, i.e., is a type of excipient and/or is for use in an amount which is not toxic to the subject.
  • one or more formulations of the dosage form include excipients, including for example and without limitation, one or more binders (binding agents), thickening agents, surfactants, diluents, release-delaying agents, colorants, flavoring agents, fillers, disintegrants/dissolution promoting agents, lubricants, plasticizers, silica flow conditioners, glidants, anti-caking agents, anti-tacking agents, stabilizing agents, anti-static agents, swelling agents and any combinations thereof.
  • binders binding agents
  • thickening agents including for example and without limitation, one or more binders (binding agents), thickening agents, surfactants, diluents, release-delaying agents, colorants, flavoring agents, fillers, disintegrants/dissolution promoting agents, lubricants, plasticizers, silica flow conditioners, glidants, anti-caking agents, anti-tacking agents, stabilizing agents, anti-static agents, swelling agents and any combinations thereof.
  • a single excipient can fulfill more than two functions at once, e.g., can act as both a binding agent and a thickening agent.
  • these terms are not necessarily mutually exclusive.
  • Examples of commonly used excipient include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition.
  • lipid vesicle e.g., liposome
  • wetting agents or auxiliary substances such as emulsifying agents, preservatives, or buffers, which increase the shelf life or effectiveness.
  • the antibody or antigen-binding fragment thereof defined herein is encapsulated in a vesicle or vesicle-like particle, such as a lipid vesicle (e.g., liposome).
  • composition may also comprise one or more additional active agents for the treatment the targeted disease/condition or for the management of symptom(s) of the targeted disease/condition (e.g., pain killers, anti-nausea agents, anti-inflammatory agents, immunotherapeutic agents, etc.).
  • additional active agents for the treatment the targeted disease/condition or for the management of symptom(s) of the targeted disease/condition (e.g., pain killers, anti-nausea agents, anti-inflammatory agents, immunotherapeutic agents, etc.).
  • the composition is a liquid or semi-liquid composition.
  • the composition of the disclosure is in the form of a liquid mixture.
  • the composition of the disclosure is in the form of an aqueous mixture.
  • the liquid mixture is selected from liquid solution, liquid suspension and liquid emulsion.
  • the aqueous mixture is an aqueous solution, aqueous suspension, and aqueous emulsion.
  • the composition of the current invention is in the form of an aqueous solution.
  • the composition further comprises a saline solution or buffer, such as phosphate-buffered saline (PBS).
  • a saline solution or buffer such as phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the pH of the solution or buffer is from 6 to 8.
  • the pH of the solution or buffer is from 6.5 to 7.5 or from 7 to 7.5, for example about 7.2.
  • the composition further comprises a surfactant or emulsifier.
  • the surfactant is a nonionic surfactant, such as a nonionic polyoxyethylene surfactant.
  • the surfactant is a polysorbate, such as Polysorbate-20 or Polysorbate-80.
  • the surfactant or emulsifier is present in at a concentration of about 0.001% to 0.1% in the composition.
  • the surfactant or emulsifier is present in at a concentration of about 0.005% to 0.05% in the composition.
  • the surfactant or emulsifier is present in at a concentration of about 0.01% in the composition.
  • the composition comprises (i) the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof described herein; (ii) a saline solution or buffer, such as PBS; and (iii) a surfactant or emulsifier, such as a Polysorbate (e.g., Polysorbate-20).
  • a saline solution or buffer such as PBS
  • a surfactant or emulsifier such as a Polysorbate (e.g., Polysorbate-20).
  • the composition or dosage form comprises from 1 mg to 30 mg of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof described herein. In a further embodiment, the composition or dosage form comprises from 2 mg to 20 mg of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof described herein. In a further embodiment, the composition or dosage form comprises from 5 mg to 15 mg of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof described herein. In a further embodiment, the composition or dosage form comprises about 6, 7, 8, 9, 10, 11 , 12, 13 or 14 mg of the combination of anti-SARS-CoV-2 antibodies or antigenbinding fragments thereof described herein. In a further embodiment, the composition or dosage form comprises about 10 mg of the combination of anti-SARS-CoV-2 antibodies or antigenbinding fragments thereof described herein.
  • the present disclosure provides a dosage form, e.g., an aerosol, comprising the composition described herein.
  • a dosage form e.g., an aerosol
  • aerosol refers to a suspension of fine solid particles or liquid droplets in air or another gas.
  • the composition or dosage form is suitable for aerosolization or nebulization.
  • the dosage form may be a nebulizer cartridge containing the composition, which is adapted for aerosolization from a nebulizer.
  • the composition may be for filling a nebulizer.
  • Such compositions or dosage forms allow delivery of the antibodies or antigen-binding fragments (or nucleic acids encoding same) to specific sites of action along the nasopharyngeal, trachea, and/or lungs, i.e., in the respiratory tract.
  • any device or system for generating an aerosol may be used for administration of the composition described herein.
  • Such devices or systems include for example nebulizers and nebulizer systems or inhalers and inhaler systems.
  • the nebulizer or inhaler may be a jet nebulizer (or atomizer), an ultrasonic nebulizer (e.g., OmronTM NE-U17 and BeurerTM Nebulizer IH30), a vibrating mesh nebulizer (e.g., Pari eFlowTM, Respironics i-NebTM, BeurerTM Nebulizer IH50, Aerogen Solo/UltraTM), a soft mist inhaler (e.g., Respimat® Soft MistTM Inhaler, Boehringer Ingelheim) or a metered dose inhaler.
  • a jet nebulizer or atomizer
  • an ultrasonic nebulizer e.g., OmronTM NE-
  • the nebulizer or inhaler is a vibrating mesh nebulizer.
  • the droplets of the aerosol delivered by the nebulizer have a Volumetric Median Diameter (VMD) of no more than 5 microns.
  • VMD Volumetric Median Diameter
  • the droplets of the aerosol delivered by the nebulizer have a VMD of between about 1 micron to about 5 microns, between about 2 microns to about 5 microns or between about 3 microns to about 5 microns.
  • the VMD is measured by laser diffraction spectroscopy (LDS).
  • composition may also comprise one or more additional active agents for the treatment the targeted disease/condition or for the management of symptom(s) of the targeted disease/condition (e.g., pain killers, anti-nausea agents, anti-inflammatory agents, immunotherapeutic agents, etc.).
  • additional active agents for the treatment the targeted disease/condition or for the management of symptom(s) of the targeted disease/condition (e.g., pain killers, anti-nausea agents, anti-inflammatory agents, immunotherapeutic agents, etc.).
  • the present disclosure provides a method for preventing a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 infection or a related disease (Coronavirus disease 2019, COVID-19), in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition described herein.
  • a betacoronavirus such as a sarbecovirus, e.g., SARS-CoV-2 infection or a related disease (Coronavirus disease 2019, COVID-19)
  • a betacoronavirus such as a sarbecovirus, e.g., SARS-CoV-2 infection or a related disease (e.g., COVID-19) in a subject.
  • the present disclosure also provides the use of the composition described herein, for the manufacture of a medicament for preventing a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 infection or a related disease (e.g., COVID-19) in a subject.
  • a betacoronavirus such as a sarbecovirus, e.g., SARS-CoV-2 infection or a related disease (e.g., COVID-19) in a subject.
  • the present disclosure provides a method for reducing the risk of developing a betacoronavirus-related disease, such as a sarbecovirus-related disease (e.g., COVID-19), or the severity of a betacoronavirus-related disease, such as a sarbecovirus-related disease (e.g., COVID-19), in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition described herein.
  • a betacoronavirus-related disease such as a sarbecovirus-related disease (e.g., COVID-19)
  • a sarbecovirus-related disease e.g., COVID-19
  • the present disclosure also provides the use of the composition described herein for reducing the risk of developing a betacoronavirus-related disease, such as a sarbecovirus-related disease (e.g., COVID-19), or the severity of a betacoronavirus-related disease, such as a sarbecovirus-related disease (e.g., COVID-19), in a subject.
  • a betacoronavirus-related disease such as a sarbecovirus-related disease (e.g., COVID-19)
  • COVID-19 sarbecovirus-related disease
  • the present disclosure also provides the composition described herein for use in reducing the risk of developing a betacoronavirus-related disease, such as a sarbecovirus-related disease (e.g., COVID-19), or the severity of a betacoronavirus-related disease, such as a sarbecovirus-related disease (e.g., COVID-19), in a subject.
  • a betacoronavirus-related disease such as a sarbecovirus-related disease (e.g., COVID-19)
  • COVID-19 sarbecovirus-related disease
  • the present disclosure provides a method in vitro or in vivo) for blocking the entry of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 in a cell, such as an ACE2-expressing cell, comprising contacting the cell and/or virus with an effective amount of the composition described herein.
  • a betacoronavirus such as a sarbecovirus, e.g., SARS-CoV-2 in a cell, such as an ACE2-expressing cell.
  • the present disclosure provides the use of the composition described herein for the manufacture of a medicament for blocking the entry of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 in a cell, such as an ACE2- expressing cell.
  • a betacoronavirus such as a sarbecovirus, e.g., SARS-CoV-2 in a cell, such as an ACE2- expressing cell.
  • the present disclosure provides the composition described herein, for use in blocking the entry of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 in a cell, such as an ACE2-expressing cell.
  • the methods and uses defined herein are for the prevention, treatment and/or management of infections by the Wuhan original SARS-CoV-2 strain.
  • the methods and uses defined herein are for the prevention, treatment and/or management of infections by variants of the Wuhan original SARS-CoV-2 strain, such as the B.1.1.7 (also known as VOC-202012/01 or alpha (a)), 501Y.V2 (also known as B.1.351 or beta (P)), P.1 (also known as B.1.1.28.1 or gamma (y)), B.1.617.2 (also known as delta (5)), or B.1.1.529 (omicron) variant, as well as other variants of concern (VOC) such as B.1.429, B.1.526,
  • B.1.1.7 also known as VOC-202012/01 or alpha (a)
  • 501Y.V2 also known as B.1.351 or beta (P)
  • P.1 also known as B.1.1.28.1 or gamma (y)
  • B.1.617.2 also
  • the methods and uses defined herein are for the prevention, treatment and/or management of infections by the SARS-CoV-2 Delta (5) variant.
  • the methods and uses defined herein are for the prevention, treatment and/or management of infections by the SARS-CoV-2 Omicron variant, e.g., Omicron sublineage BA.1 , BA1.1.529, BA1.5, BA.2, BA2.12.1 , BA2.75, BA.3, BA.4/5, BA.4/6, BQ1.1 , XBB and/or XBB1 .5.
  • described herein is a method of delivery of the composition described herein to the respiratory system of an individual infected with a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2, the method comprising administering the composition described herein in nebulized or aerosolized form to the individual.
  • a betacoronavirus such as a sarbecovirus, e.g., SARS-CoV-2
  • composition described herein in nebulized or aerosolized form for delivering the composition to the respiratory system of an individual infected with a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2.
  • a betacoronavirus such as a sarbecovirus, e.g., SARS-CoV-2.
  • the appropriate dosage of the combination/composition described herein will depend on the type of disease or condition to be treated, the severity and course of the disease or condition, whether the combination/composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the composition, and the discretion of the attending physician.
  • the combination/composition described herein may be suitably administered to the patient at one time or over a series of treatments. Preferably, it is desirable to determine the dose-response curve in vitro, and then in useful animal models prior to testing in humans.
  • the present disclosure provides dosages for the combination/composition.
  • the effective dose may be 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/ 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000 mg/kg, or may range between any two of the foregoing values.
  • a typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the dosage of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof is from 1 mg to 30 mg. In a further embodiment, the dosage of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof is from 2 mg to 20 mg.
  • the dosage of the combination of anti- SARS-CoV-2 antibodies or antigen-binding fragments thereof is from 5 mg to 15 mg. In a further embodiment, the dosage of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof is about 6, 7, 8, 9, 10, 11 , 12, 13 or 14 mg. In a further embodiment, the dosage of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof is about 10 mg.
  • treating or “treatment” in reference to viral infection or disease is meant to refer to administration of the composition after infection that leads to a reduction/improvement in one or more symptoms or pathological features associated with said viral disease (e.g., COVID-19).
  • Non-limiting examples include a decrease in viral load, reduction of cough, fever, fatigue, shortness of breath, reduction/prevention of acute respiratory distress syndrome (ARDS), reduction/prevention of multi-organ failure, septic shock, and blood clots, hospitalization, etc.
  • ARDS acute respiratory distress syndrome
  • preventing or “prevention” in reference to viral infection or disease is meant to refer to administration of the composition prior to infection that leads to protection from being infected or from developing the viral disease (e.g., COVID-19), to a delay in the development of the disease, or to a reduction of one or more symptoms or pathological features associated with the viral disease.
  • the viral disease e.g., COVID-19
  • the administration/use of the composition described herein delays the onset of one or more symptoms of a betacoronavirus or sarbecovirus-caused infection, e.g., SARS-CoV-2-caused infection (e.g., COVID-19).
  • a betacoronavirus or sarbecovirus-caused infection e.g., SARS-CoV-2-caused infection (e.g., COVID-19).
  • composition described herein may be used alone or in combination with other prophylactic agents such as antivirals, anti-inflammatory agents, vaccines, immunotherapies, etc.
  • the combination of active agents and/or compositions comprising same may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form.
  • Co-administration in the context of the present disclosure refers to the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time.
