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WO2023155902A1 - Intranasal formulations and anti-sars-cov-2-spike protein antibodies - Google Patents

Intranasal formulations and anti-sars-cov-2-spike protein antibodies Download PDF

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Publication number
WO2023155902A1
WO2023155902A1 PCT/CN2023/077013 CN2023077013W WO2023155902A1 WO 2023155902 A1 WO2023155902 A1 WO 2023155902A1 CN 2023077013 W CN2023077013 W CN 2023077013W WO 2023155902 A1 WO2023155902 A1 WO 2023155902A1
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WIPO (PCT)
Prior art keywords
pharmaceutical composition
seq
antibody
composition comprises
antigen binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2023/077013
Other languages
French (fr)
Inventor
Ailong HUANG
Guofeng CHENG
Chengyong Yang
Aishun Jin
Tingting Li
Xiaojian HAN
Yingming Wang
Chao Hu
Feiyang Luo
Bingxia Lu
Cuicui LIN
Shuhui LUO
Tingting Lu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chongqing Mingdao Haoyue Biotechnology Co Ltd
Original Assignee
Chongqing Mingdao Haoyue Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN202210151716.6A external-priority patent/CN116655778A/en
Priority claimed from CN202210151970.6A external-priority patent/CN116650638A/en
Application filed by Chongqing Mingdao Haoyue Biotechnology Co Ltd filed Critical Chongqing Mingdao Haoyue Biotechnology Co Ltd
Publication of WO2023155902A1 publication Critical patent/WO2023155902A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C07K16/104—
    • 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
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame

Definitions

  • the present disclosure relates to (i) intranasal formulations and application of proteins, such as antibodies; and (ii) antibodies capable of binding to the spike protein of multiple variants of SARS-CoV-2.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a protein, a cryoprotectant, a humectant, an emulsifier, a controlled release agent, and a buffer.
  • the disclosure provides a pharmaceutical composition, comprising: an active ingredient or a precursor thereof; no more than about 40% (w/v) of a cryoprotectant; no more than about 30% (w/v) of a humectant; no more than about 5% (w/v) of an emulsifier; no more than about 10% (w/v) of a controlled release agent; and a buffer.
  • the pharmaceutical composition is suitable for mucosal administration to a human. In some embodiments, the pharmaceutical composition is suitable for intranasal administration to a human.
  • the cryoprotectant is selected from the group consisting of trehalose, glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol, and sucrose, or a combination thereof.
  • the cryoprotectant in the pharmaceutical composition is:
  • cryoprotectant in the pharmaceutical composition is:
  • the cryoprotectant is trehalose.
  • the pharmaceutical composition comprises about 0.3% (w/v) to about 12% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 8% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 4% (w/v) of the cryoprotectant.
  • the humectant is selected from the group consisting of glycerin, mannitol, polyethylene glycol 400, polyethylene glycol 4000, D-sorbitol, chitosan, xylitol, and sodium hyaluronate, or a combination thereof.
  • the humectant in the pharmaceutical composition is:
  • the humectant in the pharmaceutical composition is:
  • the humectant is glycerin.
  • the pharmaceutical composition comprises about 0.2% (w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 4% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.7% (w/v) to about 2% (w/v) of the humectant.
  • the emulsifier is selected from the group consisting of TWEEN 80, polysorbate 20, lecithin, sorbitan esters, mono-and/or diglycerides, and sodium stearoyl lactylate, or a combination thereof. In some embodiments, the emulsifier is TWEEN 80.
  • the pharmaceutical composition comprises about 0.002% (w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.04% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.02% (w/v) of the emulsifier.
  • the controlled release agent is selected from the group consisting of hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone K30, or a combination thereof. In some embodiments, the controlled release agent is hydroxypropyl methylcellulose (HPMC) .
  • the pharmaceutical composition comprises about 0.01% (w/v) to about 5% (w/v) , or about 0.01% (w/v) to about 1% (w/v) , of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.004% (w/v) to about 0.4% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) , or about 0.1% (w/v) , of the controlled release agent.
  • the controlled release agent is polyvinylpyrrolidone K30.
  • the pharmaceutical composition comprises about 2% (w/v) to about 10% (w/v) , or about 3% (w/v) to about 6% (w/v) , of the controlled release agent.
  • the buffer is selected from the group consisting of a phosphate buffer, a tris buffer, and a glycine buffer.
  • the buffer in the pharmaceutical composition is:
  • the buffer in the pharmaceutical composition is:
  • the buffer is a phosphate buffer. In some embodiments, the buffer in the pharmaceutical composition is about 20 mM phosphate or tris.
  • the buffer has a pH of about 4-8, or about 5-7. In some embodiments, the buffer has a pH of about 5.5-6.5. In some embodiments, the buffer has a pH of about 6.0.
  • the pharmaceutical composition comprises an antibiotic.
  • the antibiotic comprises benzalkonium chloride, benzyl alcohol, chlorobutanol, or a combination thereof.
  • the antibiotic in the pharmaceutical composition is:
  • the antibiotic in the pharmaceutical composition is:
  • the antibiotic is benzalkonium chloride.
  • the pharmaceutical composition comprises about 0.002% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.02% (w/v) , or about 0.01% (w/v) , of the antibiotic.
  • the mean retention time of the active ingredient or a precursor thereof in a nasal cavity of a primate is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, or at least 12 hours, after intranasal administration of the pharmaceutical composition to the primate.
  • the primate is a rhesus macaque.
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • cryoprotectant about 2%-about 8% (w/v) of the cryoprotectant
  • the buffer has a pH of about 5.5-6.5.
  • the pharmaceutical composition comprises:
  • the buffer has a pH of about 6.0.
  • the pharmaceutical composition comprises:
  • the buffer has a pH of about 6.0.
  • the pharmaceutical composition comprises:
  • the buffer has a pH of about 6.0.
  • the pharmaceutical composition comprises:
  • the buffer has a pH of about 6.0.
  • the buffer comprises about 20 mM phosphate salt.
  • the phosphate salt is a sodium phosphate salt.
  • cryoprotectant is trehalose
  • the humectant is glycerin
  • the emulsifier is TWEEN 80
  • the controlled release agent is HPMC.
  • the pharmaceutical composition comprises about 0.01% (w/v) benzalkonium chloride.
  • the active ingredient comprises a polypeptide. In some embodiments, the active ingredient comprises an antibody or an antigen binding fragment thereof. In some embodiments, the active ingredient has a concentration of about 0.1-100 mg/ml, about 0.5-50 mg/ml, or about 1-30 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 mg/ml. In some embodiments, the antibody or antigen fragment thereof binds to a coronavirus. In some embodiments, the antibody or antigen fragment thereof binds to SARS-CoV-2.
  • the pharmaceutical composition comprises one or more of:
  • an antibody A or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 13, a HCDR2 comprising SEQ ID NO: 14, and a HCDR3 comprising SEQ ID NO: 15 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 10, a LCDR2 comprising SEQ ID NO: 11, and a LCDR3 comprising SEQ ID NO: 12;
  • an antibody A-1 or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 4, a HCDR2 comprising SEQ ID NO: 5, and a HCDR3 comprising SEQ ID NO: 6 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 1, a LCDR2 comprising SEQ ID NO: 2, and a LCDR3 comprising SEQ ID NO: 3;
  • an antibody B or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 23, a HCDR2 comprising SEQ ID NO: 24, and a HCDR3 comprising SEQ ID NO: 25 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, and a LCDR3 comprising SEQ ID NO: 22;
  • an antibody C or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 33, a HCDR2 comprising SEQ ID NO: 34, and a HCDR3 comprising SEQ ID NO: 35 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 30, a LCDR2 comprising SEQ ID NO: 31, and a LCDR3 comprising SEQ ID NO: 32;
  • an antibody D/E or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 43, a HCDR2 comprising SEQ ID NO: 44, and a HCDR3 comprising SEQ ID NO: 45 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 40, a LCDR2 comprising SEQ ID NO: 41, and a LCDR3 comprising SEQ ID NO: 42; and
  • an antibody F or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 63, a HCDR2 comprising SEQ ID NO: 64, and a HCDR3 comprising SEQ ID NO: 65 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62.
  • the antibody A or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 18 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 16 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
  • the antibody A-1 or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 8 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 7 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
  • the antibody B or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 28 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 26 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
  • the antibody C or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 38 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 36 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
  • the antibody D/E or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 48 or 52 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 46 or 50 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; and/or
  • the antibody F or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 68 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 66 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
  • (e) the antibody D/E or antigen binding fragment comprises:
  • HCR heavy chain constant region
  • LCR light chain constant region
  • HCR comprising or consisting of SEQ ID NO: 53 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a LCR comprising or consisting of SEQ ID NO: 51 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or
  • the pharmaceutical composition comprises at least two of the antibodies or antigen binding fragments. In some embodiments, the pharmaceutical composition comprises (c) the antibody B or antigen binding fragment thereof and (e) the antibody D/E or antigen binding fragment thereof. In some embodiments, the ratio of (c) the antibody B or antigen binding fragment thereof to (e) the antibody D/E or antigen binding fragment thereof is between about 1: 2 and about 1: 8. In some embodiments, the ratio of (c) the antibody B or antigen binding fragment thereof to (e) the antibody D/E or antigen binding fragment thereof is about 1: 4.
  • the pharmaceutical composition comprises (d) the antibody C or antigen binding fragment thereof.
  • the pharmaceutical composition comprising (f) the antibody F or antigen binding fragment thereof.
  • the antibody is an IgG, an IgM, or an IgA.
  • the pharmaceutical composition is administered via a nasal administration device.
  • the disclosure provides a nasal or mucosal administration device comprising the pharmaceutical composition of the disclosure.
  • the disclosure provides a kit comprising the pharmaceutical composition of the disclosure and a nasal or mucosal administration device.
  • the nasal administration device is a metered dose nasal pump device or a unit dose nasal pump device.
  • the disclosure provides an antibody or antigen binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) , a heavy chain complementarity determining region 2 (HCDR2) and a heavy chain complementarity determining region 3 (HCDR3) , wherein: the HCDR1 comprises the amino acid sequence of GFTFSGSA (SEQ ID NO: 13) ; the HCDR2 comprises the amino acid sequence of IVVGSGNT (SEQ ID NO: 14) ; and the HCDR3 comprises the amino acid sequence of AAPYCSSTSCRDGFDI (SEQ ID NO: 15) .
  • VH heavy chain variable domain
  • HCDR1 comprises the amino acid sequence of GFTFSGSA (SEQ ID NO: 13)
  • the HCDR2 comprises the amino acid sequence of IVVGSGNT (SEQ ID NO: 14)
  • the HCDR3 comprises the amino acid sequence of AAPYCSSTSCRDGFDI (SEQ ID NO: 15) .
  • the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 18, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
  • the antibody or antigen binding fragment comprises a light chain variable domain (VL) comprising a light chain complementarity determining region 1 (LCDR1) , a light chain complementarity determining region 2 (LCDR2) and a light chain complementarity determining region 3 (LCDR3) , wherein: the LCDR1 comprises the amino acid sequence of QSVRSSY (SEQ ID NO: 10) ; the LCDR2 comprises the amino acid sequence of GAS (SEQ ID NO: 11) ; and the LCDR3 comprises the amino acid sequence of QQYGRSPWT (SEQ ID NO: 12) .
  • VL light chain variable domain
  • the LCDR1 comprises the amino acid sequence of QSVRSSY (SEQ ID NO: 10)
  • the LCDR2 comprises the amino acid sequence of GAS (SEQ ID NO: 11)
  • the LCDR3 comprises the amino acid sequence of QQYGRSPWT (SEQ ID NO: 12) .
  • VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
  • the disclosure provides an antibody or antigen binding fragment thereof, comprising:
  • VH comprising a HCDR1 comprising SEQ ID NO: 23, a HCDR2 comprising SEQ ID NO: 24, and a HCDR3 comprising SEQ ID NO: 25 and a VL comprising a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, and a LCDR3 comprising SEQ ID NO: 22;
  • VH comprising a HCDR1 comprising SEQ ID NO: 33, a HCDR2 comprising SEQ ID NO: 34, and a HCDR3 comprising SEQ ID NO: 35 and a VL comprising a LCDR1 comprising SEQ ID NO: 30, a LCDR2 comprising SEQ ID NO: 31, and a LCDR3 comprising SEQ ID NO: 32;
  • VH comprising a HCDR1 comprising SEQ ID NO: 43, a HCDR2 comprising SEQ ID NO: 44, and a HCDR3 comprising SEQ ID NO: 45 and a VL comprising a LCDR1 comprising SEQ ID NO: 40, a LCDR2 comprising SEQ ID NO: 41, and a LCDR3 comprising SEQ ID NO: 42; or
  • VH comprising a HCDR1 comprising SEQ ID NO: 63, a HCDR2 comprising SEQ ID NO: 64, and a HCDR3 comprising SEQ ID NO: 65 and a VL comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62; .
  • the antibody or antigen binding fragment is for intranasal administration.
  • the antibody or antigen binding fragment is capable of binding to a SARS-CoV-2 spike protein, optionally wherein the SARS-CoV-2 spike proteins comprises the amino acid sequence set forth in SEQ ID NO: 106, SEQ ID NO: 108, or SEQ ID NO: 110.
  • the antibody is an IgG, an IgM, or an IgA.
  • the disclosure provides a polynucleotide encoding the antibody or antigen binding fragment of the disclosure.
  • the disclosure provides a vector comprising the polynucleotide of the disclosure.
  • the disclosure provides a composition comprising the antibody or antigen binding fragment, the polynucleotide, or the vector of the disclosure.
  • the disclosure provides a cell comprising the polynucleotide or the vector of the disclosure.
  • the disclosure provides a method of producing the antibody or antigen binding fragment of the disclosure, comprising culturing a cell comprising a vector encoding the antibody or antigen binding fragment thereof and harvesting the antibody or antigen binding fragment from the culture medium of the cell.
  • the disclosure provides a method of treating or preventing a SARS-CoV-2 infection in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen binding fragment of the disclosure.