  • a first agent e.g., the composition described herein
  • a second active agent e.g., an antiviral or anti-inflammatory agent
  • the agents may in an embodiment be combined/formulated in a single composition and thus administered at the same time.
  • the composition described herein is for administration prior to exposure to a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2.
  • a betacoronavirus such as a sarbecovirus, e.g., SARS-CoV-2.
  • the composition described herein is for administration after exposure to a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2.
  • the composition described herein is for administration prior to and after exposure to a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2.
  • the composition described herein is for administration prior to development of the viral disease (e.g., COVID-19). In another embodiment, the composition described herein is for administration after development of the viral disease (e.g., COVID-19). In another embodiment, the composition described herein is for administration prior to and after development of the viral disease (e.g., COVID-19).
  • the subject or patient has a weakened immune system and a reduced ability to fight viral infections such as SARS-CoV-2 infection.
  • the subject or patient is an immunosuppressed or immunocompromised subject or patient. Immunosuppression may be caused by certain diseases or conditions, such as AIDS, cancer, diabetes, malnutrition, and certain genetic disorders, or certain drugs or treatments such as anticancer drugs, radiation therapy, and stem cell or organ transplant.
  • the subject or patient is an elderly subject or patient, for example a subject or patient having 60 years old or more, 65 years old or more, 70 years old or more, 75 years old or more, or 80 years old or more, who typically develop a weaker immune response to vaccines and infections.
  • kits comprising the composition of the disclosure.
  • Kits include one or more containers comprising by way of example, and not limitation, the composition or dosage form described herein and instructions for use in accordance with any of the methods of the disclosure.
  • instructions comprise a description of administration.
  • the containers can be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
  • Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • kits are provided in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, tubes, jars, flexible packaging (e.g., sealed Mylar or plastic bags), nebulizer cartridge, and the like.
  • a kit can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the container can also have a sterile access port (e.g., the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • Kits can optionally provide additional components such as buffers and interpretive information.
  • the kit comprises a container and a label or package insert(s) on or associated with the container.
  • the kit include a device for intrapulmonary administration of the composition, such as a nebulizer, metered-dose inhaler, or dry powder inhaler.
  • Antibodies Characteristics of anti-SARS-CoV-2 antibodies IBIO1 , IBIO2 and IBIO3 present in the antibody cocktail used in the studies reported herein are depicted in Table 1 below. IBIO1 and IBIO2 are described in PCT application No. PCT/CA2021/051873, and IBIO3 is described in PCT application No. PCT/CA2022/051074.
  • Binding assay on Trimeric SARS-CoV-2 Spike Protein.
  • An indirect dose-response ELISA assay was performed after adsorption of 100 ng of trimeric Spike protein (SMT1-1 , NRCC) in each well of a 96-wells microplate (Nunc MaxisorbTM). Increasing doses of the antibody cocktail were deposited in each well.
  • Bovine Serum Albumin (BSA) was used as a negative control antigen. Binding was revealed by the addition of an anti-Human IgG/HRP secondary antibody. Signal was measured by optical density at 450 nm (Biotek Instruments, Synergy HTX).
  • SARS-CoV-2 neutralization assay (FIGs. 4 and 5). Pseudovirions expressing the SARS- CoV-2 spike protein (from the Wuhan strain) and carrying an eGFP reporter gene were produced in 293T cells. HEK cells overexpressing ACE2/TMPRSS2 were used for infection. Pseudovirions produced without Spike served as negative controls. Expression of the eGFP reporter gene follows fluorescently-labeled virus in living cells and allows quantification of viral infection. The efficiency of eGFP transduction was analyzed by flow cytometry or fluorescent plate reader (IncuCyte 53 and Mithras, respectively) 48 hours post-infection. A characterized anti-SARS-2 rabbit monoclonal antibody was used as a positive control.
  • SARS-CoV-2 neutralization assay (FIGs. 6A-B). Lentiviruses were produced in 293T cell by transfecting the 4 pg pHAGE-CMV-Luc2-IRES-zsGreen3 pg psPAX2 (packaging vector): and 3 pg pcDNA3.1 -SARS-CoV-2 spike delta19 (spike glycoprotein) and PEI transfecting agent. Viruses were harvested 48h after the transfection. The neutralization activity was measured by microneutralization assay in vitro.
  • the virus microneutralization (MN) test was performed on 293T-ACE2 cells infected with SARS-CoV-2 (2019-nCoV) Spike Pseudovirus under treatment of serial dilutions of the antibody cocktail. Rate of inhibition was determined by comparing the Relative Light Unit (RLU) of Luciferase reporter in different antibody concentrations with plate reader
  • a up to five SARS-CoV-2 virus strains (FIG. 6C).
  • the assays were conducted in triplicate in flatbottom 96-well plates using Vero’76 cells grown at 80% - 90% confluence.
  • the virus inoculum sizes were approximately 25-50 TCID50 per well.
  • Testing concentrations of the antibodies before equal volume mixture with virus solution were 50, 25, 12.5, 6.25, 3.12, 1 .56, 0.8 and 0.00 pg/ml. Therefore, the final concentrations in the reactions were 25, 12.5, 6.25, 3.12, 1.56, 0.8, 0.4 and 0.00 pg/ml.
  • the cytopathic effect (CPE) was assessed by microscopic examination on Day 3 and 5, and the lowest antibody concentration with no CPE at Day 5 was defined as the neutralization titer in pg/ml.
  • CPE cytopathic effect
  • the CPE in each well was scored. No CPE in a well was scored 0.1 , and the degree of CPE intensity in a well with virus infection without antibody treatment was scored 100.
  • the antibodies (6.3 mg of IBIO1 , 0.5 mg of IBIO2 and 3.2 mg of IBIO3) were mixed in 1 ml of phosphate-buffered saline (PBS) 1X (pH 7.2) with 0.01% polysorbate-20.
  • PBS phosphate-buffered saline
  • Droplet size characterization expressed as volumetric median droplet diameter (VMD/Dv5O) of aerosolized samples was determined by laser diffraction spectroscopy using the Malvern Spraytec device in order to characterize the volumetric droplet size generated by the Aerogen Solo/Ultra + mouthpiece combination. Testing was carried out using 0.25 mL of the IBIO123 antibody cocktail at a concentration of 10 mg/ml.
  • Anti-SARS-CoV-2 spike protein antibodies were mixed in various proportions (FIG. 2A) and the antibody cocktails were tested in a SARS-CoV-2 neutralization assay. As shown in FIG. 2B, there was some significant differences in the potency of the antibody cocktails to neutralize infection of HEK cells by pseudovirions expressing the SARS-CoV-2 spike protein. Because it exhibited one the lowest IC50, IC 8 o and IC90 among the antibody cocktails tested, cocktail J (hereinafter referred to as IBIO123) that comprises 63% of IBIO1 , 5% of IBIO2 and 32% of IBIO3 was selected for further studies.
  • IBIO123 cocktail J that comprises 63% of IBIO1 , 5% of IBIO2 and 32% of IBIO3 was selected for further studies.
  • FIG. 3 shows that antibody cocktail IBIO123 binds trimeric SARS-CoV-2 Spike protein in a dose-dependent manner and with an EC 5 o of 43.67 ng/ml. Additional SARS-CoV-2 neutralization assay performed revealed that antibody cocktail IBIO123 exhibits an IC 5 o between 7.76 ng/ml and 17.23 ng/ml (FIGs. 4 and 5), compared to 575.8 ng/ml for a reference SARS-CoV-2 Spike Neutralizing Antibody from Sino Biological (Cat. No. 40592-MM57) (FIG. 5).
  • FIGs. 6A and 6B show that antibody cocktail I BI0123 maintains the ability to neutralize pseudovirions expressing the SARS-CoV-2 spike protein from several VOCs including the highly mutated Omicron variants (albeit with reduced potency). Similar results were obtained in neutralization assays using complete viruses (FIG. 6C).
  • the antibody cocktail I BI0123 was able to completely neutralize infection by the Delta variant at all doses tested, whereas higher doses of the cocktail were needed to neutralize the Omicron variants (BA.1 , BA.2 et BA.5).
  • IBIO123 neutralization concentrations were 0.4 pg/ml on the Delta variant, 25 pg/ml on Omicron BA.1 , 15 pg/ml on Omicron BA.2 and 16.5 pg/ml on Omicron BA.5, respectively.
  • Example 3 Assessment of a nebulized formulation of antibody cocktail IBIO123
  • the formulation for nebulization comprises 10 mg of the antibody cocktail IBIO123 in 1 ml of phosphate-buffered saline (PBS) 1X (pH 7.2) with 0.01% polysorbate-20.
  • PBS phosphate-buffered saline
  • FIG. 7A and FIG. 7B show that nebulization of the formulation using a vibrating mesh nebulizer has no significant effect on the ability of the antibodies to bind to trimeric SARS-CoV-2 Spike Protein (FIG. 7A) or to neutralize infection of HEK cells by pseudovirions expressing the SARS- CoV-2 spike protein (FIG. 7B).
  • VMD/Dv50 volumetric median droplet diameter

Abstract

The present application relates to a composition comprising a combination of anti-SARS- CoV-2 antibodies or antigen-binding fragments thereof, or of nucleic acids encoding the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof. Methods and uses of the compositions for the prevention and/or treatment of betacoronavirus infections and related diseases such as COVID-19, are also described. The compositions may be administered in the respiratory tract, e.g., in nebulized or aerosolized form, to subjects infected with or at risk of being infected with a coronavirus such as SARS-CoV-2.

Description

TITLE OF INVENTION
FORMULATION OF ANTI-SARS-COV-2 NEUTRALIZING ANTIBODIES AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. provisional patent application No. 63/268,657, filed on February 28, 2022, which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
The present disclosure generally relates to viral infection, and more particularly to the prevention and/or treatment of coronavirus infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.
BACKGROUND ART
Coronaviruses are large, roughly spherical, RNA viruses with bulbous surface projections that cause diseases in mammals and birds. In humans, these viruses cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold (which is also caused by other viruses, predominantly rhinoviruses), while more lethal varieties can cause severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Coronavirus disease 2019 (COVID-19). Coronaviruses have four structural proteins, namely the Spike (S), Envelope (E), and Membrane (M) proteins, forming the viral envelope, as well as the Nucleocapsid (N) protein, holding the viral RNA genome.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the strain of coronavirus that causes COVID-19, the respiratory illness responsible for the COVID-19 pandemic. The spike protein SARS-CoV-2 is the glycoprotein responsible for allowing the virus to attach to and fuse with the membrane of a host cell; specifically, its S1 subunit contains the receptor-binding domain (RBD) that interacts with the cellular receptor angiotensin-converting enzyme 2 (ACE2) and catalyzes virus attachment, and its S2 subunit possesses the fusion machinery, which can mediate host-viral membrane fusion after S1 shedding. The main receptor involved in SARS- CoV-2 entry into human cells is the angiotensin converting enzyme 2 (ACE2). After attachment of a SARS-CoV-2 virion to a target cell, the cell's protease transmembrane protease, serine 2 (TMPRSS2) cuts open the spike protein of the virus, exposing a fusion peptide in the S2 subunit, and the host receptor ACE2.
Multiple variants of SARS-CoV-2 are circulating globally and within the United States. Four new variants that have rapidly become dominant within their countries have aroused concerns: B.1.1.7 (also known as VOC-202012/01), 501Y.V2 (B.1.351), P.1 (B.1.1.28.1) delta (B.1.617.2) and Omicron (B.1.1.529). The B.1.1.7 variant (23 mutations with 17 amino acid changes) was first described in the United Kingdom in December 2020; the 501Y.V2 variant (23 mutations with 17 amino acid changes) was initially reported in South Africa in December 2020; and the P.1 variant (approximately 35 mutations with 17 amino acid changes) was reported in Brazil in January 2021 . By February 2021 , the B.1.1.7 variant had been reported in 93 countries, the 501 Y.V2 variant in 45, and the P.1 variant in 21. All three variants have the N501Y mutation, which changes the amino acid asparagine (N) to tyrosine (Y) at position 501 in the receptor-binding domain of the spike protein. The 501Y.V2 and P.1 variants both have two additional receptor-binding-domain mutations, K417N/T and E484K. These mutations increase the binding affinity of the receptorbinding domain to the angiotensin-converting enzyme 2 (ACE2) receptor. Four key concerns stemming from the emergence of the new variants are their effects on viral transmissibility, disease severity, reinfection rates (i.e., escape from natural immunity), and vaccine effectiveness (i.e., escape from vaccine-induced immunity). Recently, two more SARS-CoV-2 variants, B.1 .427 and B.1.429, which were first detected in California, have been shown to be approximately 20% more transmissible than pre-existing variants and have been classified by the CDC as variants of concern. The B.1.617.2 delta variant comprises the following substitutions in the Spike protein that are known to affect transmissibility of the virus: D614G, T478K, P681 R and L452R. On November 26, 2021 , WHO designated the variant B.1 .1 .529 (Omicron) a variant of concern based on several mutations that may have an impact on how it behaves, for example, on how easily it spreads or the severity of illness it causes.
Studies on these variants have provided compelling evidence that they have the potential to escape naturally-induced immunity as well as the immunity induced by currently approved vaccines.