  • the antibody or antigen binding fragment is administered intranasally, subcutaneously, intravenously, or intramuscularly. In some embodiments, the antibody or antigen binding fragment is administered intranasally.
  • the disclosure provides a combination of antibodies or antigen binding fragments thereof, comprising at least two of the following antibodies or antigen binding fragments thereof:
  • an antibody A or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 13, a HCDR2 comprising SEQ ID NO: 14, and a HCDR3 comprising SEQ ID NO: 15 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 10, a LCDR2 comprising SEQ ID NO: 11, and a LCDR3 comprising SEQ ID NO: 12;
  • an antibody A-1 or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 4, a HCDR2 comprising SEQ ID NO: 5, and a HCDR3 comprising SEQ ID NO: 6 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 1, a LCDR2 comprising SEQ ID NO: 2, and a LCDR3 comprising SEQ ID NO: 3;
  • an antibody B or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 23, a HCDR2 comprising SEQ ID NO: 24, and a HCDR3 comprising SEQ ID NO: 25 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, and a LCDR3 comprising SEQ ID NO: 22;
  • an antibody C or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 33, a HCDR2 comprising SEQ ID NO: 34, and a HCDR3 comprising SEQ ID NO: 35 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 30, a LCDR2 comprising SEQ ID NO: 31, and a LCDR3 comprising SEQ ID NO: 32;
  • an antibody D/E or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 43, a HCDR2 comprising SEQ ID NO: 44, and a HCDR3 comprising SEQ ID NO: 45 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 40, a LCDR2 comprising SEQ ID NO: 41, and a LCDR3 comprising SEQ ID NO: 42; and
  • an antibody F or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 63, a HCDR2 comprising SEQ ID NO: 64, and a HCDR3 comprising SEQ ID NO: 65 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62.
  • the antibody A or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 18 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 16 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
  • the antibody A-1 or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 8 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 7 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
  • the antibody B or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 28 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 26 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
  • the antibody C or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 38 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 36 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
  • the antibody D/E or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 48 or 52 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 46 or 50 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; and/or
  • the antibody F or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 68 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 66 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
  • the antibody D/E or antigen binding fragment comprises: a heavy chain constant region (HCR) comprising or consisting of SEQ ID NO: 49 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a light chain constant region (LCR) comprising or consisting of SEQ ID NO: 47 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or a HCR comprising or consisting of SEQ ID NO: 53 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a LCR comprising or consisting of SEQ ID NO: 51 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity
  • the combination of antibodies or antigen binding fragments comprises: the antibody B or antigen binding fragment thereof, and the antibody D/E or antigen binding fragment thereof.
  • the ratio of the antibody B or antigen binding fragment thereof to the antibody D/E or antigen binding fragment thereof is between about 1: 2 and about 1: 8. In some embodiments, the ratio of the antibody B or antigen binding fragment thereof to the antibody D/E or antigen binding fragment thereof is about 1: 4.
  • the combination of antibodies or antigen binding fragments comprises the antibody C or antigen binding fragment thereof. In some embodiments, the combination of antibodies or antigen binding fragments comprises the antibody F or antigen binding fragment thereof.
  • the combination displays a synergistic effect against COVID-19 Omicron BA. 2, BA. 2.12.1, or BA. 5 strain.
  • the disclosure provides a method of delivering an active ingredient or a precursor thereof to a nasal cavity or a mucosa of a subject, comprising administering to the subject the pharmaceutical composition of the disclosure.
  • the disclosure provides a method of preventing a disease in a subject in need thereof, comprising administering the pharmaceutical composition, the antibody or antigen binding fragment, or the combination, to the subject.
  • the disclosure provides a method of treating a disease in a subject in need thereof, comprising administering the pharmaceutical composition, the antibody or antigen binding fragment, or the combination, to the subject.
  • the pharmaceutical composition, or the antibody or antigen binding fragment or the combination is administered to a mucosa of the subject.
  • the pharmaceutical composition, or the antibody or antigen binding fragment or the combination is administered intranasally to the subject.
  • about 30-150 ⁇ l per administration of the pharmaceutical composition is delivered to a nasal cavity of the subject. In some embodiments, about 70-100 ⁇ l per administration of the pharmaceutical composition is delivered to the nasal cavity of the subject.
  • about 0.15 mg to about 0.75 mg of the active ingredient or a precursor thereof is delivered to the nasal cavity per administration.
  • the antibody or antigen binding fragment, or the combination is delivered to the subject about 1-6 times per day. In some embodiments, the pharmaceutical composition, the antibody or antigen binding fragment, or the combination, is delivered to the subject about 2-3 times per day.
  • the pharmaceutical composition, the antibody or antigen binding fragment thereof, or the combination is delivered to the subject for about 7-14 days.
  • administering the pharmaceutical composition, the antibody or antigen binding fragment thereof, or the combination starts prior to the onset of any symptom of the disease. In some embodiments, administering the pharmaceutical composition, the antibody or antigen binding fragment thereof, or the combination, starts after the onset of at least one symptom of the disease.
  • the symptom is a respiratory symptom.
  • the disease comprises a respiratory symptom.
  • the respiratory symptom is allergy, nasal congestion, nasal infection, or a combination thereof.
  • the disease is a respiratory infection caused by a virus, a bacterial, a fungus, or a combination thereof.
  • the disease is a viral infection.
  • the disease is a chronic viral infection.
  • the disease is an acute viral infection.
  • the disease is a coronavirus infection.
  • the disease is SARS-CoV-2 infection.
  • the concentration of the antibody or antigen binding fragment thereof in a nasal cavity of the subject is at least 100-fold higher than the concentration of the antibody or antigen binding fragment thereof in the blood sample of the subject within 24 hours after the administration as measured by ELISA.
  • any of the features or components of embodiments discussed above or herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value discussed above or herein may be combined with another related value discussed above or herein to recite a range with the values representing the upper and lower ends of the range, and such ranges are encompassed within the scope of the present disclosure.
  • Figures 1A-1F show protective efficacy of Antibody B against the Delta variant of the SARS-Cov-2 virus (Delta variant) in a hamster model.
  • Figure 1A Animal experimental scheme.
  • Figure 1B Body weight changes.
  • Figure 1C Viral RNA (log 10 (RNA copies per ml) ) detected in the nasal washes of hamsters challenged with the Delta variant at 3 days post infection (dpi) .
  • Figure 1D Viral RNA (log10 (RNA copies per g) ) detected in the respiratory tracts of hamsters challenged with the Delta variant at 3 dpi.
  • Figure 1E Viral RNA (log 10 (RNA copies per ml) ) detected in the throat swabs of hamsters challenged with the Delta variant at 3 dpi.
  • Figure 1F The number of infectious viruses (PFU) was measured in the respiratory tract at 3 dpi by a viral plaque assay in Vero E6 cells.
  • Figure 1G shows the concentration of antibody B in the nasal cavity or in the circulating blood in a Cynomolgus monkey study.
  • Figures 2A-2B show histopathological changes in the lung at 3 dpi in hamsters challenged with Delta variant.
  • Figure 2A Representative images of the H&E-stained lung tissues of Group 1, 2, 3, and 4 hamsters at 3 days after Delta variant challenge. There was no obvious abnormality in the structure of the bronchus at any level. The alveolar wall was composed of a single layer of epithelium with a clear structure. No obvious inflammatory changes were observed (Group 1, prophylactic group) . Lung tissue showed small foci of alveolar wall thickening with a small amount of neutrophil infiltration (black arrows) ; vascular congestion and dilation were common (green arrows) (Group 2, 3-dose antibody treatment group) .
  • Lung tissue showed mild alveolar wall thickening with neutrophil infiltration (black arrows) ; occasionally, there were a few necrotic epithelial cells, lymphocytes, and neutrophils in the bronchi (purple arrows) (Group 3, 2-dose antibody treatment group) . Diffuse hemorrhage in the alveolar wall (red arrow) was seen in the lung tissue; a large number of blood cells in the bronchi were often seen (green arrow) ; multifocal thickening of the alveolar wall with neutrophil infiltration (black arrow) was seen (Group 4, control group without antibody) .
  • Figure 2B Sections were comprehensively evaluated for histological changes and inflammation progression.
  • Figures 3A-3E show protective efficacy of Antibody B against omicron in the hamster model.
  • Figure 3A Animal experimental scheme.
  • Figure 3B Viral RNA (log 10 (RNA copies per ml) ) detected in the nasal washes of hamsters challenged with Omicron variant at 3 dpi.
  • Figure 3C Viral RNA (log10 (RNA copies per g) ) detected in respiratory tracts of hamsters challenged with Omicron variant at 3 dpi.
  • Figure 3D Viral RNA (log10 (RNA copies per ml) ) detected in the throat swabs of hamsters challenged with Omicron at 3 dpi.
  • Figure 3E The number of infectious virus (PFU) was measured in the respiratory tract at 3 dpi by the viral plaque assay in Vero E6 cells.
  • PFU infectious virus
  • Figures 4A-4E show activities of Antibody B.
  • Figure 4A Binding of Antibody B to different RBDs, as measured by an ELISA.
  • Figure 4B Neutralization of pseudotyped SARS-CoV-2 wildtype (WT) and different variants by Antibody B. Inhibitory rates of infection were calculated by scanning fluorescent plaques.
  • Figure 4C Evaluation of the neutralization activity of Antibody B against live SARS-CoV-2 WT and the Delta and Omicron variants.
  • Figure 4D Binding regions of Antibody B. RBDs from WT SARS-CoV-2 and the Alpha, Beta, Gamma, Delta and Omicron variants are aligned. The lines indicate the binding regions of Antibody B.
  • Figure 4E Structure of the complex of Antibody B Fab bound onto the SARS-CoV-2 RBD.
  • the secondary structure elements of the RBD and heavy and light chains of Antibody B Fab are colored green, cyan and violet, respectively.
  • the surface of the RBD is colored green.
  • the mutated residues of the Delta variant are colored blue (middle) , while those from the Omicron are colored red (right) .
  • Figure 5 shows the weight change of golden hamsters after challenge (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001) .
  • Figure 6 shows viral RNA level in the indicated tissue at 3-day post infection (dpi) .
  • L.O.D limit of detection is 1.649.
  • Figure 8 shows the weight change of golden hamsters after challenge.
  • Figure 9 shows the viral load of tissues of golden hamsters in each group after challenge. (****p ⁇ 0.0001) .
  • L.O.D limit of detection is 1.649.
  • Figure 10 shows the virus titer of tissues of golden hamsters in each group after challenge. (****p ⁇ 0.0001)
  • Figure 11A shows the amount of Antibody B antibody in mice nasal mucosa.
  • Figure 11B shows the amount of Antibody B antibody in mice blood.
  • Figure 11C shows the amount of Antibody B antibody in mice trachea.
  • Figure 12A shows the radioactive retention in the nasal cavities of healthy adult rabbits within 24h after nasal dripping of 131 I-Antibody B antibody and Na 131 I.
  • the bar on the left represents the value for 131 I-Antibody B and the bar on the right represents the value for Na 131 I.
  • Figure 12B shows the downward trend of radioactive retention in the nasal cavities of healthy adult rabbits within 24h after nasal dripping of 131 I-Antibody B antibody and Na 131 I.
  • Figure 12C shows the radioactive retention in the nasal cavities and pharynges of healthy adult rabbits within 24h after nasal dripping of 131 I-Antibody B antibody and Na 131 I.
  • Figure 12D shows the downward trend of the radioactive retention in the nasal cavities and pharynges of healthy adult rabbits within 24h after nasal dripping of 131 I-Antibody B antibody and Na 131 I.
  • Figure 13 shows the drug content in nasal swabs of cynomolgus macaques after nasal spraying.
  • Figure 14 shows the binding ability of Antibody D to recombinant proteins of wild-type and various mutant strains of coronaviruses.
  • Figure 15 shows the binding ability of Antibody B to recombinant proteins of wild-type and various mutant strains of coronaviruses.
  • Figure 16 shows affinity results between Antibody D antibody and wild-type, Delta, Omicron BA. 1, Omicron BA. 2 and Omicron BA. 4/5) of coronaviruses.
  • Figure 17 shows affinity results between Antibody B antibody and coronaviruses Delta and Omicron (BA. 1 and BA. 2) .
  • Figure 18 shows blocking of binding of Omicron RBD/Sto ACE2 through Antibody D, Antibody B and A8G6 antibodies.
  • the upper figures show that the antibodies compete with Omicron RBD for binding to ACE2 recombinant protein.
  • the lower figures show that the antibodies compete with Omicron S for binding to ACE2 recombinant protein.
  • the figures from left to right sequentially represent Antibody B, Antibody D and A8G6.
  • the black curve represents the control group.
  • ACE2 When no antibody is added, ACE2 can bind to RBD or S coated on the chip, and the response value increases with time, the curve shows an upward trend (blue curve) ; the red curve shows that after the antibody is added in advance to bind to RBD or S on the chip, ACE2 can no longer bind to RBD or S on the chip, and the response value does not increase with time.
  • Antibody D partially competes with ACE2 to block its binding to the recombinant protein of spike protein (S) of a coronavirus; Antibody D does not compete with ACE2 and cannot block its binding to the recombinant protein of Receptor Binding Domain (RBD) of a coronavirus. 2.
  • Antibody B completely competes with ACE2, to block its binding to a coronavirus RBD or S recombinant protein.
  • A8G6 completely competes with ACE2 to block its binding to a coronavirus RBD or S recombinant protein.
  • Figure 19A shows pseudovirus neutralization ability tests of Antibody B against wild-type and various mutant strains of a coronavirus.
  • Figure 19B shows pseudovirus neutralization ability tests of Antibody B against wild-type and various mutant strains of coronaviruses.
  • Figure 20 shows pseudovirus neutralization test results of A8G6 coronavirus neutralizing antibodies.
  • Figure 21A shows 3D Synergy 95%pattern-20220811 of BA. 2 pseudovirus neutralization tests under joint use of Antibody B and Antibody D.
  • Figure 21B shows 3D Synergy 95%pattern-20220816 of BA. 2 pseudovirus neutralization tests under joint use of Antibody B and Antibody D.
  • Figure 22A The neutralizing potencies of 58G6 alone, 55A8 alone or 58G6 and 55A8 in combination against SARS-CoV-2 and the Delta, Omicron BA. 1, Omicron BA. 1 +L452R, and Omicron BA. 2 variants were measured with a pseudovirus neutralization assay. The dashed line indicates a 0%or 50%reduction in viral neutralization. Data are presented as the mean values.
  • Figure 22B Neutralization against authentic SARS-CoV-2, Delta and Omicron BA. 1 viruses.
  • Figure 23A shows BA. 2 pseudovirus neutralization test results under joint use of Antibody B and Antibody D at different ratios.
  • Figure 23B shows BA. 2.12.1 pseudovirus neutralization test results under joint use of Antibody B and Antibody D at different ratios.
  • Figure 23C shows BA. 5 pseudovirus neutralization test results under joint use of Antibody B and Antibody D at different ratios.
  • Figure 23D shows Delta pseudovirus neutralization test results under joint use of Antibody B and Antibody D at different ratios.
  • Figure 24 shows neutralization activities of A8G6 and Antibody D against live viruses of wild-type, Delta and Omicron strains.
  • Figure 25A shows an animal experimental scheme.
  • Figure 25B shows the viral RNA (log10 (RNA copies per g) ) detected in the respiratory tract of hamsters challenged with the Omicron variant at 3 dpi.
  • the order is Group 1, Group 2, Group 3, Group 4, from left to right.
  • Figure 25C shows the number of infectious viruses (PFU) in the respiratory tract measured at 3 dpi with a viral plaque assay performed with Vero E6 cells.
  • PFU infectious viruses
  • Figures 26A-26D show the results of the protective efficacy of 55A8/58G6 cocktails against Omicron in a hamster model.
  • Figure 26A shows an animal experimental scheme.
  • Figure 26B shows the viral RNA (log10 (RNA copies per g) ) detected in the respiratory tract of hamsters challenged with the Omicron variant at 3 dpi.
  • the order is Group 1, Group 2, Group 3, Group 4, Group 5, from left to right.
  • Figure 26C shows the number of infectious viruses (PFU) in the respiratory tract measured at 3 dpi with a viral plaque assay performed with Vero E6 cells.
  • the order is Group 1, Group 2, Group 3, Group 4, Group 5, from left to right.
  • Figure 26D shows the weight changes in the hamster model treated with 2-cocktail as a measure of the protective efficacy against Omicron.
  • Figures 27A-27D shows the results of the prophylactic dose of 55A8/58G6 cocktails against Omicron in the hamster model.
  • Figure 27A shows an animal experimental scheme.
  • Figure 27B shows the viral RNA (log10 (RNA copies per g) ) detected in the respiratory tract of hamsters challenged with the Omicron variant at 3 dpi (**p ⁇ 0.01; ****p ⁇ 0.0001; L.O.D (limit of detection) is 1.30) .
  • the order is Group 1, Group 2, Group 3, Group 4, Group 5, from left to right.
  • Figure 27C shows the number of infectious viruses (PFU) in the respiratory tract measured at 3 dpi with a viral plaque assay performed with Vero E6 cells (****p ⁇ 0.0001; L.O.D (limit of detection) is 1.30) .
  • the order is Group 1, Group 2, Group 3, Group 4, Group 5, from left to right.
  • Figure 27D shows weight changes in the hamster model after treatment with 2-cocktails as a measure to define the prophylactic dose effective against Omicron.
  • Figure 28A shows the content of A8G6 antibodies in nasal mucosa of mice.
  • Figure 28B shows the content of A8G6 antibodies in blood of mice.
  • Figure 28C shows the content of A8G6 antibodies in tracheas of mice.
  • Figure 29 shows mean drug concentration-time curves of nasal swabs after different times of A8G6 coronavirus neutralizing antibody nasal spray to rhesus monkeys every day.
  • Figure 30A shows Antibody B antibody concentration in rat mucosal samples 4-hours post administration.
  • LOB is the maximal value of the blank control (Group I) .
  • Figure 30B shows Antibody B antibody concentration in mouse mucosal samples 6-hours post administration. Two animals in the glycerin group and one animal in the HPMC group has antibody level that is higher than the upper detection limit.
  • LOB is the maximal value of the blank control (Group F) .
  • the term “about” in relation to a reference numerical value can include the numerical value itself and a range of values plus or minus 10%from that numerical value. For example, the amount “about 10” includes 10 and any amounts from 9 to 11.
  • coronavirus refers to any virus of the coronavirus family, including but not limited to SARS-CoV-2, MERS-CoV, and SARS-CoV.
  • SARS-CoV-2 refers to the recently emerged coronavirus.
  • SARS-CoV-2 has also been known as 2019-nCoV and Wuhan coronavirus. Without being held to theory or mechanism, it binds via the viral spike protein to human host cell receptor angiotensin-converting enzyme 2 (ACE2) .
  • ACE2 human host cell receptor angiotensin-converting enzyme 2
  • the spike protein also binds to and is cleaved by TMPRSS2, which activates the spike protein for membrane fusion of the virus.
  • CoV-S also called “S” or “S protein” refers to the spike protein of a coronavirus, and can refer to specific S proteins such as SARS-CoV-2-S, MERS-CoV S, and SARS-CoV S.
  • the wild-type SARS-CoV-2-Spike protein is a 1273 amino acid type I membrane glycoprotein which assembles into trimers that constitute the spikes or peplomers on the surface of the enveloped coronavirus particle.
  • the protein has two essential functions, host receptor binding and membrane fusion, which are attributed to the N-terminal (S1) and C-terminal (S2) halves of the S protein.
  • CoV-S binds to its cognate receptor via a receptor binding domain (RBD) present in the S1 subunit.
  • the amino acid sequence of wild-type SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 70.
  • the amino acid sequence of alpha (B. 1.1.7) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 72.
  • the amino acid sequence of beta (B. 1.351) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 74.
  • the amino acid sequence of gamma (P. 1) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 76.
  • the amino acid sequence of delta (B.1.617.2) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 78.
  • the amino acid sequence of omicron (BA. 1) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 80.
  • the term “CoV-S” includes protein variants of CoV spike protein isolated from different CoV isolates as well as recombinant CoV spike protein or a fragment thereof.
  • alpha CoV-S” or “alpha (B. 1.1.7) CoV-S” refers to the CoV spike protein of the alpha (B.1.1.7) SARS-CoV-2 spike protein.
  • beta CoV-S or “beta (B.
  • CoV-S refers to the CoV spike protein of the beta (B. 1.351) SARS-CoV-2 spike protein.
  • gamma CoV-S or “gamma (P. 1) CoV-S” refers to the CoV spike protein of the gamma (P. 1) SARS-CoV-2 spike protein.
  • delta CoV-S or “delta (B. 1.617.2) CoV-S” refers to the CoV spike protein of the delta (B. 1.617.2) SARS-CoV-2 spike protein.
  • omicron CoV-S or “omicron (BA. 1 or BA. 2) CoV-S” refers to the CoV spike protein of the omicron (BA. 1 or BA. 2) SARS-CoV-2 spike protein.
  • the term also encompasses CoV spike protein or a fragment thereof coupled to, for example, a histidine tag, mouse or human Fc, or a signal sequence such as ROR1.
  • RBD-CoV-S also called “RBD-S” or “RBD-S protein” refers to the RBD-domain spike protein of a coronavirus, and can refer to specific RBD-domains of S proteins such as RBD-SARS-CoV-2-S, RBD-MERS-CoV S, and RBD-SARS-CoV S.
  • the RBD domain of the wild-type SARS-CoV-2-Spike protein facilitates binding of the S1 subunit to its cognate receptor.
  • the amino acid sequence of the RBD domain of the wild-type SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 71.
  • the amino acid sequence of the RBD domain of the alpha (B.1.1.7) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 73.
  • the amino acid sequence of the RBD domain of the beta (B. 1.351) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 75.
  • the amino acid sequence of the RBD domain of the gamma (P. 1) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 77.
  • the amino acid sequence of the RBD domain of the delta (B. 1.617.2) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 79.
  • SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 81.
  • RBD-CoV-S includes the RBD-domain of protein variants of CoV spike protein isolated from different CoV isolates as well as recombinant CoV spike protein or a fragment thereof.
  • alpha RBD-CoV-S or “alpha (B. 1.1.7) RBD-CoV-S” refers to the RBD domain of the alpha (B. 1.1.7) SARS-CoV-2 spike protein.
  • beta RBD-CoV-S or “beta (B. 1.351) RBD-CoV-S” refers to the RBD domain of the beta (B.
  • the term “gamma CoV-S” or “gamma (P.1) CoV-S” refers to the RBD domain of the gamma (P. 1) SARS-CoV-2 spike protein.
  • the term “delta CoV-S” or “delta (B. 1.617.2) CoV-S” refers to the RBD domain of the delta (B.1.617.2) SARS-CoV-2 spike protein.
  • the term “omicron CoV-S” or “omicron (BA. 1 or BA. 2) CoV-S” refers to the RBD domain of the omicron (BA. 1 or BA. 2) SARS-CoV-2 spike protein.
  • the term also encompasses CoV spike protein or a fragment thereof coupled to, for example, a histidine tag, mouse or human Fc, or a signal sequence such as ROR1.
  • coronavirus infection refers to infection with a coronavirus such as SARS-CoV-2, MERS-CoV, or SARS-CoV.
  • coronavirus respiratory tract infections often in the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastro-intestinal symptoms such as diarrhea, organ failure (kidney failure and renal dysfunction) , septic shock, and death in severe cases.
  • alpha coronavirus infection or “alpha SARS-CoV-2 infection, ” as used herein, refers to infection with an alpha coronavirus such as alpha (B. 1.1.7) SARS-CoV-2.
  • beta coronavirus infection or “beta SARS-CoV-2 infection, ” as used herein, refers to infection with a beta coronavirus such as beta (B. 1.351) SARS-CoV-2.
  • gamma coronavirus infection or “gamma SARS-CoV-2 infection, ” as used herein, refers to infection with a gamma coronavirus such as gamma (P. 1) SARS-CoV-2.
  • delta coronavirus infection or “delta SARS-CoV-2 infection, ” as used herein, refers to infection with a delta coronavirus such as delta (B.1.617.2) SARS-CoV-2.
  • omicron coronavirus infection or “omicron SARS-CoV-2 infection, ” as used herein, refers to infection with an omicron coronavirus such as omicron (BA. 1 or BA. 2) SARS-CoV-2.
  • antigen-binding portion or “antigen-binding fragment” of an antibody or antigen-binding protein, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F (ab’) 2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide) , or a constrained FR3-CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., as defined in WO08/020079 or WO09/138519) (e.g., monovalent nanobodies, bivalent nanobodies, etc. ) , small modular immunopharmaceuticals (SMIPs) , and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment, ” as used herein.
  • the antigen-binding fragment comprises three or more CDRs of an antibody of Table 1 (e.g., HCDR1, HCDR2 and HCDR3; or LCDR1, LCDR2 and LCDR3) .
  • polypeptide and “protein” are used interchangeably in this disclosure.
  • the present disclosure includes methods for treating or preventing a viral infection in a subject.
  • the viral infection is caused by a respiratory infection virus.
  • the respiratory infection virus is influenza virus, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, or bocaviruses.
  • virus includes any virus whose infection in the body of a subject is treatable or preventable by administration of an respiratory viral antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof that bind to influenza virus antigens, respiratory syncytial virus antigens, parainfluenza virus antigens, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, or bocaviruses.
  • an respiratory viral antigen-binding proteins e.g., antibodies or antigen-binding fragments thereof that bind to influenza virus antigens, respiratory syncytial virus antigens, parainfluenza virus antigens, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, or bocaviruses.
  • virus includes any virus whose infection in the body of a subject is treatable or preventable by administration of an anti-viral antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof of the present disclosure (e.g., of Table 1) , (e.g., wherein infectivity of the virus is at least partially dependent on CoV-S) .
  • an anti-viral antigen-binding proteins e.g., antibodies or antigen-binding fragments thereof of the present disclosure (e.g., of Table 1) , (e.g., wherein infectivity of the virus is at least partially dependent on CoV-S) .
  • a “virus” is any virus that expresses spike protein (e.g., CoV-S) .
  • virus also includes a CoV-S-dependent respiratory virus which is a virus that infects the respiratory tissue of a subject (e.g., upper and/or lower respiratory tract, trachea, bronchi, lungs) and is treatable or preventable by administration of an anti-CoV-S antibody or antigen-binding fragment thereof.
  • virus includes coronavirus, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) , SARS-CoV (severe acute respiratory syndrome coronavirus) , and MERS-CoV (Middle East respiratory syndrome (MERS) coronavirus) .
  • Coronaviruses can include the genera of alphacoronaviruses, beta coronaviruses, gamma coronaviruses, delta coronaviruses, and/or omicron coronaviruses.
  • the antibodies or antigen-binding fragments provided herein can bind to and/or neutralize an alpha coronavirus, a beta coronavirus, a gamma coronavirus, a delta coronavirus, and/or an omicron coronavirus. In some embodiments, this binding and/or neutralization can be specific for a particular genus of coronavirus or for a particular subgroup of a genus.
  • “Viral infection” refers to the invasion and multiplication of a virus in the body of a subject.
  • coronavirus virions are spherical with diameters of approximately 125 nm.
  • the most prominent feature of coronaviruses is the club-shape spike projections emanating from the surface of the virion. These spikes are a defining feature of the virion and give them the appearance of a solar corona, prompting the name, coronaviruses.
  • Within the envelope of the virion is the nucleocapsid.
  • Coronaviruses have helically symmetrical nucleocapsids, which is uncommon among positive-sense RNA viruses, but far more common for negative-sense RNA viruses.
  • SARS-CoV-2, MERS-CoV, and SARS-CoV belong to the coronavirus family.
  • the initial attachment of the virion to the host cell is initiated by interactions between the S protein and its receptor.