Most neutralizing antibodies described so far target the or near the ACE2-binding interface of the RBD, making them vulnerable to escape by evolving viral mutations within the RBD. Antibody monotherapy significantly increases this risk. Dual-Ab cocktails from Regeneron (casirivimab and imdevimab, targeting adjacent, non-overlapping epitopes) and Eli Lilly (bamlanivimab and etesevimab, targeting overlapping epitopes) leaves both Abs in each cocktail potentially susceptible to evasion by single point mutations. Indeed, recent evidence suggests that these cocktails are ineffective at neutralizing Omicron (https://www.medrxiv.orq/content/10.1101/2021.12.07.21267432v4; https://www.medrxiv.Org/content/10.1101/2021.12.14.21267769v1.full-text#T1).
Thus, there is a need for the development of therapies that elicit neutralizing activity against SARS-CoV-2, including SARS-CoV-2 variants, and that minimize the risk of viral escape.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety. SUMMARY
The present disclosure provides the following items:
1 . A composition comprising a combination of anti-SARS-CoV-2 antibodies or antigenbinding fragments thereof, said combination comprising:
(a) 55% to 70% of a first antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs): a light chain CDR1 (CDR-L1) comprising the sequence RASQSVSSSYLA (SEQ ID NO:7); a light chain CDR2 (CDR-L2) comprising the sequence GASSRAT (SEQ ID NO:8); a light chain CDR3 (CDR-L3) comprising the sequence QQYGTSPWT (SEQ ID NO:9); a heavy chain CDR1 (CDR-H1) comprising the sequence GFTFTSS (SEQ ID NO: 10); a heavy chain CDR2 (CDR-H2) comprising the sequence WGSGN (SEQ ID NO:11); and a heavy chain CDR3 (CDR-H3) comprising the sequence PSCSGGRCYDGFDI (SEQ ID NO:12);
(b) 2% to 10% of a second antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising the sequence RASQGISSWLA (SEQ ID NO: 13); a CDR- L2 comprising the sequence AASSLQS (SEQ ID NO:14); a CDR-L3 comprising the sequence QQGNSFPYT (SEQ ID NO:15); a CDR-H1 comprising the sequence GYTFTRY (SEQ ID NO:16); a CDR-H2 comprising the sequence YPGDSD (SEQ ID NO:17); and a CDR-H3 comprising the sequence LPQYCSNGVCQRWFDP (SEQ ID NO:18); and
(c) 25% to 40% of a third antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising the sequence RASQSVSSSYLA (SEQ ID NO:7); a CDR-L2 comprising the sequence GASSRAT (SEQ ID NO:8); a CDR-L3 comprising the sequence QQYGSSYT (SEQ ID NO:19); a CDR-H1 comprising the sequence GITVSSN (SEQ ID NO:20); a CDR-H2 comprising the sequence YSGGS (SEQ ID NO:21); and a CDR-H3 comprising the sequence DLEMAGAFDI (SEQ ID NO:22).
2. The composition of item 1 , wherein the first antibody or antigen-binding fragment thereof comprises a light chain FR1 comprising the sequence of the FR1 depicted in FIG. 1A (SEQ ID NO:27).
3. The composition of item 1 or 2, wherein the first antibody or antigen-binding fragment thereof comprises a light chain FR2 comprising the sequence of the FR2 depicted in FIG. 1A (SEQ ID NO:28).
4. The composition of any one of items 1 to 3, wherein the first antibody or antigen-binding fragment thereof comprises a light chain FR3 comprising the sequence of the FR3 depicted in FIG. 1A (SEQ ID NO:29).
5. The composition of any one of items 1 to 4, wherein the first antibody or antigen-binding fragment thereof comprises a light chain FR4 comprising the sequence of the FR4 depicted in FIG. 1A (SEQ ID NQ:30). 6. The composition of any one of items 1 to 5, wherein the third antibody or antigen-binding fragment thereof comprises a light chain FR1 comprising the sequence of the FR1 depicted in FIG. 1C (SEQ ID NO:43).
7. The composition of any one of items 1 to 6, wherein the third antibody or antigen-binding fragment thereof comprises a light chain FR2 comprising the sequence of the FR2 depicted in FIG. 1C (SEQ ID NO:44).
8. The composition of any one of items 1 to 7, wherein the third antibody or antigen-binding fragment thereof comprises a light chain FR3 comprising the sequence of the FR3 depicted in FIG. 10 (SEQ ID NO:45).
9. The composition of any one of items 1 to 8, wherein the third antibody or antigen-binding fragment thereof comprises a light chain FR4 comprising the sequence of the FR4 depicted in FIG. 1C (SEQ ID NO:46).
10. The composition of any one of items 1 to 9, wherein the second antibody or antigenbinding fragment thereof comprises a light chain FR1 comprising the sequence of the FR1 depicted in FIG. 1 B (SEQ ID NO:35).
11 . The composition of any one of items 1 to 10, wherein the second antibody or antigenbinding fragment thereof comprises a light chain FR2 comprising the sequence of the FR2 depicted in FIG. 1 B (SEQ ID NO:36).
12. The composition of any one of items 1 to 11 , wherein the second antibody or antigenbinding fragment thereof comprises a light chain FR3 comprising the sequence of the FR3 depicted in FIG. 1 B (SEQ ID NO:37).
13. The composition of any one of items 1 to 12, wherein the second antibody or antigenbinding fragment thereof comprises a light chain FR4 comprising the sequence of the FR4 depicted in FIG. 1 B (SEQ ID NO:38).
14. The composition of any one of items 1 to 13, wherein the first antibody or antigenbinding fragment thereof comprises a heavy chain comprising the heavy chain sequence depicted in FIG. 1A (SEQ ID NO:1), and a light chain comprising the light chain sequence depicted in FIG. 1A (SEQ ID NO:2).
15. The composition of any one of items 1 to 14, wherein the third antibody or antigenbinding fragment thereof comprises a heavy chain comprising the heavy chain sequence depicted in FIG. 1C (SEQ ID NO:3), and a light chain comprising the light chain sequence depicted in FIG. 1C (SEQ ID NO:4).
16. The composition of any one of items 1 to 15, wherein the second antibody or antigenbinding fragment thereof comprises a heavy chain comprising the heavy chain sequence depicted in FIG. 1 B (SEQ ID NO:5), and a light chain comprising the light chain sequence depicted in FIG. 1 B (SEQ ID NO:6). 17. The composition of any one of items 1 to 16, wherein the first antibody or antigenbinding fragment thereof is a human antibody.
18. The composition of any one of items 1 to 17, wherein the second antibody or antigenbinding fragment thereof is a human antibody.
19. The composition of any one of items 1 to 18, wherein the third antibody or antigenbinding fragment thereof is a human antibody.
20. The composition of any one of items 1 to 19, wherein the first, second and/or third antibodies are of the IgG class.
21 . The composition of item 20, wherein the first, second and/or third antibodies are of the lgG1 subclass.
22. The composition of any one of items 1 to 21 , wherein the combination comprises about 55% to about 65% of the first antibody or antigen-binding fragment thereof.
23. The composition of item 22, wherein the combination comprises about 63% of the first antibody or antigen-binding fragment thereof.
24. The composition of any one of items 1 to 23, wherein the combination comprises about 28% to about 37% of the third antibody or antigen-binding fragment thereof.
25. The composition of item 24, wherein the combination comprises about 32% of the third antibody or antigen-binding fragment thereof.
26. The composition of any one of items 1 to 25, wherein the combination comprises about 3% to about 8% of the second antibody or antigen-binding fragment thereof.
27. The composition of item 26, wherein the combination comprises about 5% of the second antibody or antigen-binding fragment thereof.
28. The composition of any one of items 1 to 27, further comprising a pharmaceutically acceptable carrier or excipient.
29. The composition of item 28, wherein the pharmaceutically acceptable carrier or excipient is adapted for administration of the composition by the pulmonary route.
30. The composition of item 28 or 29, wherein the composition is an aqueous solution, suspension or emulsion.
31 . The composition of any one of items 28 to 30, wherein the pharmaceutically acceptable carrier or excipient comprises a saline solution or buffer.
32. The composition of any one of items 28 to 31 , wherein the pharmaceutically acceptable carrier or excipient comprises a surfactant or emulsifier.
33. The composition of item 32, wherein the surfactant or emulsifier is a polysorbate.
34. The composition of item 32 or 33, wherein the surfactant or emulsifier is present at a concentration of 0.005% to 0.05% in the composition.
35. The composition of any one of items 28 to 34, wherein the composition has a pH of about 6.5 to about 7.5. 36. A composition comprising one or more nucleic acids encoding the first, second and third antibodies or antigen-binding fragments thereof defined in any one of items 1 to 27.
37. The composition of item 36, wherein the one or more nucleic acids comprise a first nucleic acid encoding the first antibody or antigen-binding fragment thereof defined in any one of items 1 to 27, a second nucleic acid encoding the second antibody or antigen-binding fragment thereof defined in any one of items 1 to 27, and a third nucleic acid encoding the third antibody or antigen-binding fragment thereof defined in any one of items 1 to 27.
38. The composition of item 36 or 37, wherein the one or more nucleic acids are mRNA molecules.
39. The composition of any one of items 36 to 38, wherein the one or more nucleic acids are encapsulated within vesicles.
40. The composition of item 39, wherein the vesicles are lipid nanoparticles or liposomes.
41 . An inhaler or nebulization device comprising the composition of any one of items 1 to 40.
42. A method for preventing or treating a betacoronavirus infection or a related disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of any one of items 1 to 40.
43. A method for reducing the risk of developing a betacoronavirus-related disease or the severity of a betacoronavirus-related disease in a subject, the method comprising administering to the subject an effective amount of the composition of any one of items 1 to 40.
44. A method for blocking the entry of a betacoronavirus in an ACE2-expressing cell, the method comprising contacting the cell and/or the virus with an effective amount of the composition of any one of items 1 to 40.
45. The method of any one of items 42 to 44, wherein the betacoronavirus is a sarbecovirus.
46. The method of item 45, wherein the sarbecovirus is SARS-CoV-2.
47. The method of item 46, wherein the SARS-CoV-2 is a variant of the Wuhan original
SARS-CoV-2 strain, such as an Omicron variant.
48. The method of any one of items 42 to 47, wherein the composition is administered with (i) at least one additional anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, or nucleic acid(s) encoding said at least one additional anti-SARS-CoV-2 antibody or antigenbinding fragment thereof; and/or (ii) at least one antiviral or anti-inflammatory drug.
49. The method of any one of items 42 to 48, wherein the subject is an immunosuppressed or immunocompromised subject.
50. Use of the composition of any one of items 1 to 40 for preventing or treating a betacoronavirus infection or a related disease in a subject.
51 . Use of the composition of any one of items 1 to 40 for the manufacture of a medicament for preventing or treating a betacoronavirus infection or a related disease in a subject. 52. Use of the composition of any one of items 1 to 40 for reducing the risk of developing a betacoronavirus-related disease or the severity of a betacoronavirus-related disease in a subject.
53. Use of the composition of any one of items 1 to 40 for the manufacture of a medicament for reducing the risk of developing a betacoronavirus-related disease or the severity of a betacoronavirus-related disease in a subject.
54. Use of the composition of any one of items 1 to 40 for blocking the entry of a betacoronavirus in an ACE2-expressing cell.
55. Use of the composition of any one of items 1 to 40 for the manufacture of a medicament for blocking the entry of a betacoronavirus in an ACE2-expressing cell.
56. The use of any one of items 50 to 55, wherein the betacoronavirus is a sarbecovirus.
57. The use of item 56, wherein the sarbecovirus is SARS-CoV-2.
58. The use of item 57, wherein the SARS-CoV-2 is a variant of the Wuhan original SARS-
CoV-2 strain, such as an Omicron variant.
59. The use of any one of items 50 to 58, wherein the composition is for administration with (i) at least one additional anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, or nucleic acid(s) encoding said at least one additional anti-SARS-CoV-2 antibody or antigenbinding fragment thereof; and/or (ii) at least one antiviral or anti-inflammatory drug.
60. The use of any one of 50 to 59, wherein the subject is an immunosuppressed or immunocompromised subject.
61 . The composition of any one of items 1 to 40 for use in preventing or treating a betacoronavirus infection or a related disease in a subject.
62. The composition of any one of items 1 to 40 for use in reducing the risk of developing a betacoronavirus-related disease or the severity of a betacoronavirus-related disease in a subject.
63. The composition of any one of items 1 to 40 for use in blocking the entry of a betacoronavirus in an ACE2-expressing cell.
64. The composition for use of any one of items 61 to 63, wherein the betacoronavirus is a sarbecovirus.
65. The composition for use of item 64, wherein the sarbecovirus is SARS-CoV-2.
66. The composition for use of item 65, wherein the SARS-CoV-2 is a variant of the Wuhan original SARS-CoV-2 strain, such as an Omicron variant.
67. The composition for use of any one of items 61 to 66, wherein the composition is for administration with (i) at least one additional anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, or nucleic acid(s) encoding said at least one additional anti-SARS-CoV-2 antibody or antigen-binding fragment thereof; and/or (ii) at least one antiviral or anti-inflammatory drug.. Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
In the appended drawings:
FIG. 1A shows the amino acid sequence of the heavy and light chain variable regions of Antibody IBIO1 described herein. The amino acid corresponding to the CDR1 , CDR2 and CDR3 according to the Clothia numbering scheme are underlined.
FIG. 1B shows the amino acid sequence of the heavy and light chain variable regions of Antibody IBIO2 described herein. The amino acid corresponding to the CDR1 , CDR2 and CDR3 according to the Clothia numbering scheme are underlined.
FIG. 1C shows the amino acid sequence of the heavy and light chain variable regions of Antibody IBIO3 described herein. The amino acid corresponding to the CDR1 , CDR2 and CDR3 according to the Clothia numbering scheme are underlined.