  • the sites of receptor binding domains (RBD) within the S1 region of a coronavirus S protein vary depending on the virus, with some having the RBD at the C-terminus of S1.
  • the S-protein/receptor interaction is the primary determinant for a coronavirus to infect a host species and also governs the tissue tropism of the virus.
  • Many coronaviruses utilize peptidases as their cellular receptor. Following receptor binding, the virus must next gain access to the host cell cytosol. This is generally accomplished by acid-dependent proteolytic cleavage of S protein by a cathepsin, TMPRRS2 or another protease, followed by fusion of the viral and cellular membranes.
  • the present disclosure provides pharmaceutical compositions comprising: an active ingredient (e.g., a protein) , a cryoprotectant, a humectant, an emulsifier, a controlled release agent, and a buffer.
  • an active ingredient e.g., a protein
  • a cryoprotectant e.g., a humectant
  • an emulsifier e.g., a controlled release agent
  • such a pharmaceutical composition can retain the active ingredient or precursor thereof at the target site (e.g., nasal cavity) for a long time.
  • the mean retention time of the active ingredient or precursor thereof in the target site (e.g., nasal cavity) of a primate, when formulated in the pharmaceutical composition of the disclosure is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, or at least 12 hours, after administration of the pharmaceutical composition to the primate.
  • the mean retention time is at least 1 hour. In some embodiments, the mean retention time is at least 2 hours.
  • the mean retention time is at least 2 hours. In some embodiments, the mean retention time is at least 3 hours. In some embodiments, the mean retention time is at least 4 hours. In some embodiments, the mean retention time is at least 5 hours. In some embodiments, the mean retention time is at least 6 hours. In some embodiments, the mean retention time is at least 7 hours. In some embodiments, the mean retention time is at least 8 hours. In some embodiments, the mean retention time is at least 9 hours. In some embodiments, the mean retention time is at least 10 hours. In some embodiments, the mean retention time is at least 11 hours. In some embodiments, the mean retention time is at least 12 hours. In some embodiments, the primate is a human. In some embodiments, the primate is a rhesus macaque.
  • cryoprotectant refers to a substance that can slow or prevent ice nucleation, ice-crystal growth, ice formation, or a combination thereof, in a composition. Accordingly, the cryoprotectant improves the post thaw viability of the active ingredient in the composition.
  • the cryoprotectant is selected from the group consisting of dimethyl sulfoxide (DMSO) , glycerol, a polyethylene glycol (PEG) , a polysaccharide, a sugar, and an amino acid, or any combination thereof.
  • the cryoprotectant is selected from the group consisting of trehalose, maltose, sucrose, glucose, lactose, dextran, mannitol, and sorbitol, or any combination thereof. Additional disclosure of the cryoprotectant can be found, for example, in U.S. Pat. No. 9,707,204, which is incorporated by reference in its entirety.
  • the cryoprotectant comprises trehalose.
  • the cryoprotectant is selected from the group consisting of trehalose, glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol, and sucrose, or a combination thereof. In some embodiments, the cryoprotectant is trehalose.
  • the pharmaceutical composition comprises no more than 40% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 40% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 35% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.0001%(w/v) to about 30% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 25% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 20%(w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 0.1% (w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3%(w/v) to about 0.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 1% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 1% (w/v) to about 2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2%(w/v) to about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 5% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 10%(w/v) to about 15% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 15% (w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 2% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 1%(w/v) to about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 5%(w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 10% (w/v) to about 20% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 0.1% (w/v) to about 0.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 5% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 2% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 5% (w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 2% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 0.3%(w/v) to about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2%(w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 20% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 0.1% (w/v) to about 2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2%(w/v) to about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 15% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 2% (w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2%(w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 15% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 1%(w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 20% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 0.1% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 20% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 0.1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2%(w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.4% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.6% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.7% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.8% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 0.9%(w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1.1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1.2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1.3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1.7% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 2.2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 4% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 4.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 5.5% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 6% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 6.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 7% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 7.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 8% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 8.5%(w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 9% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 9.5% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 11% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 12% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 13% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 14%(w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 16% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 17% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 18% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 19% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 20% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 4% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3.8%(w/v) to about 4.2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3.5% (w/v) to about 4.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2.5%(w/v) to about 6% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 8% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 1% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3%(w/v) to about 6% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 7% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 8% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3%(w/v) to about 10% (w/v) of the cryoprotectant.
  • the pharmaceutical composition comprises about 2.5% (w/v) to about 6% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 6% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 6% (w/v) of the cryoprotectant.
  • the cryoprotectant is trehalose, with a concentration of 0.0001-40%(w/v) . In some embodiments, the cryoprotectant is trehalose, with a concentration of 2%-8%(w/v) .
  • the cryoprotectant is glycerol, with a concentration of 0.0001-40%(w/v) . In some embodiments, the cryoprotectant is glycerol, with a concentration of 1-20% (w/v) .
  • the cryoprotectant is dimethyl sulfoxide, with a concentration of 0.0001-10% (w/v) . In some embodiments, the cryoprotectant is dimethyl sulfoxide, with a concentration of 1-10% (w/v) .
  • the cryoprotectant is ethylene glycol, with a concentration of 0.0001-40% (w/v) . In some embodiments, the cryoprotectant is ethylene glycol, with a concentration of 4-30% (w/v) .
  • the cryoprotectant is polyethylene glycol, with a concentration of 0.0001-20% (w/v) . In some embodiments, the cryoprotectant is polyethylene glycol, with a concentration of 5-15% (w/v) .
  • the cryoprotectant is sucrose, with a concentration of 0.5 M. In some embodiments, the cryoprotectant is sucrose, with a concentration of 0.1-0.4 M.
  • the term “humectant” refers to a substance that promotes the retention of moisture in a composition.
  • the humectant is selected from the group consisting of glycerin, diglycerin, betaine, diols, propylene glycol, butylene glycol, pentylene glycol, propanediol, 1, 2-hexanediol, D-ribose, glucose, sorbitol, dextrose, urea, 2-Pyrrolidone-5-Carboxylic Acid and related salts, inorganic salts of lactic acid, ectoin, lactic acid, betaine, glycolic acid, and lactobionic acid, or any combination thereof. Additional disclosure of the humectant can be found, for example, in US 2020/0375870, which is incorporated by reference in its entirety.
  • the humectant is selected from the group consisting of glycerin, mannitol, polyethylene glycol 400, polyethylene glycol 4000, D-sorbitol, chitosan, xylitol, and sodium hyaluronate, or a combination thereof.
  • the humectant comprises glycerin.
  • the humectant is glycerin.
  • cryoprotectant in the pharmaceutical composition comprises or is glycerol
  • the humectant does not comprise glycerin
  • the cryoprotectant when the humectant in the pharmaceutical composition comprises or is glycerin, the cryoprotectant does not comprise glycerol.
  • the pharmaceutical composition comprises no more than 30% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.0001%(w/v) to about 30% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 25% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 20%(w/v) of the humectant.
  • the pharmaceutical composition comprises about 0.1% (w/v) to about 20% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 0.5%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 1% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 1% (w/v) to about 2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 5% (w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 10% (w/v) to about 15% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 15% (w/v) to about 20%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5%(w/v) to about 2% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 1% (w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 10%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 5%(w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 10% (w/v) to about 20% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 0.1% (w/v) to about 0.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2%(w/v) to about 1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 5% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 2%(w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 5% (w/v) to about 20% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1%(w/v) to about 1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 2% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 0.3% (w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 5%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 20% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 0.1%(w/v) to about 2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 10%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 15% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 2% (w/v) to about 20% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 15% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 1% (w/v) to about 20%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5%(w/v) to about 20% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 0.1% (w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3%(w/v) to about 20% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 20% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 0.1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.4% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.6% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.7% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 0.8% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.9% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.3%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.4% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 1.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.6% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.7% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.8% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.9% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2.1% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 2.2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2.3%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2.4% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 3.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 4% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 4.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 5.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 6% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 6.5%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 7%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 7.5% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 8% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 8.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 9% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 9.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 11% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 12% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 13% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 14%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 15%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 16% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 17% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 18% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 19% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 20% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 1.7% (w/v) to about 2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.6% (w/v) to about 2.1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.5% (w/v) to about 2.2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.4% (w/v) to about 2.3%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.3% (w/v) to about 2.4% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 1.2% (w/v) to about 2.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.1% (w/v) to about 2.7% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.6% (w/v) to about 1.8% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.5% (w/v) to about 1.9% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 1.4% (w/v) to about 2%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.3% (w/v) to about 2.1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.2% (w/v) to about 2.2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.1% (w/v) to about 2.3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 2.4% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 1.9% (w/v) to about 2.1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.8% (w/v) to about 2.2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.7%(w/v) to about 2.3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.6% (w/v) to about 2.4% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.5% (w/v) to about 2.5% (w/v) of the humectant.
  • the pharmaceutical composition comprises about 1.4%(w/v) to about 2.6% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.3% (w/v) to about 2.7% (w/v) of the humectant.
  • the humectant is glycerin, with a concentration of 0.0001-30%(w/v) . In some embodiments, the humectant is glycerin, with a concentration of 2-6% (w/v) .
  • the humectant is mannitol, with a concentration of 0.0001-7%(w/v) . In some embodiments, the humectant is mannitol, with a concentration of 3-5% (w/v) .
  • the humectant is polyethylene glycol 400, with a concentration of 0.0001-20% (w/v) . In some embodiments, the humectant is polyethylene glycol 400, with a concentration of 10-18% (w/v) .
  • the humectant is polyethylene glycol 4000, with a concentration of 5-15% (w/v) . In some embodiments, the humectant is polyethylene glycol 4000, with a concentration of 5-10% (w/v) .
  • the humectant is D-sorbitol, with a concentration of 3-15% (w/v) . In some embodiments, the humectant is D-sorbitol, with a concentration of 3-6% (w/v) .
  • the humectant is chitosan, with a concentration of 0.01-3% (w/v) . In some embodiments, the humectant is chitosan, with a concentration of 0.01-1% (w/v) .
  • the humectant is xylitol, with a concentration of 0.0001-10% (w/v) . In some embodiments, the humectant is xylitol, with a concentration of 1-10% (w/v) .
  • the humectant is sodium hyaluronate, with a concentration of 0.1-2.0% (w/v) . In some embodiments, the humectant is sodium hyaluronate, with a concentration of 0.1-1.0% (w/v) .
  • emulsifier refers to a substance that promotes the formation and stabilization of an emulsion.
  • the emulsifier is selected from the group consisting of a polysorbate, sodium dodecyl sulfate, a phospholipid, a glycolipid, a triglyceride, lecithin, sodium stearate, potassium stearate, ammonium stearate, sodium oleate, potassium oleate, ammonium oleate, sodium palmitate, potassium palmitate, and ammonium palmitate, or any combination thereof.
  • the emulsifier is selected from the group consisting of TWEEN 80, TWEEN 60, Vitamin E, Pluronic F68, Pluronic F127, Poloxamer 407, glycerol monostearate, Ascorbyl palmitate lecithin, egg yolk, a phospholipid, a phosphatidylcholine, and a polyethylene glycol-phosphatidyl ethanolamine conjugate, or any combination thereof. Additional disclosure of the emulsifier can be found, for example, in US 2018/0206504 and US 2011/0144578, which are incorporated by reference in their entireties.
  • the emulsifier is selected from the group consisting of TWEEN 80, polysorbate 20, lecithin, sorbitan esters, mono-and/or diglycerides, and sodium stearoyl lactylate, or a combination thereof.
  • the emulsifier comprises TWEEN 80.
  • the emulsifier is TWEEN 80.
  • the pharmaceutical composition comprises no more than 5% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0001%(w/v) to about 5% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 2%(w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.0001% (w/v) to about 1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 0.5% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.001% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.002% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.003% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.005% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.01% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.01% (w/v) to about 0.02% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.03% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.03% (w/v) to about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.15%(w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.15% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.003% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.005% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.003% (w/v) to about 0.01%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.02% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.03% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.03%(w/v) to about 0.1% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.05% (w/v) to about 0.15% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.15%(w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.005%(w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.002% (w/v) to about 0.01% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.02% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.03%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.1% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.03% (w/v) to about 0.15% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.05%(w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.01% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.002% (w/v) to about 0.02% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.03%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.15% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.03%(w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.02% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.03% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.003% (w/v) to about 0.05%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.15% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.03%(w/v) to about 0.3% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.05% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.03%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.15%(w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.01% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.03% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.1% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.003% (w/v) to about 0.15%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.1% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.002% (w/v) to about 0.15% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005%(w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.15%(w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.002% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.3% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.003% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.4% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.001% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0015%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0025% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0035% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.004% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.0045% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.006% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.007%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.008% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.009% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.011% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.013% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.014% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.016%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.017% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.018% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.019% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.021% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.022% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.023% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.024% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.025%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.03% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.035% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.04% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.045% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.055% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.06% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.065% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.07%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.075% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.08% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.085% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.09% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.095% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.12% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.2%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.25% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.35% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.4% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.009% (w/v) to about 0.011% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.008% (w/v) to about 0.012% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.013% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.006% (w/v) to about 0.014% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.015% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.004% (w/v) to about 0.016% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.017% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.018% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.02% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.019% (w/v) to about 0.021% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.018% (w/v) to about 0.022% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.017% (w/v) to about 0.023% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.016% (w/v) to about 0.024% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.025% (w/v) of the emulsifier.
  • the pharmaceutical composition comprises about 0.014% (w/v) to about 0.026% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.013% (w/v) to about 0.027% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.028% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.011% (w/v) to about 0.029% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.03% (w/v) of the emulsifier.
  • the emulsifier is TWEEN 80, with a concentration of ⁇ 5 % (w/v) . In some embodiments, the emulsifier is TWEEN 80, with a concentration of 0.005-2 % (w/v) .
  • the emulsifier is Polysorbate 20, with a concentration of ⁇ 5 %(w/v) . In some embodiments, the emulsifier is Polysorbate 20, with a concentration of 0.005-2 %(w/v) .