FIG. 2A shows the proportion of antibody I BI01 , antibody IBIO2 and antibody IBIO3 in the antibody cocktails A-J tested in the study reported in FIG. 2B.
FIG. 2B is a graph showing the IC50, IC8o and IC99 of the antibody cocktails A-J in a SARS- CoV-2 Spike Neutralization Assay.
FIG. 3 is a graph showing the results of a dose-response direct ELISA assay of antibody cocktail J (IBIO123) binding on trimeric SARS-CoV-2 Spike Protein (100 ng). EC50 and correlation determined by the 5-parameter logistic non-linear regression curve fit analysis.
FIG. 4 is a graph showing the results of a dose-response neutralization assay of SARS- CoV-2 viral particles using antibody cocktail J (IBIO123).
FIG. 5 is a graph showing the results of a dose-response neutralization assay of SARS- CoV-2 viral particles using antibody cocktail J (IBIO123) prior and after virus filtering of the composition, or a control SARS-CoV-2 Spike Neutralizing Antibody from Sino Biological (Cat. No. 40592-MM57).
FIG. 6A and 6B show the results of a dose-response neutralization assay of SARS-CoV-2 variants of concern (VOCs) using the cell-virus fusion assay. Relative viral infectivity is shown at increasing antibody concentrations. Inhibition dose-response nonlinear regression curve fit (three parameters) was used to analyze the results.
FIG. 6C shows the results of a dose-response neutralization assay of IBIO123 on complete SARS-CoV-2 virus variants (Delta, Omicron BA.1 , Omicron BA.2 and Omicron BA.5). The cytopathic effect (CPE) score was set at 100 for the virus control and at 0.1 for the medium control.
FIG. 7A is a graph showing the results of a dose-response ELISA with initial (pre- nebulization, circles) and nebulized (squares) IBIO123 antibody cocktail on 100 ng on SARS- CoV-2 Wuhan Trimeric Spike glycoprotein (SMT1-1) (CNRC). EC5o values were calculated using the 5-parameter logistic sigmoidal non-linear regression curve-fit model.
FIG. 7B is a graph showing the results of a dose-response neutralization assay of SARS- CoV-2 Wuhan pseudovirus with initial (pre-nebulization, circles) and nebulized (post-nebulization, squares) IBIO123 antibody cocktail. IC5o values were determined using the log(inhibitor) vs. response (three parameters) dose-response inhibition curve-fit model.
FIG. 8 is a graph depicting the mutations in the Spike protein from SARS-CoV-2 VOCs Omicron sublineages BA.1 , BA.2 and BA.3 (from William A. Haseltine, Birth of The Omicron Family: BA.1 , BA.2, BA.3. Each As Different As Alpha Is From Delta, Forbes, Jan 26, 2022).
DETAILED DISCLOSURE
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The terms "comprising", "having", "including", and "containing" are to be construed as open- ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (“e.g.”, "such as") provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.
No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.
Herein, the term "about" has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.
Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives. Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1- 4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).
The present disclosure provides a composition, such as an aerosol composition, comprising a combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof, said combination comprising at least two of the following antibodies:
(a) a first antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs): a light chain CDR1 (CDR-L1) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQSVSSSYLA (SEQ ID NO:7); a light chain CDR2 (CDR-L2) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GASSRAT (SEQ ID NO:8); a light chain CDR3 (CDR- L3) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQYGTSPWT (SEQ ID NO:9); a heavy chain CDR1 (CDR-H1) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GFTFTSS (SEQ ID NQ:10); a heavy chain CDR2 (CDR-H2) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence WGSGN (SEQ ID NO: 11); and a heavy chain CDR3 (CDR-H3) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence PSCSGGRCYDGFDI (SEQ ID NO:12);
(b) a second antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQGISSWLA (SEQ ID NO: 19); a CDR- L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence AASSLQS (SEQ ID NO:20); a CDR-L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQGNSFPYT (SEQ ID NO:21); a CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GYTFTRY (SEQ ID NO:22); a CDR-H2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence YPGDSD (SEQ ID NO:23); and a CDR-H3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence LPQYCSNGVCQRWFDP (SEQ ID NO:24); and
(c) a third antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQSVSSSYLA (SEQ ID NO:13); a CDR- L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GASSRAT (SEQ ID NO:14); a CDR-L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQYGSSYT (SEQ ID NO:15); a CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GITVSSN (SEQ ID NO: 16); a CDR-H2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence YSGGS (SEQ ID NO:17); and a CDR-H3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence DLEMAGAFDI (SEQ ID NO: 18).
The present disclosure provides a composition, such as an aerosol composition, comprising a combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof, said combination comprising:
(a) 5% to 90% of a first antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs): a light chain CDR1 (CDR-L1) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQSVSSSYLA (SEQ ID NO:7); a light chain CDR2 (CDR- L2) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GASSRAT (SEQ ID NO:8); a light chain CDR3 (CDR-L3) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQYGTSPWT (SEQ ID NO:9); a heavy chain CDR1 (CDR-H1) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GFTFTSS (SEQ ID NQ:10); a heavy chain CDR2 (CDR-H2) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence WGSGN (SEQ ID NO: 11); and a heavy chain CDR3 (CDR-H3) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence PSCSGGRCYDGFDI (SEQ ID NO:12);
(b) 5% to 90% of a second antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQGISSWLA (SEQ ID NO: 19); a CDR-L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence AASSLQS (SEQ ID NQ:20); a CDR- L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQGNSFPYT (SEQ ID NO:21); a CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GYTFTRY (SEQ ID NO:22); a CDR-H2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence YPGDSD (SEQ ID NO:23); and a CDR-H3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence LPQYCSNGVCQRWFDP (SEQ ID NO:24); and
(c) 5% to 90% of a third antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQSVSSSYLA (SEQ ID NO: 13); a CDR-L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GASSRAT (SEQ ID NO:14); a CDR- L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQYGSSYT (SEQ ID NO:15); a CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GITVSSN (SEQ ID NO:16); a CDR-H2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence YSGGS (SEQ ID NO:17); and a CDR-H3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence DLEMAGAFDI (SEQ ID NO: 18).
The present disclosure also provides a composition comprising a combination of anti- SARS-CoV-2 antibodies or antigen-binding fragments thereof, said combination comprising:
(a) 55% to 70% of a first antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs): a light chain CDR1 (CDR-L1) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQSVSSSYLA (SEQ ID NO:7); a light chain CDR2 (CDR- L2) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GASSRAT (SEQ ID NO:8); a light chain CDR3 (CDR-L3) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQYGTSPWT (SEQ ID NO:9); a heavy chain CDR1 (CDR-H1) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GFTFTSS (SEQ ID NQ:10); a heavy chain CDR2 (CDR-H2) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence WGSGN (SEQ ID NO: 11); and a heavy chain CDR3 (CDR-H3) comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence PSCSGGRCYDGFDI (SEQ ID NO:12);
(b) 2% to 10% of a second antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQGISSWLA (SEQ ID NO: 19); a CDR-L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence AASSLQS (SEQ ID NQ:20); a CDR- L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQGNSFPYT (SEQ ID NO:21); a CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GYTFTRY (SEQ ID NO:22); a CDR-H2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence YPGDSD (SEQ ID NO:23); and a CDR-H3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence LPQYCSNGVCQRWFDP (SEQ ID NO:24); and
(c) 25% to 40% of a third antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence RASQSVSSSYLA (SEQ ID NO: 13); a CDR-L2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GASSRAT (SEQ ID NO:14); a CDR- L3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence QQYGSSYT (SEQ ID NO:15); a CDR-H1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence GITVSSN (SEQ ID NO:16); a CDR-H2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence YSGGS (SEQ ID NO:17); and a CDR-H3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence DLEMAGAFDI (SEQ ID NO: 18). It is to be understood that the total amount of the first, second and third antibodies or antigen-binding fragments thereof in the combination cannot exceed 100%.
The term “antibody or antigen-binding fragment thereof’ as used herein refers to any type of antibody/antibody fragment including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, humanized antibodies, CDR-grafted antibodies, chimeric antibodies and antibody fragments so long as they exhibit the desired antigenic specificity/binding activity. Antibody fragments comprise a portion of a full-length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain Fv, scFv), single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, H regions ( H, VH-VH), anticalins, PepBodies, antibody-T- cell epitope fusions (Troybodies) or Peptibodies.
The term "monoclonal antibody" as used herein refers to an antibody from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are substantially similar and bind the same epitope(s), except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. Such monoclonal antibody typically includes an antibody comprising a variable region that binds a target, wherein the antibody was obtained by a process that includes the selection of the antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that the selected antibody can be further altered, for example, to improve affinity for the target, to humanize the antibody, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered variable region sequence is also a monoclonal antibody of this disclosure. In addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including the hybridoma method (e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 , (Elsevier, N. Y., 1981), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Sidhu et al., J. Mol. Biol. 338(2) :299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al. J. Immunol. Methods 284(1 -2): 119-132 (2004) and technologies for producing human or human-like antibodies from animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO98/24893, WO96/34096, WO96/33735, and WO91/10741 , Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immune, 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591 ,669 (all of GenPharm); 5,545,807; WO 97/17852, U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661 ,016, and Marks et al., Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).
The monoclonal antibodies herein specifically include "chimeric" or “recombinant” antibodies in which a portion of the light and/or heavy chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). Chimeric antibodies of interest herein include "humanized" antibodies. In an embodiment, the antibody is a monoclonal antibody, preferably a human antibody.
The first, second and/or third antibody of the present disclosure may be of any class or isotype, e.g., IgG, IgM, IgA, IgD or IgE. In an embodiment, the first, second and/or third antibody of the present disclosure is an IgA. In an embodiment, the first, second and/or third antibody of the present disclosure is an IgG. The IgG may be of any subclass, e.g., lgG1 , lgG2, lgG3, or lgG4. In an embodiment, the first, second and/or third antibody of the present disclosure is an igGi .
In an embodiment, the first, second and/or third antibody of the present disclosure is a multispecific antibody, such as a bispecific antibody. In such multispecific (e.g., bispecific) antibodies, at least one of the antigen-binding domains comprise one of the combinations of CDRs or variable regions described herein. In an embodiment, the multispecific (e.g., bispecific) antibody comprises two of the combinations of CDRs or variable regions described herein. The multispecific (e.g., bispecific) antibody may also comprise a binding domain that binds to another antigen (i.e., other than the Spike protein from a betacoronavirus), for example a binding domain that binds to ACE2. The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions (HVRs) both in the lightchain and heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework region (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a p-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the p-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC). From N-terminal to C-terminal, both light and heavy chain variable regions comprise alternating FRs and CDRs: FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each region may be made in accordance with the definitions of Kabat, Chothia (Al- Lazikani eta!., J Mol Biol. 1997; 273(4):927-48), or lMGT (Lefranc, M.-P., Immunology Today, 18, 509 (1997)), for example. "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a singlechain Fv species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs are involved in conferring the antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
"Hypervariable region" or "HVR" refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (Al-Lazikani et al., supra).
The term "complementarity determining regions" or "CDRs" when used herein refers to parts of immunological receptors that make contact with a specific ligand and determine its specificity. The CDRs of immunological receptors are the most variable part of the receptor protein, giving receptors their diversity, and are carried on six loops at the distal end of the receptor's variable domains, three loops coming from each of the two variable domains of the receptor.
As used herein, the term "framework region" refers to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved (i.e., other than the CDRs) among different immunoglobulins in a single species, as defined by Kabat et al. supra) or Chothia (Al- Lazikani et al., supra). As used herein, a "human framework region" is a framework region that is substantially identical to the framework region of a naturally occurring human antibody.
The sequences of the CDR and FR as defined herein are defined according to the Clothia numbering scheme. However, the skilled person would understand that the amino acids forming the CDRs and FRs regions in the sequences of the antibodies defined herein may vary depending on the numbering scheme used. Other numbering schemes include the AbM, Kabat, Contact and IMGT schemes.
In an embodiment, one or two residues in the above-noted CDRs sequences are substituted. In a further embodiment, one residue in the above-noted CDRs sequences is substituted. In another embodiment, the antibody or antigen-binding fragment thereof comprises the above-noted CDRs sequences.
In an embodiment, the first antibody or antigen-binding fragment thereof comprises a light chain FR1 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR1 depicted in FIG. 1A.
In an embodiment, the first antibody or antigen-binding fragment thereof comprises a light chain FR2 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR2 depicted in FIG. 1A.
In an embodiment, the first antibody or antigen-binding fragment thereof comprises a light chain FR3 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR3 depicted in FIG. 1A.
In an embodiment, the first antibody or antigen-binding fragment thereof comprises a light chain FR4 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR4 depicted in FIG. 1A.
In an embodiment, the second antibody or antigen-binding fragment thereof comprises a light chain FR1 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR1 depicted in FIG. 1B.
In an embodiment, the second antibody or antigen-binding fragment thereof comprises a light chain FR2 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR2 depicted in FIG. 1B. In an embodiment, the second antibody or antigen-binding fragment thereof comprises a light chain FR3 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR3 depicted in FIG. 1B.
In an embodiment, the second antibody or antigen-binding fragment thereof comprises a light chain FR4 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR4 depicted in FIG. 1B.
In an embodiment, the third antibody or antigen-binding fragment thereof comprises a light chain FR1 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR1 depicted in FIG. 1C.
In an embodiment, the third antibody or antigen-binding fragment thereof comprises a light chain FR2 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR2 depicted in FIG. 1C.
In an embodiment, the third antibody or antigen-binding fragment thereof comprises a light chain FR3 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR3 depicted in FIG. 1C.
In an embodiment, the third antibody or antigen-binding fragment thereof comprises a light chain FR4 comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the sequence of the FR4 depicted in FIG. 1C.
In some embodiments, the first antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the heavy chain sequence depicted in FIG. 1A (SEQ ID NO:1), and a light chain comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the light chain sequence depicted in FIG. 1A (SEQ ID NO:2).
In some embodiments, the second antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the heavy chain sequence depicted in FIG. 1B (SEQ ID NO:5), and a light chain comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the light chain sequence depicted in FIG. 1B (SEQ ID NO:6).
In some embodiments, the third antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the heavy chain sequence depicted in FIG. 1C (SEQ ID NO:3), and a light chain comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the light chain sequence depicted in FIG. 1C (SEQ ID NO:4).
Variations in the antibodies or antigen-binding fragments thereof described herein, can be made, for example, using any of the techniques and guidelines for conservative and non- conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the antibody that results in a change in the amino acid sequence as compared with the native sequence antibody. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the antibody or antigen-binding fragment thereof. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the antibody or antigen-binding fragment thereof with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence. In embodiment, the variant exhibits at least 50%, 55% or 60%, preferably at least 65, 70, 75, 80, 90, 95, 96, 97, 98 or 99% sequence identity with the sequence of the antibody or antigen-binding fragment thereof described herein and maintains the ability to specifically bind to SARS-CoV-2 Spike protein and/or to neutralize SARS-CoV-2 infection.
"Identity" refers to sequence identity between two polypeptides. Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
Covalent modifications of antibodies or antigen-binding fragments thereof are included within the scope of this disclosure. Covalent modifications include reacting targeted amino acid residues of the antibody or antigen-binding fragment thereof with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the antibody or antigen-binding fragment thereof. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Other types of covalent modification of the antibody or antigen-binding fragment thereof included within the scope of this disclosure include altering the native glycosylation pattern of the antibody or antigen-binding fragment thereof (Beck et al., Curr. Pharm. Biotechnol. 9: 482-501 , 2008; Walsh, Drug Discov. Today 15: 773-780, 2010), and linking the antibody or antigen-binding fragment thereof to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301 ,144; 4,670,417; 4,791 ,192 or 4,179,337.
The first, second, and/or third antibody or antigen-binding fragment thereof of the present disclosure may further comprise one or more modifications that confer additional biological properties to the antigenic peptide such as protease resistance, plasma protein binding, increased plasma half-life, intracellular penetration, etc. Such modifications include, for example, covalent attachment of molecules/moiety to the antibody or antigen-binding fragment thereof such as fatty acids (e.g., C6-Ci8), attachment of proteins such as albumin (see, e.g., U.S. Patent No. 7,268,113); sugars/polysaccharides (glycosylation), biotinylation or PEGylation (see, e.g., U.S. Patent Nos. 7,256,258 and 6,528,485). The above description of modification of the antigenic peptide does not limit the scope of the approaches nor the possible modifications that can be engineered. Thus, in another aspect, the present disclosure provides a conjugate comprising the antibody or antigen-binding fragment thereof described herein and one or more additional molecules or agents (hereinafter secondary molecules or agents). The antigenic peptide may be conjugated to any type of synthetic or natural secondary molecules or agents, such as peptides, proteins, saccharides/polysaccharides, lipids, naturally-occurring or synthetic polymers/co- polymers, etc. to modify one or more properties of the antibody or antigen-binding fragment thereof.
In an embodiment, the conjugate comprises a covalent link or bond between the antibody or antigen-binding fragment thereof and the molecule conjugated thereto. The molecule may be conjugated directly to the antibody or antigen-binding fragment thereof, or indirectly via a linker. The linker may be a polypeptide linker comprising one or more amino acids or another type of chemical linker (e.g., a carbohydrate linker, a lipid linker, a fatty acid linker, a polyether linker, PEG, etc.
In another embodiment, the molecule may be conjugated/attached to the side chain of one the amino acids of the antibody or antigen-binding fragment thereof. Methods for conjugating moieties to side chains of amino acids are well known in the art. For example, chemical groups that react with primary amines (-NH2) present in the side-chain of lysine residues such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters may be used to conjugate the molecule to the antibody or antigen-binding fragment thereof. Most of these groups conjugate to amines by either acylation or alkylation. Cysteine residues present in the antibody or antigen-binding fragment thereof may also be used to attach the molecule.
In an embodiment, the first, second, and/or third antibody or antigen-binding fragment thereof of the present disclosure is labelled or conjugated with one or more moieties. The antibody or antigen-binding fragment thereof may be labeled with one or more labels such as a biotin label, a fluorescent label, an enzyme label, a coenzyme label, a chemiluminescent label, or a radioactive isotope label. In an embodiment, the antibody or antigen-binding fragment thereof is labelled with a detectable label, for example a fluorescent moiety (fluorophore). Useful detectable labels include fluorescent compounds (e.g., fluorescein isothiocyanate, Texas red, rhodamine, fluorescein, Alexa Fluor® dyes, and the like), radiolabels, enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in a protein detection assays), streptavidin/biotin, and colorimetric labels such as colloidal gold, colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.). Chemiluminescent compounds may also be used. Such labelled antibodies or antigen-binding fragments thereof may be useful, for example, for the detection of SARS-CoV-2 and/or SARS-CoV-2-infected cells in vivo or in vitro, e.g., by flow cytometry, immunohistochemistry, etc. The antibody or antigen-binding fragment thereof can also be conjugated to detectable or affinity tags that facilitate detection and/or purification of the antibody or antigen-binding fragment thereof. Such tags are well known in the art. Examples of detectable or affinity tags include polyhistidine tags (His-tags), polyarginine tags, polyaspartate tags, polycysteine tags, polyphenylalanine tags, glutathione S-transferase (GST) tags, Maltose binding protein (MBP) tags, calmodulin binding peptide (CBP) tags, Streptavidin/Biotin-based tags, HaloTag®, Profinity eXact® tags, epitope tags (such as FLAG, hemagglutinin (HA), HSV, S/S1 , c-myc, KT3, T7, V5, E2, and Glu-Glu epitope tags), reporter tags such as p-galactosidase (|3-gal), alkaline phosphatase (AP), chloramphenicol acetyl transferase (CAT), and horseradish peroxidase (HRP) tags (see, e.g., Kimple et al., Curr Protoc Protein Sci. 2013; 73: Unit-9.9).
In an embodiment, the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 55% to about 65% of the first antibody or antigen-binding fragment thereof. In a further embodiment, the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 60% to about 65% or about 62 to about 64% of the first antibody or antigen-binding fragment thereof. In a further embodiment, the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 63% of the first antibody or antigen-binding fragment thereof.
In an embodiment, the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 3% to 8% of the second antibody or antigen-binding fragment thereof. In a further embodiment, the combination of anti-SARS-CoV-2 antibodies or antigenbinding fragments thereof comprises about 3% to 7% or about 4% to 6% of the second antibody or antigen-binding fragment thereof. In a further embodiment, the combination of anti-SARS-CoV- 2 antibodies or antigen-binding fragments thereof comprises about 5% of the second antibody or antigen-binding fragment thereof.
In an embodiment, the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 28% to about 37% of the third antibody or antigen-binding fragment thereof. In a further embodiment, the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 30% to about 35% or about 31% to about 33% of the third antibody or antigen-binding fragment thereof. In a further embodiment, the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof comprises about 32% of the third antibody or antigen-binding fragment thereof.
In some embodiments, the first, second, and/or third antibody or antigen-binding fragment thereof described herein binds to an epitope in the viral envelope spike protein (S) of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope in the receptor binding domain (RBD) of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 S protein. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope that is outside the RBD of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 S protein. In some embodiments, the antibody or antigen-binding fragment thereof neutralizes a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2. In some embodiments, the antibody or antigen-binding fragment thereof inhibits viral and cell membrane fusion. In some embodiments, the antibody or antigen-binding fragment thereof binds to the S1 subunit of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 S protein. In some embodiments, the antibody or antigenbinding fragment thereof binds to the S2 subunit of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 S protein. In some embodiments, the antibody or antigen-binding fragment thereof also binds to the S protein of one at least two betacoronaviruses, such as SARS-CoV-1 and SARS-CoV-2.
The amino acid sequence of the full-length Spike protein from SARS-CoV-2 (Wuhan strain, NCBI Reference Sequence YP_009724390.1 , SEQ ID NO: 47) is depicted below:
1 MFVFLVLL PL VSSQCVNLTT RTQL PPAYTN S FTRGVYYPD KVFRS SVLHS TQDLFL PFFS
61 NVTWFHAIHV SGTNGTKRFD NPVL PFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLL IV
121 NNATNWIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY S SANNCTFEY VSQPFLMDLE
181 GKQGNFKNLR EFVFKNIDGY FKIYSKHT PI NLVRDLPQGF SALEPLVDL P IGINITRFQT
241 LLALHRSYLT PGDSS SGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK
301 CTLKSFTVEK GIYQTSNFRV QPTES IVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN
361 CVADYSVLYN SAS FSTFKCY GVS PTKLNDL CFTNVYADS F VIRGDEVRQI APGQTGKIAD
421 YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGST PC
481 NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV WLSFELLHA PATVCGPKKS TNLVKNKCVN
541 FNFNGLTGTG VLTESNKKFL PFQQFGRDIA DTTDAVRDPQ TLE ILDITPC SFGGVSVITP
601 GTNTSNQVAV LYQDVNCTEV PVAIHADQLT PTWRVYSTGS NVFQTRAGCL IGAEHVNNSY
661 ECDI PIGAGI CASYQTQTNS PRRARSVASQ S IIAYTMSLG AENSVAYSNN S IAI PTNFTI
721 SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTGI AVEQDKNTQE
781 VFAQVKQIYK T PPIKDFGGF NFSQIL PDPS KPSKRSFIED LLFNKVTLAD AGFIKQYGDC
841 LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG T ITSGWTFGA GAALQI PFAM
901 QMAYRFNGIG VTQNVLYENQ KL IANQFNSA IGKIQDSLS S TASALGKLQD WNQNAQALN
961 TLVKQLSSNF GAISSVLNDI LSRLDKVEAE VQIDRLITGR LQSLQTYVTQ QL IRAAEIRA
1021 SANLAATKMS ECVLGQSKRV DFCGKGYHLM S FPQSAPHGV VFLHVTYVPA QEKNFTTAPA
1081 ICHDGKAHFP REGVFVSNGT HWFVTQRNFY E PQIITTDNT FVSGNCDWI GIVNNTVYDP
1141 LQPELDSFKE ELDKYFKNHT S PDVDLGDIS GINASWNIQ KEIDRLNEVA KNLNESLIDL
1201 QELGKYEQYI KWPWYIWLGF IAGL IAIVMV T IMLCCMTSC CSCLKGCCSC GSCCKFDEDD
12 61 SE PVLKGVKL HYT
Residues 1-12 correspond to the signal peptide, residues 13-685 correspond to the Spike protein subunit S1 and residues 686-1273 correspond to the Spike protein subunit S2. The receptor-binding domain (RBD) is defined by residues 319-541 (receptor-binding motif = residues 437-508). Residues 816-837 define the fusion peptide 1 , residues 835-855 define the fusion peptide 2, residues 920-970 define the heptad repeat 1 and residues 1163-1202 define the heptad repeat 2.
SARS-CoV2 variants comprise mutations in the Spike protein including L5F, S13I, L18F, T19R, T20N, P26S, A67V, del69-70, G75V, T76I, D80Y, D80A, T95I, S98F, R102I, D138Y, G142D, del142-144, del144, W152C, E154K, EFR156-158G, F157L, R190S, ins214EPE, D215G, A222V, del246-252, D253G, W258L, N354D, F342L, V367F, K417N, K417T, A435S, W436R, N439K, N440K, G446V, L452R, Y453F, K458R, G476S, S477N, S477G, T478K, V483A, E484K, E484Q, F490S, N501Y, N501S, N501T, A570D, Q613H, D614G, A626S, A653V, H655Y, Q677H, Q677P, P681 H, P681 R, A701V, T716I, D796H, D796Y, T859N, F888L, D950N, S982A, T1027I, Q1071H, E1092K, H1101Y, D1118H, V1176F, G1219V, and V1122L.
The Delta variant comprises the following Spike protein mutations: T19R, (V70F*), T95I, G142D, E156-, F157-, R158G, (A222V*), (W258L*), (K417N*), L452R, T478K, D614G, P681 R, D950N.
The Omicron variant (sublineage BA.1) comprises the following Spike protein mutations: A67V, del69-70, T95I, del142-144, Y145D, del211 , L212I, ins214EPE, G339D, S371 L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681 H, N764K, D796Y, N856K, Q954H, N969K, L981 F. FIG. 8 depicts the mutations in Omicron sublineages BA.1 , BA.2 and BA.3.
In an embodiment, the combination of antibodies or antigen-binding fragments thereof described herein binds to and neutralizes the Spike protein from a SARS-CoV2 variant. In an embodiment, the combination of antibodies or antigen-binding fragments thereof described herein binds to and neutralizes the Spike protein from the SARS-CoV2 Delta variant. In an embodiment, the combination of antibodies or antigen-binding fragments thereof described herein binds to and neutralizes the Spike protein from the SARS-CoV2 Omicron variant.