  • the emulsifier is Lecithin, with a concentration of ⁇ 5 % (w/v) . In some embodiments, the emulsifier is Lecithin, with a concentration of 0.005-2 % (w/v) .
  • the emulsifier is Sorbitan esters, with a concentration of ⁇ 5 %(w/v) . In some embodiments, the emulsifier is Sorbitan esters, with a concentration of 0.005-2 %(w/v) .
  • the emulsifier is Mono-and/or di-glycerides, with a concentration of ⁇ 5 % (w/v) . In some embodiments, the emulsifier is Mono-and/or di-glycerides, with a concentration of 0.005-2 % (w/v) .
  • the emulsifier is Sodium stearoyl lactylate, with a concentration of ⁇ 5 % (w/v) . In some embodiments, the emulsifier is Sodium stearoyl lactylate, with a concentration of 0.005-2 % (w/v) .
  • controlled release agent refers to a substance that is capable of controlling the release of the active ingredient in a composition.
  • the controlled release agent is selected from the group consisting of acetate succinate, a polyvinyl derivative, polyethylene oxide, polyacrylic acid, modified starch, cross-linked high amylose starch, hydroxypropyl starch, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, cellulose, microcrystalline cellulose, carboxymethylethyl cellulose, cellulose acetate, methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, cellulose phthalate, cellulose acetate, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate succinate, cellulose acetate butyrate, cellulose acetate trimellitate, poloxamer, povidone, alginic acid, sodium alginate, polyethylene glycol, polyethylene glycol alginate, gum (for example, xanthan gum) , polymethacrylate, a copolymer of methacrylic acid
  • the controlled release agent is selected from the group consisting of hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone K30, or a combination thereof.
  • the controlled release agent comprises hydroxypropyl methylcellulose.
  • the controlled release agent is hydroxypropyl methylcellulose.
  • the pharmaceutical composition comprises no more than 10% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 10% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 9% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 8% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 7% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 6% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.001% (w/v) to about 5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 4%(w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.001% (w/v) to about 3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.002% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.005% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.01% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.01% (w/v) to about 0.02% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.05% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.15%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 0.2% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.2% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.005% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.002% (w/v) to about 0.01% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.02% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.05% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.15%(w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.1% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 2% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.001% (w/v) to about 0.01% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.02% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.05% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.15% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.05% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 0.5%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 2% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.001% (w/v) to about 0.02% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.05% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.15% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02%(w/v) to about 0.2% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.05% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 2%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.05% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.002% (w/v) to about 0.1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.15% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.5% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.1% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.1%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.15% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.2% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.01% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.15%(w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.002% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05%(w/v) to about 2% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.001% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 1%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 2% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.001% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.5% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.002%(w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 1%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 2% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.001% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.004% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.006% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.008% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.009% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.014% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.016% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.018% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.022% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.025% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.03% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.035% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.04% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.045% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.06% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.07% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.08% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.09% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.12% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.14% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.17% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.25% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.4% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.6% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.7% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.8% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.9% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 1.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 1.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 1.7% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.019% (w/v) to about 0.021% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.018% (w/v) to about 0.022% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.017% (w/v) to about 0.023% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.025% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.013% (w/v) to about 0.027% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.01% (w/v) to about 0.03% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.05% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.09% (w/v) to about 0.11% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.08% (w/v) to about 0.12% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.07% (w/v) to about 0.13% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.06% (w/v) to about 0.15% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.17% (w/v) of the controlled release agent.
  • the pharmaceutical composition comprises about 0.04% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.03% (w/v) to about 0.25%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.4% (w/v) of the controlled release agent.
  • the controlled release agent is hydroxypropyl methylcellulose, with a concentration of 0.1-5.0% (w/v) . In some embodiments, the controlled release agent is hydroxypropyl methylcellulose, with a concentration of 0.1-1% (w/v) .
  • the controlled release agent is polyvinylpyrrolidone K30, with a concentration of 2-10% (w/v) . In some embodiments, the controlled release agent is polyvinylpyrrolidone K30, with a concentration of 3-6% (w/v) .
  • buffering agent refers to an acid or base component (usually a weak acid or weak base) that is capable of maintaining the pH of a composition at or near a pre-determined value.
  • a buffering agent is present in a mixture of a weak acid and its conjugate base or a in a mixture of a weak base and its conjugated acid.
  • a “buffer, ” when used in connection with the composition, refers to the buffering agent that is dissolved/mixed into the composition.
  • the buffering agent is selected from the group consisting of sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, magnesium oxide, magnesium aluminate, magnesium carbonate, magnesium silicate, magnesium citrate, aluminum hydroxide, aluminum hydroxide/magnesium carbonate, potassium carbonate, potassium citrate, aluminum hydroxide/sodium bicarbonate coprecipitate, aluminum magnesium hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogenphosphate, dipotassium hydrogenphosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, calcium acetate, calcium glycerophosphate, calcium hydroxide, calcium lactate, calcium carbonate, calcium gluconate, calcium bicarbonate, calcium citrate, potassium phosphate, and sodium phosphate, or any combination thereof.
  • the buffering agent is selected from the group consisting of citric acid, sodium citrate, sodium acetate, acetic acid, sodium phosphate, phosphoric acid, sodium ascorbate, tartaric acid, maleic acid, glycine, sodium lactate, lactic acid, ascorbic acid, imidazole, sodium bicarbonate and carbonic acid, sodium succinate and succinic acid, histidine, and sodium benzoate and benzoic acid, and any combination thereof.
  • the buffer is a carbonate buffer, a citrate buffer, a phosphate buffer, an acetate buffer, a hydrochloric acid buffer, a lactic acid buffer, and a tartric acid buffer.
  • the buffer is selected from the group consisting of a phosphate buffer, a tris buffer, and a glycine buffer.
  • the buffer agent comprises phosphate buffered saline. In some embodiments, the buffer agent is phosphate buffered saline.
  • the buffer has a pH of about 4-8. In some embodiments, the buffer has a pH of about 4-4.5. In some embodiments, the buffer has a pH of about 4.5-5. In some embodiments, the buffer has a pH of about 5-5.5. In some embodiments, the buffer has a pH of about 5.5-6. In some embodiments, the buffer has a pH of about 6-6.5. In some embodiments, the buffer has a pH of about 6.5-7. In some embodiments, the buffer has a pH of about 7-7.5. In some embodiments, the buffer has a pH of about 7.5-8. In some embodiments, the buffer has a pH of about 4-5. In some embodiments, the buffer has a pH of about 4.5-5.5.
  • the buffer has a pH of about 5-6. In some embodiments, the buffer has a pH of about 5.5-6.5. In some embodiments, the buffer has a pH of about 6-7. In some embodiments, the buffer has a pH of about 6.5-7.5. In some embodiments, the buffer has a pH of about 7-8. In some embodiments, the buffer has a pH of about 4-5.5. In some embodiments, the buffer has a pH of about 4.5-6. In some embodiments, the buffer has a pH of about 5-6.5. In some embodiments, the buffer has a pH of about 5.5-7. In some embodiments, the buffer has a pH of about 6-7.5. In some embodiments, the buffer has a pH of about 6.5-8.
  • the buffer has a pH of about 4-6. In some embodiments, the buffer has a pH of about 4.5-6.5. In some embodiments, the buffer has a pH of about 5-7. In some embodiments, the buffer has a pH of about 5.5-7.5. In some embodiments, the buffer has a pH of about 6-8. In some embodiments, the buffer has a pH of about 4-6.5. In some embodiments, the buffer has a pH of about 4.5-7. In some embodiments, the buffer has a pH of about 5-7.5. In some embodiments, the buffer has a pH of about 5.5-8. In some embodiments, the buffer has a pH of about 4-7. In some embodiments, the buffer has a pH of about 4.5-7.5. In some embodiments, the buffer has a pH of about 5-8. In some embodiments, the buffer has a pH of about 4-7.5. In some embodiments, the buffer has a pH of about 4.5-8.
  • the buffer has a pH of about 4. In some embodiments, the buffer has a pH of about 4.1. In some embodiments, the buffer has a pH of about 4.2. In some embodiments, the buffer has a pH of about 4.3. In some embodiments, the buffer has a pH of about 4.4. In some embodiments, the buffer has a pH of about 4.5. In some embodiments, the buffer has a pH of about 4.6. In some embodiments, the buffer has a pH of about 4.7. In some embodiments, the buffer has a pH of about 4.8. In some embodiments, the buffer has a pH of about 4.9. In some embodiments, the buffer has a pH of about 5. In some embodiments, the buffer has a pH of about 5.1.
  • the buffer has a pH of about 5.2. In some embodiments, the buffer has a pH of about 5.3. In some embodiments, the buffer has a pH of about 5.4. In some embodiments, the buffer has a pH of about 5.5. In some embodiments, the buffer has a pH of about 5.6. In some embodiments, the buffer has a pH of about 5.7. In some embodiments, the buffer has a pH of about 5.8. In some embodiments, the buffer has a pH of about 5.9. In some embodiments, the buffer has a pH of about 6. In some embodiments, the buffer has a pH of about 6.1. In some embodiments, the buffer has a pH of about 6.2. In some embodiments, the buffer has a pH of about 6.3.
  • the buffer has a pH of about 6.4. In some embodiments, the buffer has a pH of about 6.5. In some embodiments, the buffer has a pH of about 6.6. In some embodiments, the buffer has a pH of about 6.7. In some embodiments, the buffer has a pH of about 6.8. In some embodiments, the buffer has a pH of about 6.9. In some embodiments, the buffer has a pH of about 7. In some embodiments, the buffer has a pH of about 7.1. In some embodiments, the buffer has a pH of about 7.2. In some embodiments, the buffer has a pH of about 7.3. In some embodiments, the buffer has a pH of about 7.4. In some embodiments, the buffer has a pH of about 7.5.
  • the buffer has a pH of about 7.6. In some embodiments, the buffer has a pH of about 7.7. In some embodiments, the buffer has a pH of about 7.8. In some embodiments, the buffer has a pH of about 7.9. In some embodiments, the buffer has a pH of about 8.
  • the buffer has a pH of about 5.9-6.1. In some embodiments, the buffer has a pH of about 5.8-6.2. In some embodiments, the buffer has a pH of about 5.7-6.3. In some embodiments, the buffer has a pH of about 5.6-6.4. In some embodiments, the buffer has a pH of about 5.5-6.5. In some embodiments, the buffer has a pH of about 5.4-6.6. In some embodiments, the buffer has a pH of about 5.3-6.7. In some embodiments, the buffer has a pH of about 5.2-6.8. In some embodiments, the buffer has a pH of about 5.1-6.9. In some embodiments, the buffer has a pH of about 5-7.
  • the pharmaceutical composition comprises about 1 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 5 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 10 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 15 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 20 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM to about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 50 mM of the buffering agent.
  • the pharmaceutical composition comprises about 50 mM to about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 100 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 200 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 300 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 400 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 10 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 15 mM of the buffering agent.
  • the pharmaceutical composition comprises about 10 mM to about 20 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 50 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 100 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 200 mM to about 400 mM of the buffering agent.
  • the pharmaceutical composition comprises about 300 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 15 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 20 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM to about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 200 mM of the buffering agent.
  • the pharmaceutical composition comprises about 50 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 100 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 200 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 20 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 100 mM of the buffering agent.
  • the pharmaceutical composition comprises about 20 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 50 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 100 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 100 mM of the buffering agent.
  • the pharmaceutical composition comprises about 15 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 50 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 200 mM of the buffering agent.
  • the pharmaceutical composition comprises about 15 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 400 mM of the buffering agent.
  • the pharmaceutical composition comprises about 20 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 400 mM of the buffering agent.
  • the pharmaceutical composition comprises about 10 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 500 mM of the buffering agent.
  • the pharmaceutical composition comprises about 15 mM to about 25 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 7 mM to about 40 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 2 mM to about 100 mM of the buffering agent.
  • the pharmaceutical composition comprises about 5 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 6 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 7 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 8 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 9 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 12 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 14 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 16 mM of the buffering agent.
  • the pharmaceutical composition comprises about 18 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 22 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 25 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 35 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 40 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 45 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 50 mM of the buffering agent.
  • the pharmaceutical composition comprises about 60 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 70 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 80 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 90 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 120 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 140 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 160 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 180 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 200 mM of the buffering agent.
  • the buffer is a phosphate buffer, with a concentration of 10-100 mM. In some embodiments, the buffer is a phosphate buffer, with a concentration of 10-40 mM.
  • the buffer is a tris buffer, with a concentration of 10-100 mM. In some embodiments, the buffer is a tris buffer, with a concentration of 10-40 mM.
  • the buffer is a glycine buffer, with a concentration of 0.1-0.5 M. In some embodiments, the buffer is a glycine buffer, with a concentration of 0.1-0.2 M.
  • the pharmaceutical composition comprises an antibiotic.
  • the antibiotic is selected from the group consisting of benzalkonium chloride, benzyl alcohol, and chlorobutanol, or a combination thereof.
  • the antibiotic comprises benzalkonium chloride.
  • the antibiotic is benzalkonium chloride.
  • the pharmaceutical composition comprises about 0.001% (w/v) to about 3.0% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 2.5% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 2.0% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 1.5% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 1.0% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.5% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.4% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.3% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.001% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.002% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.005% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.007% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.01% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.012% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.012% (w/v) to about 0.015% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.07% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.15% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.15% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.005% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.007% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.01% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.012% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.015% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.012% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.07% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.2% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.001% (w/v) to about 0.007% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.01% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.012% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.015% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.05% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.015% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.07% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.01% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.012% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.005% (w/v) to about 0.015% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.15% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.05% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.012% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.015% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.07% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.012% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.015% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.05% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.007% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.05% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.005% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.07% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.005% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.15% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.007% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.2% (w/v) of the antibiotic.
  • the pharmaceutical composition comprises about 0.01% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.009%(w/v) to about 0.011% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.008% (w/v) to about 0.012% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.015%(w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.03% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.05%(w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.1% (w/v) of the antibiotic.
  • the antibiotic is benzalkonium chloride, with a concentration of 0.002-0.02% (w/v) . In some embodiments, the antibiotic is benzalkonium chloride, with a concentration of 0.005-0.02% (w/v) .
  • the antibiotic is benzyl alcohol, with a concentration of 0.001-3.0%(v/v) . In some embodiments, the antibiotic is benzyl alcohol, with a concentration of 1.0-3.0%(v/v) .
  • the antibiotic is chlorobutanol, with a concentration of 0.5-2.0%(w/v) . In some embodiments, the antibiotic is chlorobutanol, with a concentration of 1.0-2.0%(w/v) .
  • the active ingredient comprises a protein.
  • the protein comprises an antibody.
  • the protein comprises the antigen binding fragment of an antibody.
  • the protein is capable of treating a respiratory disease.
  • the protein is an antibody or comprises the antigen binding fragment of the antibody.
  • the antibody neutralizes SARS-CoV-2.
  • the protein is an anti-CoV-S antibodies or antigen-binding fragment thereof described herein.
  • the antibody is an antibody described in Table 1.
  • the protein is or comprises Antibody B.
  • the active ingredient has a concentration of about 0.1-100 mg/ml in the pharmaceutical composition. In some embodiments, the active ingredient has a concentration of about 0.1 to about 0.2 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 0.5 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 1 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 2 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 3 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 4 mg/ml.
  • the active ingredient has a concentration of about 4 to about 5 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 6 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 7 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 8 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 10 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 20 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 50 to about 100 mg/ml.
  • the active ingredient has a concentration of about 0.1 to about 0.5 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 1 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 2 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 3 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 4 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 5 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 7 mg/ml.
  • the active ingredient has a concentration of about 6 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 7 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 8 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 10 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 20 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 1 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 2 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 3 mg/ml.
  • the active ingredient has a concentration of about 1 to about 4 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 5 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 6 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 7 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 8 to about 50 mg/ml.
  • the active ingredient has a concentration of about 10 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 2 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 3 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 4 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 5 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 8 mg/ml.
  • the active ingredient has a concentration of about 5 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 6 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 7 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 8 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 3 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 4 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 5 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 6 mg/ml.
  • the active ingredient has a concentration of about 2 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 6 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 7 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 4 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 5 mg/ml.
  • the active ingredient has a concentration of about 0.5 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 6 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 5 mg/ml.
  • the active ingredient has a concentration of about 0.2 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 6 mg/ml.
  • the active ingredient has a concentration of about 0.2 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 8 mg/ml.
  • the active ingredient has a concentration of about 0.5 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 50 mg/ml.
  • the active ingredient has a concentration of about 2 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 100 mg/ml.
  • the active ingredient has a concentration of about 5 mg/ml in the pharmaceutical composition. In some embodiments, the active ingredient has a concentration of about 4.5 to about 5.5 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 25 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 50 mg/ml.
  • Non-limiting examples of various components that may be used in the pharmaceutical composition is listed in Table 3 below.
  • the pharmaceutical composition of the disclosure can be for other purposes –for example, mucosal administration.
  • the pharmaceutical composition is for application to mucosa of a subject.
  • Mucosa is the soft tissue that lines the body’s canals and organs in the digestive, respiratory and reproductive systems.
  • the mucosa is located in the nose, mouth, throat, ear, genital, or anus.
  • the mucosa is located in the esophagus, lungs, stomach, intestine, bladder, or uterus.
  • the mucosa is located in the mouth, esophagus, or stomach. In some embodiments, the mucosa is located in the nose, mouth, pharynx, trachea, or lung. In some embodiments, the mucosa is nasal mucosa.
  • the pharmaceutical composition is suitable for intranasal administration to a subject (e.g., a mammal) .
  • a subject e.g., a mammal
  • the mammal is a human.
  • the cryoprotectant is trehalose between about 1% (w/v) and about 8%(w/v)
  • the humectant is glycerin between about 0.01% (w/v) and about 0.1% (w/v)
  • the emulsifier is TWEEN 80 between about 1% (w/v) and about 3% (w/v)
  • the controlled release agent is hydroxypropyl methylcellulose between about 0.01% (w/v) and about 0.5% (w/v)
  • the buffer is phosphate buffered saline between about 10 mM and about 30 mM
  • the protein is an antibody described herein, such as in Table 1, such as Antibody B.
  • the disclosure provides a pharmaceutical composition which provides an efficacious dose of an antibody between about 0.0001 mg/kg to about 2 mg/kg per dose. In some embodiments, the disclosure provides a pharmaceutical composition which provides a low yet efficacious dose of an antibody which is about 0.4 mg per dose to a human.
  • antigen-binding proteins such as antibodies and antigen-binding fragments thereof, that specifically bind to CoV spike protein or an antigenic fragment thereof.
  • antigen-binding proteins can be used as active ingredients in a pharmaceutical composition for the prevention or treatment of coronavirus infection.
  • examples include: wild-type-CoV-S antigen-binding proteins, such as wild-type-CoV-S antibodies and wild-type-CoV-S antigen-binding fragments thereof, that specifically bind to wild-type-CoV spike protein or an antigenic fragment thereof; alpha-CoV-S antigen-binding proteins (alpha (B. 1.1.7) -CoV-S antigen-binding proteins) , such as alpha-CoV-S antibodies (alpha (B.
  • beta-CoV-S antigen-binding proteins (beta (B. 1.351) -CoV-S antigen-binding proteins) , such as beta-CoV-S antibodies (beta (B. 1.351) -CoV-S antibodies) and beta-CoV-S antigen-binding fragments (beta (B.
  • -CoV-S antigen-binding fragments thereof, that specifically bind to beta-CoV spike protein or an antigenic fragment thereof; gamma-CoV-S antigen-binding proteins (gamma (P.1) -CoV-S antigen-binding proteins) , such as gamma-CoV-S antibodies (gamma (P. 1) -CoV-S antibodies) and gamma-CoV-S antigen-binding fragments (gamma (P. 1) -CoV-S antigen-binding fragments) thereof, that specifically bind to gamma-CoV spike protein or an antigenic fragment thereof; delta-CoV-S antigen-binding proteins (delta (B.
  • 1.617.2) -CoV-S antigen-binding proteins such as delta-CoV-S antibodies (delta (B. 1.617.2) -CoV-S antibodies) and delta-CoV-S antigen-binding fragments (delta (B. 1.617.2) -CoV-S antigen-binding fragments) thereof, that specifically bind to delta-CoV spike protein or an antigenic fragment thereof; omicron-CoV-S antigen-binding proteins (omicron (BA. 1 or BA. 2) -CoV-S antigen-binding proteins) , such as omicron-CoV-S antibodies (omicron (BA. 1 or BA.
  • omicron-CoV-S antibodies and omicron-CoV-S antigen-binding fragments (omicron (BA. 1 or BA. 2) -CoV-S antigen-binding fragments) thereof, that specifically bind to omicron-CoV spike protein or an antigenic fragment thereof.
  • antigen-binding proteins such as antibodies and antigen-binding fragments thereof, that specifically bind to the RBD-domain of the CoV spike protein.
  • antibody refers to immunoglobulin molecules comprising four polypeptide chains, two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds (i.e., “full antibody molecules” ) , as well as multimers thereof (e.g. IgM) .
  • Exemplary antibodies include, for example, those listed in Table 1.
  • Each heavy chain comprises a heavy chain variable region ( “HVR” or “V H ” ) and a heavy chain constant region (comprised of domains C H 1, C H 2 and C H 3) .
  • Each light chain is comprised of a light chain variable region ( “LVR or “V L ” ) and a light chain constant region (C L ) .
  • V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) .
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • Heavy chain CDRs can also be referred to as HCDRs or CDR-Hs, and numbered as described above (e.g., HCDR1, HCDR2, and HCDR3 or CDR-H1, CDR-H2, and CDR-H3) .
  • light chain CDRs can be referred to as LCDRs or CDR-Ls, and numbered LCDR1, LCDR2, and LCDR3, or CDR-L1, CDR-L2, and CDR-L3.
  • the assignment of amino acids to each domain is in accordance with the definitions of the international ImMunoGeneTics information (see www. imgt. org) .
  • the FRs of the antibody are identical to the human germline sequences, or are naturally or artificially modified.
  • Exemplary human germline sequences include, but are not limited to, VH3-66 and Vkl-33.
  • the present disclosure provides anti-CoV-S antibodies or antigen-binding fragments thereof (e.g., anti-SARS-CoV-2-S antibodies or antigen-binding fragments thereof) comprising HCDR and LCDR sequences of Table 1 within a VH3-66 or Vkl-33 variable heavy chain or light chain region.
  • the present disclosure further provides anti-CoV-S antibodies or antigen-binding fragments thereof (e.g., anti-SARS-CoV-2-S antibodies or antigen-binding fragments thereof) comprising HCDR and LCDR sequences of Table 1.
  • the present disclosure further provides anti-CoV-S antibodies or antigen-binding fragments thereof (e.g., anti-SARS-CoV-2-S antibodies or antigen-binding fragments thereof) comprising HVR and LVR sequences of Table 1 within a combination of a light chain described herein, and a heavy chain described herein.
  • the present disclosure includes monoclonal anti-CoV-S antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, as well as monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins.
  • monoclonal antibody refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts.
  • a “plurality” of such monoclonal antibodies and fragments in a composition refers to a concentration of identical (i.e., as discussed above, in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts) antibodies and fragments which is above that which would normally occur in nature, e.g., in the blood of a host organism such as a mouse or a human.
  • an anti-CoV-S antigen-binding protein e.g., antibody or antigen-binding fragment comprises a heavy chain constant domain, e.g., of the type IgA (e.g., IgA1 or IgA2) , IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 and IgG4) or IgM.
  • antibody or antigen-binding fragment comprises a heavy chain constant domain of the type IgG.
  • the IgG is a IgG1, IgG2, IgG3, or IgG4 type. In some embodiments, the IgG is IgG1. In some embodiments, the IgG is IgG2. In some embodiments, the IgG is IgG3. In some embodiments, the IgG is IgG4.
  • an antigen-binding protein e.g., antibody or antigen-binding fragment comprises a light chain constant domain, e.g., of the type kappa or lambda.
  • the antibody is a human antibody.
  • human antigen-binding protein such as an antibody, as used herein, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences whether in a human cell or grafted into a non-human cell, e.g., a mouse cell. See e.g., US8502018, US6596541 or US5789215.
  • the human mAbs of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) , for example in the CDRs and, in particular, CDR3.
  • human antibody is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences.
  • the term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal.
  • the term is not intended to include antibodies isolated from or generated in a human subject. See below.
  • the present disclosure includes anti-CoV-S chimeric antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, and methods of use thereof.
  • a “chimeric antibody” is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species.
  • recombinant antigen-binding proteins such as antibodies or antigen-binding fragments thereof, refers to such molecules created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression.
  • the term includes antibodies expressed in a nonhuman mammal (including transgenic non-human mammals, e.g., transgenic mice) , or a cell (e.g., CHO cells) expression system, or a non-human cell expression system, or isolated from a recombinant combinatorial human antibody library.
  • a recombinant antibody shares a sequence with an antibody isolated from an organism (e.g., a mouse or a human) , but has been expressed via recombinant DNA technology.
  • Such antibodies may have post-translational modifications (e.g., glycosylation) that differ from the antibody as isolated from the organism.
  • the active ingredient comprises a recombinant and/or chimeric antigen-binding protein.
  • Recombinant anti-CoV-S antigen-binding proteins e.g., antibodies and antigen-binding fragments, disclosed herein may also be produced in an E. coli/T7 expression system.
  • nucleic acids encoding the anti-CoV-S antibody immunoglobulin molecules of the disclosure e.g., as found in Table 1 may be inserted into a pET-based plasmid and expressed in the E. coli/T7 system.
  • the present disclosure includes methods for expressing an antibody or antigen-binding fragment thereof or immunoglobulin chain thereof in a host cell (e.g., bacterial host cell such as E.
  • a bacterial host cell such as an E. coli, includes a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside) .
  • IPTG isopropyl-beta-D-thiogalactopyranoside
  • Transformation can be by any known method for introducing polynucleotides (e.g., DNA or RNA, including mRNA) into a host cell.
  • Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) in liposomes, lipid nanoparticle technology, biolistic injection and direct microinjection of the DNA into nuclei.
  • nucleic acid molecules may be introduced into mammalian cells by viral vectors such as lentivirus or adeno-associated virus.
  • an antibody or antigen-binding fragment thereof of the present disclosure can be introduced to a subject in nucleic acid form (e.g, DNA or RNA, including mRNA) , such that the subject’s own cells produce the antibody.
  • nucleic acid form e.g, DNA or RNA, including mRNA
  • the present disclosure further provides modifications to nucleotide sequences encoding the anti-CoV-S antibodies described herein that result in increased antibody expression, increased antibody stability, increased nucleic acid (e.g., mRNA) stability, or improved affinity or specificity of the antibodies for the CoV spike protein.
  • the present disclosure includes recombinant methods for making an anti-CoV-S antigen-binding protein, such as an antibody or antigen-binding fragment thereof of the present disclosure, or an immunoglobulin chain thereof, comprising (i) introducing one or more polynucleotides described herein encoding light and/or heavy immunoglobulin chains, or CDRs, of the antigen-binding protein, wherein the polynucleotide is in a vector; and/or integrated into a host cell chromosome and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., CHO or Pichia or Pichiapastoris) under condition favorable to expression of the polynucleotide and, (iii) optionally, isolating the antigen-binding protein, (e.g., antibody or fragment) or chain from the host cell and/or medium in which the host cell is grown.
  • a host cell e.g., CHO or Pichi
  • a polynucleotide can be integrated into a host cell chromosome through targeted insertion with a vector such as adeno-associated virus (AAV) , e.g., after cleavage of the chromosome using a gene editing system (e.g., CRISPR (for example, CRISPR-Cas9) , TALEN, megaTAL, zinc finger, or Argonaute) .
  • AAV adeno-associated virus
  • Targeted insertions can take place, for example, at host cell loci such as an albumin or immunoglobulin genomic locus.