A further aspect of the present disclosure provides nucleic acids encoding the first, second, and/or third antibody or antigen-binding fragment described herein. The isolated nucleic acid may be a synthetic DNA, a non-naturally occurring mRNA, or a cDNA, for example. The nucleic acid may be inserted within a plasmid, vector, or transcription or expression cassette. The nucleic acids encoding the first, second, and/or third antibody or antigen-binding fragment described herein may be made and the expressed antibodies or antigen-binding fragments described may be tested using conventional techniques well known in the art. In some embodiments, the nucleic acid encoding the first, second, and/or third antibody or antigen-binding fragment described herein can be maintained in the vector in a host cell. In another aspect, provided herein is a nucleic acid comprising a sequence encoding the amino acid sequence of any one of SEQ ID NOs:1-6. In some embodiments, the nucleic acid is an expression vector. In some embodiments, the nucleic acid sequence encoding the antibody can be maintained in the vector in a host cell. In embodiments, the nucleic acid(s) (DNA, mRNA) encoding the first, second and third antibodies or antigen-binding fragments described herein is/are comprised within vesicles such as lipid nanoparticles (e.g., liposomes) or any other suitable vehicle. In an embodiment, the nucleic acid(s) is/are mRNA and is/are encapsulated into nanoparticulate delivery vehicles (see, e.g., Van Hoecke and Roose, Journal of Translational Medicine, volume 17, Article number: 54 (2019); Sanz and Alvarez-Vallina, Antibodies (Basel). 2021 Sep 26;10(4):37).
In another aspect, the present disclosure provides a cell, for example a recombinant host cell, expressing the first, second, and/or third antibody or antigen-binding fragment described herein. Methods of preparing antibodies or antigen-binding fragments comprise expressing the encoding nucleic acid(s) in a host cell under conditions to produce the antibodies or antigenbinding fragments, and recovering the antibodies or antigen-binding fragments. The process of recovering the antibodies or antigen-binding fragments may comprise isolation and/or purification of the antibodies or antigen-binding fragments. The method of production may comprise formulating the antibodies or antigen-binding fragments into a composition including at least one additional component, such as a pharmaceutically acceptable excipient. In another aspect, provided herein is a cell expressing one or more antibodies of the disclosure. In some embodiments, the cell comprises one or more nucleic acid sequences encoding the amino acid sequence of any one of SEQ ID NOs: 1-6.
The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Preferably host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. To produce the antibody or antigen-binding fragment thereof recombinantly, the nucleic acid or nucleic acids encoding the light and heavy chains of the antibody or antigen-binding fragment thereof are introduced in a cell which is able to produce the recombinant antibody. Examples thereof include CHO-K1 (ATCC CCL-61), DUkXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies®, Cat #11619), rat myeloma cell YB2/3HL.P2.G11.16Ag.2O (also called YB2/0), mouse myeloma cell NSO, mouse myeloma cell SP2/0-Ag14 (ATCC No. CRL1581), mouse P3- X63-Ag8653 cell (ATCC No. CRL1580), CHO cell in which a dihydrofolate reductase gene is defective, lectin resistance-acquired Led 3, CHO cell in which a1 ,6-fucosyltransaferse gene is defective, rat YB2/3HL.P2.G11.16Ag.2O cell (ATCC No. CRL1662), CHO-3E7 cells (expressing a truncated but functional form of EBNA1 , U.S. Patent No. 8,637,315) or the like. After introduction of the expression vector, transformants which stably express a recombinant antibody are selected by culturing them in a medium for animal cell culture containing an agent such as G418 sulfate or the like. Examples of the medium for animal cell culture include RPMI1640 medium (manufactured by Invitrogen®), GIT medium (manufactured by Nihon Pharmaceutical®), EX- CELL301® medium (manufactured by JRH®), IMDM medium (manufactured by Invitrogen®), Hybridoma-SFM medium (manufactured by Invitrogen®), media obtained by adding various additives such as FBS to these media, or the like. The recombinant antibody can be produced and accumulated in a culture supernatant by culturing the obtained transformants in a medium. The expression level and antigen binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA or the like. Also, in the transformant, the expression level of the recombinant antibody can be increased by using DHFR amplification system or the like. The recombinant antibody can be purified from the culture supernatant of the transformant by using a protein A column. In addition, the recombinant antibody can be purified by combining the protein purification methods such as gel filtration, ion-exchange chromatography, ultrafiltration or the like. The molecular weight of the H chain or the L chain of the purified recombinant antibody or the antibody molecule as a whole is determined by polyacrylamide gel electrophoresis, Western blotting, or the like.
Suitable vectors comprising nucleic acid(s) encoding the antibody or antigen-binding fragment described herein can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, phage, phagemids, adenoviral, AAV, lentiviral, for example. Techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells, and gene expression, are well known in the art.
The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
Introducing such nucleic acids into a host cell can be accomplished using techniques well known in the art. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, for example. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. In one embodiment, the nucleic acid of the invention is integrated into the genome, e.g., chromosome, of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.
In an embodiment, the composition further comprises the above-mentioned first, second, and third antibodies or antigen-binding fragments thereof and a carrier or excipient, in a further embodiment a pharmaceutically acceptable carrier or excipient. Such compositions may be prepared in a manner well known in the pharmaceutical art by mixing the antibodies or antigenbinding fragments thereof having a suitable degree of purity with one or more optional pharmaceutically acceptable carriers or excipients (see Remington: The Science and Practice of Pharmacy, by Loyd Allen, Jr, 2012, 22nd edition, Pharmaceutical Press; Handbook of Pharmaceutical Excipients, by Rowe et al., 2012, 7th edition, Pharmaceutical Press). The carrier/excipient can be suitable for administration of the antibodies or antigen-binding fragments thereof by any conventional administration route, for example, for oral, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration. In an embodiment, the carrier/excipient is adapted for administration of the antibodies or antigen-binding fragments thereof by the intravenous or subcutaneous route. In an embodiment, the carriers/excipients are adapted for administration of the antibodies or antigen-binding fragments thereof by the intravenous route. In another embodiment, the carriers/excipients are adapted for administration of the antibodies or antigen-binding fragments thereof by the subcutaneous route. In an embodiment, the carriers/excipients are adapted for administration of the antibodies or antigen-binding fragments thereof by the pulmonary route.
An "excipient" as used herein has its normal meaning in the art and is any ingredient that is not an active ingredient (drug) itself. Excipients include for example binders, lubricants, diluents, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizing agent, release-delaying agents and other components. "Pharmaceutically acceptable excipient" as used herein refers to any excipient that does not interfere with effectiveness of the biological activity of the active ingredients and that is not toxic to the subject, i.e., is a type of excipient and/or is for use in an amount which is not toxic to the subject. Excipients are well known in the art, and the present system is not limited in these respects. In certain embodiments, one or more formulations of the dosage form include excipients, including for example and without limitation, one or more binders (binding agents), thickening agents, surfactants, diluents, release-delaying agents, colorants, flavoring agents, fillers, disintegrants/dissolution promoting agents, lubricants, plasticizers, silica flow conditioners, glidants, anti-caking agents, anti-tacking agents, stabilizing agents, anti-static agents, swelling agents and any combinations thereof. As those of skill would recognize, a single excipient can fulfill more than two functions at once, e.g., can act as both a binding agent and a thickening agent. As those of skill will also recognize, these terms are not necessarily mutually exclusive. Examples of commonly used excipient include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or auxiliary substances, such as emulsifying agents, preservatives, or buffers, which increase the shelf life or effectiveness. In an embodiment, the antibody or antigen-binding fragment thereof defined herein is encapsulated in a vesicle or vesicle-like particle, such as a lipid vesicle (e.g., liposome).
The composition may also comprise one or more additional active agents for the treatment the targeted disease/condition or for the management of symptom(s) of the targeted disease/condition (e.g., pain killers, anti-nausea agents, anti-inflammatory agents, immunotherapeutic agents, etc.).
According to some embodiments, the composition is a liquid or semi-liquid composition. According to some embodiments, the composition of the disclosure is in the form of a liquid mixture. According to some embodiments, the composition of the disclosure is in the form of an aqueous mixture. According to some embodiments, the liquid mixture is selected from liquid solution, liquid suspension and liquid emulsion. According to some embodiments, the aqueous mixture is an aqueous solution, aqueous suspension, and aqueous emulsion. According to some embodiments, the composition of the current invention is in the form of an aqueous solution.
In an embodiment, the composition further comprises a saline solution or buffer, such as phosphate-buffered saline (PBS). In an embodiment, the pH of the solution or buffer is from 6 to 8. In a further embodiment, the pH of the solution or buffer is from 6.5 to 7.5 or from 7 to 7.5, for example about 7.2.
In an embodiment, the composition further comprises a surfactant or emulsifier. In an embodiment, the surfactant is a nonionic surfactant, such as a nonionic polyoxyethylene surfactant. In a further embodiment, the surfactant is a polysorbate, such as Polysorbate-20 or Polysorbate-80. In an embodiment, the surfactant or emulsifier is present in at a concentration of about 0.001% to 0.1% in the composition. In a further embodiment, the surfactant or emulsifier is present in at a concentration of about 0.005% to 0.05% in the composition. In a further embodiment, the surfactant or emulsifier is present in at a concentration of about 0.01% in the composition.
In an embodiment, the composition comprises (i) the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof described herein; (ii) a saline solution or buffer, such as PBS; and (iii) a surfactant or emulsifier, such as a Polysorbate (e.g., Polysorbate-20).
In an embodiment, the composition or dosage form comprises from 1 mg to 30 mg of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof described herein. In a further embodiment, the composition or dosage form comprises from 2 mg to 20 mg of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof described herein. In a further embodiment, the composition or dosage form comprises from 5 mg to 15 mg of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof described herein. In a further embodiment, the composition or dosage form comprises about 6, 7, 8, 9, 10, 11 , 12, 13 or 14 mg of the combination of anti-SARS-CoV-2 antibodies or antigenbinding fragments thereof described herein. In a further embodiment, the composition or dosage form comprises about 10 mg of the combination of anti-SARS-CoV-2 antibodies or antigenbinding fragments thereof described herein.
In another aspect, the present disclosure provides a dosage form, e.g., an aerosol, comprising the composition described herein. The term "aerosol" as used herein refers to a suspension of fine solid particles or liquid droplets in air or another gas.
Thus, in an embodiment, the composition or dosage form is suitable for aerosolization or nebulization. More specifically, the dosage form may be a nebulizer cartridge containing the composition, which is adapted for aerosolization from a nebulizer. Alternatively, the composition may be for filling a nebulizer. Such compositions or dosage forms allow delivery of the antibodies or antigen-binding fragments (or nucleic acids encoding same) to specific sites of action along the nasopharyngeal, trachea, and/or lungs, i.e., in the respiratory tract.
Any device or system for generating an aerosol may be used for administration of the composition described herein. Such devices or systems include for example nebulizers and nebulizer systems or inhalers and inhaler systems. The nebulizer or inhaler may be a jet nebulizer (or atomizer), an ultrasonic nebulizer (e.g., Omron™ NE-U17 and Beurer™ Nebulizer IH30), a vibrating mesh nebulizer (e.g., Pari eFlow™, Respironics i-Neb™, Beurer™ Nebulizer IH50, Aerogen Solo/Ultra™), a soft mist inhaler (e.g., Respimat® Soft Mist™ Inhaler, Boehringer Ingelheim) or a metered dose inhaler. In an embodiment, the nebulizer or inhaler is a vibrating mesh nebulizer. According to some embodiments, the droplets of the aerosol delivered by the nebulizer have a Volumetric Median Diameter (VMD) of no more than 5 microns. In further embodiments, the droplets of the aerosol delivered by the nebulizer have a VMD of between about 1 micron to about 5 microns, between about 2 microns to about 5 microns or between about 3 microns to about 5 microns. In an embodiment, the VMD is measured by laser diffraction spectroscopy (LDS).
The composition may also comprise one or more additional active agents for the treatment the targeted disease/condition or for the management of symptom(s) of the targeted disease/condition (e.g., pain killers, anti-nausea agents, anti-inflammatory agents, immunotherapeutic agents, etc.).
In another aspect, the present disclosure provides a method for preventing a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 infection or a related disease (Coronavirus disease 2019, COVID-19), in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition described herein. The present disclosure also provides the use of the composition described herein for preventing a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 infection or a related disease (e.g., COVID-19) in a subject. The present disclosure also provides the use of the composition described herein, for the manufacture of a medicament for preventing a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 infection or a related disease (e.g., COVID-19) in a subject.
In another aspect, the present disclosure provides a method for reducing the risk of developing a betacoronavirus-related disease, such as a sarbecovirus-related disease (e.g., COVID-19), or the severity of a betacoronavirus-related disease, such as a sarbecovirus-related disease (e.g., COVID-19), in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition described herein. The present disclosure also provides the use of the composition described herein for reducing the risk of developing a betacoronavirus-related disease, such as a sarbecovirus-related disease (e.g., COVID-19), or the severity of a betacoronavirus-related disease, such as a sarbecovirus-related disease (e.g., COVID-19), in a subject. The present disclosure also provides the composition described herein for use in reducing the risk of developing a betacoronavirus-related disease, such as a sarbecovirus-related disease (e.g., COVID-19), or the severity of a betacoronavirus-related disease, such as a sarbecovirus-related disease (e.g., COVID-19), in a subject.
In another aspect, the present disclosure provides a method in vitro or in vivo) for blocking the entry of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 in a cell, such as an ACE2-expressing cell, comprising contacting the cell and/or virus with an effective amount of the composition described herein. The present disclosure provides the use of the composition described herein for blocking the entry of a betacoronavirus, such as a sarbecovirus, e.g., SARS- CoV-2 in a cell, such as an ACE2-expressing cell. The present disclosure provides the use of the composition described herein for the manufacture of a medicament for blocking the entry of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 in a cell, such as an ACE2- expressing cell. The present disclosure provides the composition described herein, for use in blocking the entry of a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2 in a cell, such as an ACE2-expressing cell.
In an embodiment, the methods and uses defined herein are for the prevention, treatment and/or management of infections by the Wuhan original SARS-CoV-2 strain. In another embodiment, the methods and uses defined herein are for the prevention, treatment and/or management of infections by variants of the Wuhan original SARS-CoV-2 strain, such as the B.1.1.7 (also known as VOC-202012/01 or alpha (a)), 501Y.V2 (also known as B.1.351 or beta (P)), P.1 (also known as B.1.1.28.1 or gamma (y)), B.1.617.2 (also known as delta (5)), or B.1.1.529 (omicron) variant, as well as other variants of concern (VOC) such as B.1.429, B.1.526,
B.1.525, and A.23.1 (see, e.g., https://www.cdc.qov/coronavirus/2019-ncov/variants/variant- classifications.
Figure imgf000033_0001
In an embodiment, the methods and uses defined herein are for the prevention, treatment and/or management of infections by the SARS-CoV-2 Delta (5) variant. In an embodiment, the methods and uses defined herein are for the prevention, treatment and/or management of infections by the SARS-CoV-2 Omicron variant, e.g., Omicron sublineage BA.1 , BA1.1.529, BA1.5, BA.2, BA2.12.1 , BA2.75, BA.3, BA.4/5, BA.4/6, BQ1.1 , XBB and/or XBB1 .5.
In certain embodiments, described herein, is a method of delivery of the composition described herein to the respiratory system of an individual infected with a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2, the method comprising administering the composition described herein in nebulized or aerosolized form to the individual.
In certain embodiments, described herein, is the use of the composition described herein in nebulized or aerosolized form for delivering the composition to the respiratory system of an individual infected with a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2.
For the prevention, treatment or reduction in the severity of a given disease or condition (viral disease such as COVID-19), the appropriate dosage of the combination/composition described herein will depend on the type of disease or condition to be treated, the severity and course of the disease or condition, whether the combination/composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the composition, and the discretion of the attending physician. The combination/composition described herein may be suitably administered to the patient at one time or over a series of treatments. Preferably, it is desirable to determine the dose-response curve in vitro, and then in useful animal models prior to testing in humans. The present disclosure provides dosages for the combination/composition. For example, depending on the type and severity of the disease, about 1 pg/kg to to 1000 mg per kg (mg/kg) of body weight per day. Further, the effective dose may be 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/ 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000 mg/kg, or may range between any two of the foregoing values. A typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. In an embodiment, the dosage of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof is from 1 mg to 30 mg. In a further embodiment, the dosage of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof is from 2 mg to 20 mg. In a further embodiment, the dosage of the combination of anti- SARS-CoV-2 antibodies or antigen-binding fragments thereof is from 5 mg to 15 mg. In a further embodiment, the dosage of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof is about 6, 7, 8, 9, 10, 11 , 12, 13 or 14 mg. In a further embodiment, the dosage of the combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof is about 10 mg.
As used herein the term “treating” or “treatment” in reference to viral infection or disease is meant to refer to administration of the composition after infection that leads to a reduction/improvement in one or more symptoms or pathological features associated with said viral disease (e.g., COVID-19). Non-limiting examples include a decrease in viral load, reduction of cough, fever, fatigue, shortness of breath, reduction/prevention of acute respiratory distress syndrome (ARDS), reduction/prevention of multi-organ failure, septic shock, and blood clots, hospitalization, etc.
As used herein the term “preventing” or “prevention” in reference to viral infection or disease is meant to refer to administration of the composition prior to infection that leads to protection from being infected or from developing the viral disease (e.g., COVID-19), to a delay in the development of the disease, or to a reduction of one or more symptoms or pathological features associated with the viral disease.
In an embodiment, the administration/use of the composition described herein delays the onset of one or more symptoms of a betacoronavirus or sarbecovirus-caused infection, e.g., SARS-CoV-2-caused infection (e.g., COVID-19).
The composition described herein may be used alone or in combination with other prophylactic agents such as antivirals, anti-inflammatory agents, vaccines, immunotherapies, etc. The combination of active agents and/or compositions comprising same may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form. Co-administration in the context of the present disclosure refers to the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time. For example, a first agent (e.g., the composition described herein) may be administered to a patient before, concomitantly, before and after, or after a second active agent (e.g., an antiviral or anti-inflammatory agent) is administered. The agents may in an embodiment be combined/formulated in a single composition and thus administered at the same time.
In an embodiment, the composition described herein is for administration prior to exposure to a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2. In another embodiment, the composition described herein is for administration after exposure to a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2. In another embodiment, the composition described herein is for administration prior to and after exposure to a betacoronavirus, such as a sarbecovirus, e.g., SARS-CoV-2.
In an embodiment, the composition described herein is for administration prior to development of the viral disease (e.g., COVID-19). In another embodiment, the composition described herein is for administration after development of the viral disease (e.g., COVID-19). In another embodiment, the composition described herein is for administration prior to and after development of the viral disease (e.g., COVID-19).
In an embodiment, the subject or patient has a weakened immune system and a reduced ability to fight viral infections such as SARS-CoV-2 infection. In another embodiment, the subject or patient is an immunosuppressed or immunocompromised subject or patient. Immunosuppression may be caused by certain diseases or conditions, such as AIDS, cancer, diabetes, malnutrition, and certain genetic disorders, or certain drugs or treatments such as anticancer drugs, radiation therapy, and stem cell or organ transplant. In an embodiment, the subject or patient is an elderly subject or patient, for example a subject or patient having 60 years old or more, 65 years old or more, 70 years old or more, 75 years old or more, or 80 years old or more, who typically develop a weaker immune response to vaccines and infections.
In another aspect, the disclosure provides kits comprising the composition of the disclosure. Kits include one or more containers comprising by way of example, and not limitation, the composition or dosage form described herein and instructions for use in accordance with any of the methods of the disclosure. Generally, instructions comprise a description of administration. The containers can be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
The kits are provided in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, tubes, jars, flexible packaging (e.g., sealed Mylar or plastic bags), nebulizer cartridge, and the like. A kit can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Kits can optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In an embodiment, the kit include a device for intrapulmonary administration of the composition, such as a nebulizer, metered-dose inhaler, or dry powder inhaler.
EXAMPLES
The present disclosure is illustrated in further details by the following non-limiting examples.
Example 1 : Materials and Methods
Antibodies. Characteristics of anti-SARS-CoV-2 antibodies IBIO1 , IBIO2 and IBIO3 present in the antibody cocktail used in the studies reported herein are depicted in Table 1 below. IBIO1 and IBIO2 are described in PCT application No. PCT/CA2021/051873, and IBIO3 is described in PCT application No. PCT/CA2022/051074.
Table 1 : Characteristics of antibodies IBIO-1 , IBIO-2 and IBIO-3
Figure imgf000036_0001
Binding assay (ELISA) on Trimeric SARS-CoV-2 Spike Protein. An indirect dose-response ELISA assay was performed after adsorption of 100 ng of trimeric Spike protein (SMT1-1 , NRCC) in each well of a 96-wells microplate (Nunc Maxisorb™). Increasing doses of the antibody cocktail were deposited in each well. Bovine Serum Albumin (BSA) was used as a negative control antigen. Binding was revealed by the addition of an anti-Human IgG/HRP secondary antibody. Signal was measured by optical density at 450 nm (Biotek Instruments, Synergy HTX).
SARS-CoV-2 neutralization assay (FIGs. 4 and 5). Pseudovirions expressing the SARS- CoV-2 spike protein (from the Wuhan strain) and carrying an eGFP reporter gene were produced in 293T cells. HEK cells overexpressing ACE2/TMPRSS2 were used for infection. Pseudovirions produced without Spike served as negative controls. Expression of the eGFP reporter gene follows fluorescently-labeled virus in living cells and allows quantification of viral infection. The efficiency of eGFP transduction was analyzed by flow cytometry or fluorescent plate reader (IncuCyte 53 and Mithras, respectively) 48 hours post-infection. A characterized anti-SARS-2 rabbit monoclonal antibody was used as a positive control.
SARS-CoV-2 neutralization assay (FIGs. 6A-B). Lentiviruses were produced in 293T cell by transfecting the 4 pg pHAGE-CMV-Luc2-IRES-zsGreen3 pg psPAX2 (packaging vector): and 3 pg pcDNA3.1 -SARS-CoV-2 spike delta19 (spike glycoprotein) and PEI transfecting agent. Viruses were harvested 48h after the transfection. The neutralization activity was measured by microneutralization assay in vitro. The virus microneutralization (MN) test was performed on 293T-ACE2 cells infected with SARS-CoV-2 (2019-nCoV) Spike Pseudovirus under treatment of serial dilutions of the antibody cocktail. Rate of inhibition was determined by comparing the Relative Light Unit (RLU) of Luciferase reporter in different antibody concentrations with plate reader
A
Figure imgf000037_0001
up to five SARS-CoV-2 virus strains (FIG. 6C). The assays were conducted in triplicate in flatbottom 96-well plates using Vero’76 cells grown at 80% - 90% confluence. The virus inoculum sizes were approximately 25-50 TCID50 per well. Testing concentrations of the antibodies before equal volume mixture with virus solution were 50, 25, 12.5, 6.25, 3.12, 1 .56, 0.8 and 0.00 pg/ml. Therefore, the final concentrations in the reactions were 25, 12.5, 6.25, 3.12, 1.56, 0.8, 0.4 and 0.00 pg/ml. The cytopathic effect (CPE) was assessed by microscopic examination on Day 3 and 5, and the lowest antibody concentration with no CPE at Day 5 was defined as the neutralization titer in pg/ml. To assess degrees of inhibitory effects of various antibody concentrations, the CPE in each well was scored. No CPE in a well was scored 0.1 , and the degree of CPE intensity in a well with virus infection without antibody treatment was scored 100.
Preparation of antibody cocktail for nebulization. The antibodies (6.3 mg of IBIO1 , 0.5 mg of IBIO2 and 3.2 mg of IBIO3) were mixed in 1 ml of phosphate-buffered saline (PBS) 1X (pH 7.2) with 0.01% polysorbate-20.
Droplet size characterization. Droplet size characterization expressed as volumetric median droplet diameter (VMD/Dv5O) of aerosolized samples was determined by laser diffraction spectroscopy using the Malvern Spraytec device in order to characterize the volumetric droplet size generated by the Aerogen Solo/Ultra + mouthpiece combination. Testing was carried out using 0.25 mL of the IBIO123 antibody cocktail at a concentration of 10 mg/ml.
Example 2: Assessment of anti-SARS-CoV-2 spike protein antibody mixtures
Anti-SARS-CoV-2 spike protein antibodies were mixed in various proportions (FIG. 2A) and the antibody cocktails were tested in a SARS-CoV-2 neutralization assay. As shown in FIG. 2B, there was some significant differences in the potency of the antibody cocktails to neutralize infection of HEK cells by pseudovirions expressing the SARS-CoV-2 spike protein. Because it exhibited one the lowest IC50, IC8o and IC90 among the antibody cocktails tested, cocktail J (hereinafter referred to as IBIO123) that comprises 63% of IBIO1 , 5% of IBIO2 and 32% of IBIO3 was selected for further studies.
FIG. 3 shows that antibody cocktail IBIO123 binds trimeric SARS-CoV-2 Spike protein in a dose-dependent manner and with an EC5o of 43.67 ng/ml. Additional SARS-CoV-2 neutralization assay performed revealed that antibody cocktail IBIO123 exhibits an IC5o between 7.76 ng/ml and 17.23 ng/ml (FIGs. 4 and 5), compared to 575.8 ng/ml for a reference SARS-CoV-2 Spike Neutralizing Antibody from Sino Biological (Cat. No. 40592-MM57) (FIG. 5).
The results depicted in FIGs. 6A and 6B show that antibody cocktail I BI0123 maintains the ability to neutralize pseudovirions expressing the SARS-CoV-2 spike protein from several VOCs including the highly mutated Omicron variants (albeit with reduced potency). Similar results were obtained in neutralization assays using complete viruses (FIG. 6C). The antibody cocktail I BI0123 was able to completely neutralize infection by the Delta variant at all doses tested, whereas higher doses of the cocktail were needed to neutralize the Omicron variants (BA.1 , BA.2 et BA.5). IBIO123 neutralization concentrations were 0.4 pg/ml on the Delta variant, 25 pg/ml on Omicron BA.1 , 15 pg/ml on Omicron BA.2 and 16.5 pg/ml on Omicron BA.5, respectively.