  • insertion can be at a random locus, e.g., using a vector such as lentivirus.
  • an antigen-binding protein e.g., antibody or antigen-binding fragment
  • co-expression of the chains in a single host cell leads to association of the chains, e.g., in the cell or on the cell surface or outside the cell if such chains are secreted, so as to form the antigen-binding protein (e.g., antibody or antigen-binding fragment) .
  • the methods include those wherein only a heavy immunoglobulin chain or only a light immunoglobulin chain (e.g., any of those discussed herein including mature fragments and/or variable domains thereof) is expressed.
  • Such chains are useful, for example, as intermediates in the expression of an antibody or antigen-binding fragment that includes such a chain.
  • the present disclosure also includes anti-CoV-S antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, comprising a heavy chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by a polynucleotide comprising a nucleotide sequence described herein and a light chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by a nucleotide sequence described herein which are the product of such production methods, and, optionally, the purification methods set forth herein.
  • the product of the method is an anti-CoV-S antigen-binding protein which is an antibody or fragment comprising an HVR comprising an amino acid sequence set forth in Table 1 and an LVR comprising an amino acid sequence set forth in Table 1, wherein the HVR and LVR sequences are selected from a single antibody listed in Table 1.
  • the product of the method is an anti-CoV-S antigen-binding protein which is an antibody or fragment comprising HCDR1, HCDR2, and HCDR3 comprising amino acid sequences set forth in Table 1 and LCDR1, LCDR2, and LCDR3 comprising amino acid sequences set forth in Table 1, wherein the six CDR sequences are selected from a single antibody listed in Table 1.
  • the product of the method is an anti-CoV-S antigen-binding protein which is an antibody or fragment comprising a heavy chain comprising an HC amino acid sequence set forth in Table 1 and a light chain comprising an LC amino acid sequence set forth in Table 1.
  • Eukaryotic and prokaryotic host cells may be used as hosts for expression of an anti-CoV-S antigen-binding protein.
  • host cells are well known in the art and many are available from the American Type Culture Collection (ATCC) .
  • ATCC American Type Culture Collection
  • host cells include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS) , human hepatocellular carcinoma cells (e.g., Hep G2) , A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines.
  • Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells.
  • Other cell lines that may be used are insect cell lines (e.g., Spodopterafrugiperda or Trichoplusia ni) , amphibian cells, bacterial cells, plant cells and fungal cells.
  • Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri) , Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chryso
  • the term “specifically binds” refers to those antigen-binding proteins (e.g., mAbs) having a binding affinity to an antigen, such as a CoV-S protein (e.g., SARS-CoV-2-S) or the RBD-domain of a CoV-S protein (e.g., RBD-SARS-CoV-2-S) , expressed as KD, of at least about 10 - 8 M, as measured by real-time, label free bio-layer interferometry assay, for example, at 25°C or 37°C, e.g., an HTX biosensor, or by surface plasmon resonance, e.g., BIACORETM, or by solution-affinity ELISA.
  • the present disclosure includes antigen-binding proteins that specifically bind to a CoV-S protein, or to the RBD-domain of a CoV-S protein.
  • An antigen-binding fragment of an antibody in some embodiments, comprises at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences.
  • the V H and V L domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain V H -V H , V H -V L or V L -V L dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric V H or V L domain.
  • an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (V) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (Viii) V L -C H 1; (ix) V L - C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V
  • variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
  • a hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
  • an antigen-binding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric V H or V L domain (e.g., by disulfide bond (s) ) .
  • the present disclosure provides an isolated protein (e.g., an antibody or antigen binding fragment thereof) .
  • isolated antigen-binding proteins, antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides and vectors are at least partially free of other biological molecules from the cells or cell culture from which they are produced.
  • biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium.
  • An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof.
  • isolated is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.
  • isolated antigen-binding proteins, antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides and vectors are essentially free of other biological molecules from the cells or cell culture from which they are produced.
  • isolated antigen-binding proteins, antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides and vectors are free of other biological molecules from the cells or cell culture from which they are produced.
  • epitope refers to an antigenic determinant (e.g., a CoV-S polypeptide) that interacts with a specific antigen-binding site of an antigen-binding protein, e.g., a variable region of an antibody molecule, known as a paratope.
  • a specific antigen-binding site of an antigen-binding protein e.g., a variable region of an antibody molecule, known as a paratope.
  • a single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects.
  • epitope also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional.
  • Epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction.
  • Epitopes may be linear or conformational, that is, composed of non-linear amino acids.
  • epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, In some embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
  • Methods for determining the epitope of an antigen-binding protein include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63) , peptide cleavage analysis, crystallographic studies and NMR analysis.
  • methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496) .
  • Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding protein e.g., antibody or fragment or polypeptide
  • an antigen-binding protein e.g., antibody or fragment or polypeptide
  • the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antigen-binding protein, e.g., antibody or fragment or polypeptide, to the deuterium-labeled protein.
  • the CoV-S protein/antigen-binding protein complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface.
  • amino acids that form part of the protein/antigen-binding protein interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface.
  • the target protein After dissociation of the antigen-binding protein (e.g., antibody or fragment or polypeptide) , the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antigen-binding protein interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.
  • the antigen-binding protein e.g., antibody or fragment or polypeptide
  • Compet refers to an antigen-binding protein (e.g., antibody or antigen-binding fragment thereof) that binds to an antigen (e.g., CoV-S) and inhibits or blocks the binding of another antigen-binding protein (e.g., antibody or antigen-binding fragment thereof) to the antigen.
  • the term also includes competition between two antigen-binding proteins e.g., antibodies, in both orientations, i.e., a first antibody that binds and blocks binding of second antibody and vice versa.
  • the first antigen-binding protein (e.g., antibody) and second antigen-binding protein (e.g., antibody) may bind to the same epitope.
  • the first and second antigen-binding proteins may bind to different, but, for example, overlapping epitopes, wherein binding of one inhibits or blocks the binding of the second antibody, e.g., via steric hindrance.
  • Competition between antigen-binding proteins may be measured by methods known in the art, for example, by a real-time, label-free bio-layer interferometry assay.
  • Epitope mapping (e.g., via alanine scanning or hydrogen-deuterium exchange (HDX) ) can be used to determine whether two or more antibodies are non-competing (e.g., on a spike protein receptor binding domain (RBD) monomer) , competing for the same epitope, or competing but with diverse micro-epitopes (e.g., identified through HDX) .
  • HDX hydrogen-deuterium exchange
  • competition between a first and second anti-CoV-S antigen-binding protein is determined by measuring the ability of an immobilized first anti-CoV-S antigen-binding protein (e.g., antibody) (not initially complexed with CoV-S protein) to bind to soluble CoV-S protein complexed with a second anti-CoV-S antigen-binding protein (e.g., antibody) .
  • the degree of competition can be expressed as a percentage of the reduction in binding.
  • Such competition can be measured using a real time, label-free bio-layer interferometry assay, e.g., on an Octet RED384 biosensor (Pall ForteBio Corp. ) , ELISA (enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance) .
  • Binding competition between anti-CoV-S antigen-binding proteins can be determined using a real time, label-free bio-layer interferometry assay on an Octet RED384 biosensor (Pall ForteBio Corp. ) .
  • the anti-CoV-S mAb can be first captured onto anti-hFc antibody coated Octet biosensor tips (Pall ForteBio Corp., #18-5060) by submerging the tips into a solution of anti-CoV-S mAb (subsequently referred to as “mAb 1” ) .
  • the antibody captured biosensor tips can then be saturated with a known blocking isotype control mAb (subsequently referred to as “blocking mAb” ) by dipping into a solution of blocking mAb.
  • blocking mAb a blocking isotype control mAb
  • the biosensor tips can then be subsequently dipped into a co-complexed solution of CoV-S polypeptide and a second anti-CoV-S mAb (subsequently referred to as “mAb2” ) , that had been pre-incubated for a period of time and binding of mAbl to the CoV-S polypeptide can be determined.
  • the biosensor tips can be washed in buffer in between every step of the experiment. The real-time binding response can be monitored during the course of the experiment and the binding response at the end of every step can be recorded.
  • the competition assay is conducted at 25 °C and pH about 7, e.g., 7.4, e.g., in the presence of buffer, salt, surfactant and a non-specific protein (e.g., bovine serum albumin) .
  • a non-specific protein e.g., bovine serum albumin
  • an antibody or antigen-binding fragment of the disclosure which is modified in some way retains the ability to specifically bind to CoV-S, e.g., retains at least 10%of its CoV-S binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis.
  • an antibody or antigen-binding fragment of the disclosure retains at least 20%, 50%, 70%, 80%, 90%, 95%or 100%or more of the CoV-S binding affinity as the parental antibody.
  • an antibody or antigen-binding fragment of the disclosure can include conservative or non-conservative amino acid substitutions (referred to as “conservative alternates” or “function conserved alternates” of the antibody) that do not substantially alter its biologic activity.
  • an “alternate” of a polypeptide” refers to a polypeptide comprising an amino acid sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to a referenced amino acid sequence that is set forth herein (e.g., SEQ ID NO: 2, 10, 18, 20, 22, 30, 38, 40, 42, 50, 58, or 60) ; when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expect threshold: 10; word size: 3; max matches in a query range: 0; BLOSUM 62 matrix;
  • an “alternate” of a polynucleotide refers to a polynucleotide comprising a nucleotide sequence that is at least about 70-99.9% (e.g., at least about 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9%) identical to a referenced nucleotide sequence that is set forth herein (e.g., SEQ ID NO: 1, 9, 17, 19, 21, 29, 37, 39, 41, 49, 57, or 59) ; when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expect threshold: 10; word size: 28; max matches in a query range: 0
  • Anti-CoV-S antigen-binding proteins include a heavy chain immunoglobulin variable region having at least 70% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the HVR amino acid sequences set forth in Table 1; and/or a light chain immunoglobulin variable region having at least 70% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the LVR amino acid sequences set forth in Table 1.
  • a heavy chain immunoglobulin variable region having at least 70% (e.g., at least 80%, 81%, 82%, 83%
  • an alternate anti-CoV-S antigen-binding protein may include a polypeptide comprising an amino acid sequence that is set forth herein except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or more) mutations such as, for example, missense mutations (e.g., conservative substitutions) , non-sense mutations, deletions, or insertions.
  • the present disclosure includes antigen-binding proteins which include an immunoglobulin light chain alternate comprising an LVR amino acid sequence set forth in Table 1 but having one or more of such mutations and/or an immunoglobulin heavy chain alternate comprising an HVR amino acid sequence set forth in Table 1 but having one or more of such mutations.
  • an alternate anti-CoV-S antigen-binding protein includes an immunoglobulin light chain alternate comprising LCDR1, LCDR2 and LCDR3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g., conservative substitutions) and/or an immunoglobulin heavy chain alternate comprising HCDR1, HCDR2, and HCDR3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g., conservative substitutions) .
  • Substitutions can be in a CDR, framework, or constant region.
  • the disclosure further provides alternate anti-CoV-S antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof, comprising one or more alternate CDRs (e.g., any one or more of LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and/or HCDR3) that are set forth herein with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%sequence identity to, e.g., the heavy chain and light chain CDRs of Table 1.
  • alternate CDRs e.g., any one or more of LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and/or HCDR3
  • Embodiments of the present disclosure also include alternate antigen-binding proteins, e.g., anti-CoV-S antibodies and antigen-binding fragments thereof, that comprise immunoglobulin VHs and VLs; or HCs and LCs, which comprise an amino acid sequence having 70%or more (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) overall amino acid sequence identity or similarity to the amino acid sequences of the corresponding VHs, VLs, HCs or LCs specifically set forth herein, but wherein the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of such immunoglobulins are not alternates and comprise CDR amino acid sequence set forth in Table 1.
  • the CDRs within alternate antigen-binding proteins are not, themselves, alternates.
  • Conservatively/Function-conservative modified alternate anti-CoV-S antibodies and antigen-binding fragments thereof are also part of the present disclosure.
  • a “conservatively modified alternate” or a “conservative substitution” refers to an alternate wherein there is one or more substitutions of amino acids in a polypeptide with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc. ) . Such changes can frequently be made without significantly disrupting the biological activity of the antibody or fragment.
  • Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4’ Ed. ) ) .
  • substitutions of structurally or functionally similar amino acids are less likely to significantly disrupt biological activity.
  • Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
  • a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45.
  • Function-conservative alternates of the anti-CoV-S antibodies and antigen-binding fragments thereof are also part of the present disclosure. Any of the alternates of the anti-CoV-S antibodies and antigen-binding fragments thereof (as discussed herein) may be “function-conservative alternates” . Such function-conservative alternates may, in some cases, also be characterized as conservatively modified alternates. “Function-conservative alternates, ” as used herein, refers to alternates of the anti-CoV-S antibodies or antigen-binding fragments thereof in which one or more amino acid residues have been changed without significantly altering one or more functional properties of the antibody or fragment. In some embodiments, a function-conservative alternate anti-CoV-S antibody or antigen-binding fragment thereof of the present disclosure comprises an alternate amino acid sequence and exhibits one or more of the following functional properties:
  • ⁇ Inhibits growth of coronavirus (e.g., SARS-CoV-2, SARS-CoV, and/or MERS-CoV) in ACE2-and/or TMPRSS2-expressing cells (e.g., Calu-3 cells) ;
  • coronavirus e.g., SARS-CoV-2, SARS-CoV, and/or MERS-CoV
  • ACE2-and/or TMPRSS2-expressing cells e.g., Calu-3 cells
  • ⁇ Inhibits the binding or interaction of coronavirus or coronavirus pseudovirus with ACE2-and/or TMPRSS2-expressing cells.