Example 3: Assessment of a nebulized formulation of antibody cocktail IBIO123
The formulation for nebulization comprises 10 mg of the antibody cocktail IBIO123 in 1 ml of phosphate-buffered saline (PBS) 1X (pH 7.2) with 0.01% polysorbate-20. The results presented in FIG. 7A and FIG. 7B show that nebulization of the formulation using a vibrating mesh nebulizer has no significant effect on the ability of the antibodies to bind to trimeric SARS-CoV-2 Spike Protein (FIG. 7A) or to neutralize infection of HEK cells by pseudovirions expressing the SARS- CoV-2 spike protein (FIG. 7B).
Experiments were performed to characterize the volumetric median droplet diameter (VMD/Dv50) generated following nebulization of the formulation using the vibrating mesh nebulizer. It is generally accepted that the droplet size of medical aerosol should be controlled within 1 to 6 pm because the droplets could deposit in the mouth and throat when the size is larger than 6 pm, and may be exhaled when the size is smaller than 2 pm. The test was performed using 9 different nebulizer devices each comprising a volume of 0.25 ml of the formulation. The results are reported in the Table 2 below.
Table 2: Aerosol particle size distribution (APSD) expressed as VMD in the nebulized antibody cocktail IBIO123 formulation
Figure imgf000038_0001
Figure imgf000039_0001
These results show that the formulation is suitable for nebulization.
Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise.

Claims

WHAT IS CLAIMED IS:
1 . A composition comprising a combination of anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof, said combination comprising:
(a) 55% to 70% of a first antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs): a light chain CDR1 (CDR-L1) comprising the sequence RASQSVSSSYLA (SEQ ID NO:7); a light chain CDR2 (CDR-L2) comprising the sequence GASSRAT (SEQ ID NO:8); a light chain CDR3 (CDR-L3) comprising the sequence QQYGTSPWT (SEQ ID NO:9); a heavy chain CDR1 (CDR-H1) comprising the sequence GFTFTSS (SEQ ID NO: 10); a heavy chain CDR2 (CDR-H2) comprising the sequence WGSGN (SEQ ID NO:11); and a heavy chain CDR3 (CDR-H3) comprising the sequence PSCSGGRCYDGFDI (SEQ ID NO:12);
(b) 2% to 10% of a second antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising the sequence RASQGISSWLA (SEQ ID NO:19); a CDR-L2 comprising the sequence AASSLQS (SEQ ID NQ:20); a CDR-L3 comprising the sequence QQGNSFPYT (SEQ ID NO:21); a CDR-H1 comprising the sequence GYTFTRY (SEQ ID NO:22); a CDR-H2 comprising the sequence YPGDSD (SEQ ID NO:23); and a CDR-H3 comprising the sequence LPQYCSNGVCQRWFDP (SEQ ID NO:24); and
(c) 25% to 40% of a third antibody or antigen-binding fragment thereof comprising the following CDRs: a CDR-L1 comprising the sequence RASQSVSSSYLA (SEQ ID NO: 13); a CDR-L2 comprising the sequence GASSRAT (SEQ ID NO:14); a CDR-L3 comprising the sequence QQYGSSYT (SEQ ID NO:15); a CDR-H1 comprising the sequence GITVSSN (SEQ ID NO:16); a CDR-H2 comprising the sequence YSGGS (SEQ ID NO:17); and a CDR-H3 comprising the sequence DLEMAGAFDI (SEQ ID NO:18).
2. The composition of claim 1 , wherein the first antibody or antigen-binding fragment thereof comprises (a) a light chain framework region (FR) 1 comprising the sequence of SEQ ID NO:27; (b) a light chain FR2 comprising the sequence of SEQ ID NO:28; (c) a light chain FR3 comprising the sequence of SEQ ID NO:29; and/or (d) a light chain FR4 comprising the sequence of SEQ ID NQ:30.
3. The composition of claim 1 or 2, wherein the first antibody or antigen-binding fragment thereof comprises a heavy chain FR1 comprising the sequence of SEQ ID NO: 23; (b) a heavy chain FR2 comprising the sequence of SEQ ID NO:24; (c) a heavy chain FR3 comprising the sequence of SEQ ID NO:25; and/or (d) a heavy chain FR4 comprising the sequence of SEQ ID NO:26.
4. The composition of any one of claims 1 to 3, wherein the second antibody or antigen-binding fragment thereof comprises (a) a light chain FR1 comprising the sequence of SEQ ID NO:35; (b) a light chain FR2 comprising the sequence of SEQ ID NO:36; (c) a light chain FR3 comprising the sequence of SEQ ID NO:37; and/or (d) a light chain FR4 comprising the sequence of SEQ ID NO:38.
5. The composition of any one of claims 1 to 4, wherein the second antibody or antigen-binding fragment thereof comprises a heavy chain FR1 comprising the sequence of SEQ ID NO:31 ; (b) a heavy chain FR2 comprising the sequence of SEQ ID NO:32; (c) a heavy chain FR3 comprising the sequence of SEQ ID NO:33; and/or (d) a heavy chain FR4 comprising the sequence of SEQ ID NO:34.
6. The composition of any one of claims 1 to 5, wherein the third antibody or antigen-binding fragment thereof comprises (a) a light chain FR1 comprising the sequence of SEQ ID NO:43; (b) a light chain FR2 comprising the sequence of SEQ ID NO:44; (c) a light chain FR3 comprising the sequence of SEQ ID NO:45; and/or (d) a light chain FR4 comprising the sequence of SEQ ID NO:46.
7. The composition of any one of claims 1 to 6, wherein the third antibody or antigen-binding fragment thereof comprises a heavy chain FR1 comprising the sequence of SEQ ID NO:39; (b) a heavy chain FR2 comprising the sequence of SEQ ID NQ:40; (c) a heavy chain FR3 comprising the sequence of SEQ ID NO:41 ; and/or (d) a heavy chain FR4 comprising the sequence of SEQ ID NO:42.
8. The composition of any one of claims 1 to 7, wherein the first antibody or antigen-binding fragment thereof comprises a heavy chain comprising the sequence of SEQ ID NO:1 , and a light chain comprising the sequence of SEQ ID NO:2.
9. The composition of any one of claims 1 to 8, wherein the second antibody or antigen-binding fragment thereof comprises a heavy chain comprising the sequence of SEQ ID NO:3, and a light chain comprising the sequence of SEQ ID NO:4.
10. The composition of any one of claims 1 to 9, wherein the third antibody or antigen-binding fragment thereof comprises a heavy chain comprising the sequence of SEQ ID NO:5, and a light chain comprising the sequence of SEQ ID NO:6.
11 . The composition of any one of claims 1 to 10, wherein the first antibody or antigen-binding fragment thereof is a human antibody.
12. The composition of any one of claims 1 to 11 , wherein the second antibody or antigenbinding fragment thereof is a human antibody.
13. The composition of any one of claims 1 to 12, wherein the third antibody or antigen-binding fragment thereof is a human antibody.
14. The composition of any one of claims 1 to 13, wherein the first, second and/or third antibodies are of the IgG class.
15. The composition of claim 14, wherein the first, second and/or third antibodies are of the lgG1 subclass.
16. The composition of any one of claims 1 to 15, wherein the combination comprises about 55% to about 65% of the first antibody or antigen-binding fragment thereof.
17. The composition of claim 16, wherein the combination comprises about 63% of the first antibody or antigen-binding fragment thereof.
18. The composition of any one of claims 1 to 17, wherein the combination comprises about 28% to about 37% of the third antibody or antigen-binding fragment thereof.
19. The composition of claim 18, wherein the combination comprises about 32% of the third antibody or antigen-binding fragment thereof.
20. The composition of any one of claims 1 to 19, wherein the combination comprises about 3% to about 8% of the second antibody or antigen-binding fragment thereof.
21 . The composition of claim 20, wherein the combination comprises about 5% of the second antibody or antigen-binding fragment thereof.
22. The composition of any one of claims 1 to 21 , further comprising a pharmaceutically acceptable carrier or excipient.
23. The composition of claim 22, wherein the pharmaceutically acceptable carrier or excipient is adapted for administration of the composition by the pulmonary route.
24. The composition of claim 22 or 23, wherein the composition is an aqueous solution, suspension or emulsion.
25. The composition of any one of claims 22 to 24, wherein the pharmaceutically acceptable carrier or excipient comprises a saline solution or buffer.
26. The composition of any one of claims 22 to 25, wherein the pharmaceutically acceptable carrier or excipient comprises a surfactant or emulsifier.
27. The composition of claim 26, wherein the surfactant or emulsifier is a polysorbate.
28. The composition of claim 26 or 27, wherein the surfactant or emulsifier is present at a concentration of 0.005% to 0.05% in the composition.
29. The composition of any one of claims 22 to 28, wherein the composition has a pH of about 6.5 to about 7.5.
30. A composition comprising one or more nucleic acids encoding the first, second and third antibodies or antigen-binding fragments thereof defined in any one of claims 1 to 15.
31 . The composition of claim 30, wherein the one or more nucleic acids comprise a first nucleic acid encoding the first antibody or antigen-binding fragment thereof defined in any one of claims 1 to 15, a second nucleic acid encoding the second antibody or antigen-binding fragment thereof defined in any one of claims 1 to 15, and a third nucleic acid encoding the third antibody or antigen-binding fragment thereof defined in any one of claims 1 to 15.
32. The composition of claim 30 or 31 , wherein the one or more nucleic acids are mRNA molecules.
33. The composition of any one of claims 30 to 32, wherein the one or more nucleic acids are encapsulated within vesicles.
34. The composition of claim 33, wherein the vesicles are lipid nanoparticles or liposomes.
35. An inhaler or nebulization device comprising the composition of any one of claims 1 to 34.
36. A method for preventing or treating a betacoronavirus infection or a related disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of any one of claims 1 to 34.
37. A method for reducing the risk of developing a betacoronavirus-related disease or the severity of a betacoronavirus-related disease in a subject, the method comprising administering to the subject an effective amount of the composition of any one of claims 1 to 34.
38. A method for blocking the entry of a betacoronavirus in an ACE2-expressing cell, the method comprising contacting the cell and/or the virus with an effective amount of the composition of any one of claims 1 to 34.
39. The method of any one of claims 36 to 38, wherein the betacoronavirus is a sarbecovirus.
40. The method of claim 39, wherein the sarbecovirus is SARS-CoV-2.
41. The method of claim 40, wherein the SARS-CoV-2 is a variant of the Wuhan original SARS-
CoV-2 strain.
42. The method of claim 41 , wherein the SARS-CoV-2 variant is an Omicron variant.
43. The method of any one of claims 36 to 42, wherein the composition is administered with (i) at least one additional anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, or nucleic acid(s) encoding said at least one additional anti-SARS-CoV-2 antibody or antigen-binding fragment thereof; and/or (ii) at least one antiviral or anti-inflammatory drug.
44. The method of any one of claims 36 to 43, wherein the subject is an immunosuppressed or immunocompromised subject.
45. Use of the composition of any one of claims 1 to 34 for preventing or treating a betacoronavirus infection or a related disease in a subject.
46. Use of the composition of any one of claims 1 to 34 for the manufacture of a medicament for preventing or treating a betacoronavirus infection or a related disease in a subject.
47. Use of the composition of any one of claims 1 to 34 for reducing the risk of developing a betacoronavirus-related disease or the severity of a betacoronavirus-related disease in a subject.
48. Use of the composition of any one of claims 1 to 34 for the manufacture of a medicament for reducing the risk of developing a betacoronavirus-related disease or the severity of a betacoronavirus-related disease in a subject.
49. Use of the composition of any one of claims 1 to 34 for blocking the entry of a betacoronavirus in an ACE2-expressing cell.
50. Use of the composition of any one of claims 1 to 34 for the manufacture of a medicament for blocking the entry of a betacoronavirus in an ACE2-expressing cell.
51 . The use of any one of claims 45 to 50, wherein the betacoronavirus is a sarbecovirus.
52. The use of claim 51 , wherein the sarbecovirus is SARS-CoV-2.
53. The use of claim 52, wherein the SARS-CoV-2 is a variant of the Wuhan original SARS- CoV-2 strain.
54. The use of claim 53, wherein the SARS-CoV-2 variant is an Omicron variant.
55. The use of any one of claims 45 to 54, wherein the composition is for administration with (i) at least one additional anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, or nucleic acid(s) encoding said at least one additional anti-SARS-CoV-2 antibody or antigen-binding fragment thereof; and/or (ii) at least one antiviral or anti-inflammatory drug.
56. The use of any one of 45 to 55, wherein the subject is an immunosuppressed or immunocompromised subject.
57. The composition of any one of claims 1 to 34 for use in preventing or treating a betacoronavirus infection or a related disease in a subject.
58. The composition of any one of claims 1 to 34 for use in reducing the risk of developing a betacoronavirus-related disease or the severity of a betacoronavirus-related disease in a subject.
59. The composition of any one of claims 1 to 34 for use in blocking the entry of a betacoronavirus in an ACE2-expressing cell.
60. The composition for use according to any one of claims 57 to 59, wherein the betacoronavirus is a sarbecovirus.
61 . The composition for use according to claim 60, wherein the sarbecovirus is SARS-CoV-2.
62. The composition for use according to claim 61 , wherein the SARS-CoV-2 is a variant of the Wuhan original SARS-CoV-2 strain.
63. The composition for use according to claim 62, wherein the SARS-CoV-2 variant is an Omicron variant.
64. The composition for use according to any one of claims 57 to 63, wherein the composition is for administration with (i) at least one additional anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, or nucleic acid(s) encoding said at least one additional anti-SARS-CoV-2 antibody or antigen-binding fragment thereof; and/or (ii) at least one antiviral or anti-inflammatory drug.
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