  • ⁇ Protects a mouse engineered to express the human TMPRSS2 and/or ACE2 protein from death caused by coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) , for example, wherein the mice are infected with an otherwise lethal dose of the virus, optionally when combined with a second therapeutic agent;
  • coronavirus infection e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV
  • ⁇ Protects a mouse engineered to express the human TMPRSS2 and/or ACE2 protein from weight loss caused by coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) , for example, wherein the mice are infected with a dose of the virus that would otherwise cause weight loss, optionally when combined with a second therapeutic agent;
  • coronavirus infection e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV
  • ⁇ Protects a hamster from death caused by coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) , for example, wherein the animal is infected with an otherwise lethal dose of the virus, optionally when combined with a second therapeutic agent;
  • coronavirus infection e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV
  • ⁇ Protects a hamster from weight loss caused by coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) , for example, wherein the animal is infected with a dose of the virus that would otherwise cause weight loss, optionally when combined with a second therapeutic agent; and/or
  • coronavirus infection e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV
  • a “neutralizing” or “antagonist” anti-CoV-S antigen-binding protein refers to a molecule that inhibits an activity of CoV-S to any detectable degree, e.g., inhibits the ability of CoV-S to bind to a receptor such as ACE2, to be cleaved by a protease such as TMPRSS2, or to mediate viral entry into a host cell or viral reproduction in a host cell.
  • the neutralizing anti-CoV-S antigen-binding protein inhibits the ability of CoV-S to bind to a receptor such as ACE2 by between about 15%to about 25%, or between about 25%to about 35%, or between about 35%to about 45%, or between about 45%to about 55%, or between about 55%to about 65%, or between about 65%to about 75%, or between about 65%to about 80%, or between about 75%to about 85%, or between about 85%to about 95%, or between about 95%to about 99%, or between about 90%to about 99%, or between about 95%to 100%.
  • Table 1 refers to antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that comprise the heavy chain or VH (or an alternate thereof) and light chain or VL (or an alternate thereof) as set forth below; or that comprise a VH that comprises the CDRs thereof (HCDR1 (or an alternate thereof) , HCDR2 (or an alternate thereof) and HCDR3 (or an alternate thereof) ) and a VL that comprises the CDRs thereof (LCDR1 (or an alternate thereof) , LCDR2 (or an alternate thereof) and LCDR3 (or an alternate thereof) ) , e.g., wherein the immunoglobulin chains, variable regions and/or CDRs comprise the specific amino acid sequences described below.
  • the immunoglobulin chains, variable regions and/or CDRs comprise the specific amino acid sequences described below.
  • the antibodies described herein also include embodiments wherein the VH is fused to a wild-type IgG4 (e.g., wherein residue 108 is S) or to IgG4 alternates (e.g., wherein residue 108 is P) .
  • Antibodies and antigen-binding fragments of the present disclosure comprise immunoglobulin chains including the amino acid sequences set forth herein as well as cellular and in vitro post-translational modifications to the antibody.
  • the present disclosure includes antibodies and antigen-binding fragments thereof that specifically bind to CoV-S comprising heavy and/or light chain amino acid sequences set forth herein (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3) as well as antibodies and fragments wherein one or more amino acid residues is glycosylated, one or more Asn residues is deamidated, one or more residues (e.g., Met, Trp and/or His) is oxidized, the N-terminal Gln is pyroglutamate (pyroE) and/or the C-terminal Lysine is missing.
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3 as well as antibodies and fragments wherein one or more amino
  • SARS-CoV-2-S anti-SARS-CoV-2-Spike protein
  • Additional antibodies or antigen binding fragments can be found, for example, in: Chinese Pat. No. 111909261B, Chinese Pat. No. 111909263B, Chinese Pat. No. 111925444B, Chinese Pat. No. 111909260B, Chinese Pat. No. 111925440B, Chinese Pat. No. 111925441B, Chinese Pat. Publ. No. 114989293A, Chinese Pat. No. CN111925442B, Chinese Pat. No. CN111925443B, WO2022037616, WO2022036788, WO2022037033, the content of each of which is incorporated by reference in its entirety.
  • Further antibodies or antigen binding fragments include those that comprises the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of any one of the antibodies disclosed in these patents and applications.
  • the antibody or antigen binding fragment thereof comprises a heavy chain variable domain (VH) comprising:
  • an HCDR1 comprising the amino acid sequence of GFTFSGSA (SEQ ID NO: 13) ;
  • an HCDR2 comprising the amino acid sequence of IVVGSGNT (SEQ ID NO: 14) ;
  • an HCDR3 comprising the amino acid sequence of AAPYCSSTSCRDGFDI (SEQ ID NO: 15) .
  • the antibody or antigen binding fragment thereof comprises a light chain variable domain (VL) comprising:
  • LCDR1 comprising the amino acid sequence of QSVRSSY (SEQ ID NO: 10) ;
  • LCDR2 comprising the amino acid sequence of GAS (SEQ ID NO: 11) ;
  • LCDR3 comprising the amino acid sequence of QQYGRSPWT (SEQ ID NO: 12) .
  • the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 18, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto. In some embodiments, the VH comprises, consists essentially of, or consists of a sequence having at least 95%identity to SEQ ID NO: 18. In some embodiments, the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto. In some embodiments, the VL comprises, consists essentially of, or consists of a sequence having at least 95%identity to SEQ ID NO: 16.
  • the antibody or antigen binding fragment is capable of binding to a SARS-CoV-2 spike protein.
  • the SARS-CoV-2 spike proteins comprises the amino acid sequence set forth in SEQ ID NO: 76, SEQ ID NO: 78, or SEQ ID NO: 80, or a sequence having at least 95%, at least 98%, or at least 99%sequence identity thereto.
  • the antibody is an IgG, an IgM, or an IgA.
  • the antigen binding fragment is an Fab fragment, an F (ab’) 2 fragment, an Fd fragment, an Fv fragment, or a single-chain Fv (scFv) molecules.
  • the antibody or antigen binding fragment is for intranasal, subcutaneous, intravenous, or intramuscular administration. In some embodiments, the antibody or antigen binding fragment is for intranasal administration.
  • the disclosure further provides method of producing the antibody or antigen binding fragment of the disclosure, comprising culturing a cell comprising a vector encoding the antibody or antigen binding fragment thereof and harvesting the antibody or antigen binding fragment from the culture medium of the cell.
  • the disclosure provides a combination of antibodies or antigen binding fragments of the disclosure.
  • the combination displays a synergistic effect against one or more coronavirus strains.
  • the combination displays a synergistic effect against COVID-19 Omicron BA. 2, BA. 2.12.1, or BA. 5 strain.
  • the combination comprises at least two different antibodies or antigen binding fragments, each comprising the HCDRs and LCDRs of one of the antibodies listed in Table 1.
  • the combination comprises one of the following embodiments in Table 2 below.
  • the combination comprises at least two antibodies and/or antigen binding fragments, each comprising the HCDRs or LCDRs of the antibodies according to one of combinations #1 to #10 of Table 2.
  • the combination comprises at least three antibodies and/or antigen binding fragments, each comprising the HCDRs and LCDRs of the antibodies according to one of combinations #11 to #20 of Table 2.
  • the combination comprises (i) an antibody or antigen binding fragment comprising the HCDRs and LCDRs of antibody B in Table 1; and (ii) an antibody or antigen binding fragment comprising the HCDRs and LCDRs of antibody D/E in Table 1. See, for examples, one of combinations #6, #12, #17, and #19 in Table 2.
  • the ratio of (i) an antibody or antigen binding fragment comprising the HCDRs and LCDRs of antibody B in Table 1 to (ii) an antibody or antigen binding fragment comprising the HCDRs and LCDRs of antibody D/E is between about 1: 1 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 2.
  • the ratio is between about 1: 2 to about 1: 3. In some embodiments, the ratio is between about 1: 3 to about 1: 4. In some embodiments, the ratio is between about 1: 4 to about 1: 5. In some embodiments, the ratio is between about 1: 5 to about 1: 6. In some embodiments, the ratio is between about 1: 6 to about 1: 7. In some embodiments, the ratio is between about 1: 7 to about 1: 8. In some embodiments, the ratio is between about 1: 8 to about 1: 9. In some embodiments, the ratio is between about 1: 9 to about 1: 10. In some embodiments, the ratio is between about 1: 10 to about 1: 15. In some embodiments, the ratio is between about 1: 15 to about 1: 20.
  • the ratio is between about 1: 1 to about 1: 3. In some embodiments, the ratio is between about 1: 2 to about 1: 4. In some embodiments, the ratio is between about 1: 3 to about 1: 5. In some embodiments, the ratio is between about 1: 4 to about 1: 6. In some embodiments, the ratio is between about 1: 5 to about 1: 7. In some embodiments, the ratio is between about 1: 6 to about 1: 8. In some embodiments, the ratio is between about 1: 7 to about 1: 9. In some embodiments, the ratio is between about 1: 8 to about 1: 10. In some embodiments, the ratio is between about 1: 9 to about 1: 15. In some embodiments, the ratio is between about 1: 10 to about 1: 20.
  • the ratio is between about 1: 1 to about 1: 4. In some embodiments, the ratio is between about 1: 2 to about 1: 5. In some embodiments, the ratio is between about 1: 3 to about 1: 6. In some embodiments, the ratio is between about 1: 4 to about 1: 7. In some embodiments, the ratio is between about 1: 5 to about 1: 8. In some embodiments, the ratio is between about 1: 6 to about 1: 9. In some embodiments, the ratio is between about 1: 7 to about 1: 10. In some embodiments, the ratio is between about 1: 8 to about 1: 15. In some embodiments, the ratio is between about 1: 9 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 5.
  • the ratio is between about 1: 2 to about 1: 6. In some embodiments, the ratio is between about 1: 3 to about 1: 7. In some embodiments, the ratio is between about 1: 4 to about 1: 8. In some embodiments, the ratio is between about 1: 5 to about 1: 9. In some embodiments, the ratio is between about 1: 6 to about 1: 10. In some embodiments, the ratio is between about 1: 7 to about 1: 15. In some embodiments, the ratio is between about 1: 8 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 6. In some embodiments, the ratio is between about 1: 2 to about 1: 7. In some embodiments, the ratio is between about 1: 3 to about 1: 8.
  • the ratio is between about 1: 4 to about 1: 9. In some embodiments, the ratio is between about 1: 5 to about 1: 10. In some embodiments, the ratio is between about 1: 6 to about 1: 15. In some embodiments, the ratio is between about 1: 7 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 7. In some embodiments, the ratio is between about 1: 2 to about 1: 8. In some embodiments, the ratio is between about 1: 3 to about 1: 9. In some embodiments, the ratio is between about 1: 4 to about 1: 10. In some embodiments, the ratio is between about 1: 5 to about 1: 15. In some embodiments, the ratio is between about 1: 6 to about 1: 20.
  • the ratio is between about 1: 1 to about 1: 8. In some embodiments, the ratio is between about 1: 2 to about 1: 9. In some embodiments, the ratio is between about 1: 3 to about 1: 10. In some embodiments, the ratio is between about 1: 4 to about 1: 15. In some embodiments, the ratio is between about 1: 5 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 9. In some embodiments, the ratio is between about 1: 2 to about 1: 10. In some embodiments, the ratio is between about 1: 3 to about 1: 15. In some embodiments, the ratio is between about 1: 4 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 10.
  • the ratio is between about 1: 2 to about 1: 15. In some embodiments, the ratio is between about 1: 3 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 15. In some embodiments, the ratio is between about 1: 2 to about 1: 20. In some embodiments, the ratio is about 1: 1. In some embodiments, the ratio is about 1: 1.1. In some embodiments, the ratio is about 1: 1.3. In some embodiments, the ratio is about 1: 1.5. In some embodiments, the ratio is about 1: 1.7. In some embodiments, the ratio is about 1: 2. In some embodiments, the ratio is about 1: 2.2. In some embodiments, the ratio is about 1: 2.5.
  • the ratio is about 1: 2.7. In some embodiments, the ratio is about 1: 3. In some embodiments, the ratio is about 1: 3.5. In some embodiments, the ratio is about 1: 4. In some embodiments, the ratio is about 1: 4.5. In some embodiments, the ratio is about 1: 5. In some embodiments, the ratio is about 1: 5.5. In some embodiments, the ratio is about 1: 6. In some embodiments, the ratio is about 1: 6.5. In some embodiments, the ratio is about 1: 7. In some embodiments, the ratio is about 1: 7.5. In some embodiments, the ratio is about 1: 8. In some embodiments, the ratio is about 1: 8.5. In some embodiments, the ratio is about 1: 9.
  • the ratio is about 1: 9.5. In some embodiments, the ratio is about 1: 10. In some embodiments, the ratio is about 1: 12. In some embodiments, the ratio is about 1: 14. In some embodiments, the ratio is about 1: 16. In some embodiments, the ratio is about 1: 18. In some embodiments, the ratio is about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 8. In some embodiments, the ratio is between about 1: 2 to about 1: 6. In some embodiments, the ratio is between about 1: 3 to about 1: 5. In some embodiments, the ratio is between about 1: 3.5 to about 1: 4.5. In some embodiments, the ratio is about 1: 4.
  • a protein described herein is admixed with the cryoprotectant, humectant, emulsifier, controlled release agent, and/or buffer described herein.
  • antigen-binding protein is admixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington’s Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984) ; Hardman, et al.
  • the pharmaceutical composition is sterile. Such compositions are part of the present disclosure.
  • the scope of the present disclosure includes desiccated, e.g., freeze-dried, compositions comprising an anti-CoV-S antigen-binding proteins, e.g., antibody or antigen-binding fragment thereof (e.g., of Table 1) , or a pharmaceutical composition thereof that includes a pharmaceutically acceptable carrier but substantially lacks water.
  • the scope of the disclosure also includes proteins provided as part of an aqueous solution or a suspension.
  • routes of administration can vary.
  • routes of administration include intranasal, inhalation, insufflation, and oral.
  • routes of administration include parenteral, intramuscular, and subcutaneous.
  • the disclosure provides intranasal administration of an antibody described herein.
  • the disclosure provides intranasal administration of an antigen-binding protein, e.g., antibody or antigen-binding fragment thereof (e.g., of Table 1) , described herein.
  • the disclosure provides inhaled administration of an antigen-binding protein, e.g., antibody or antigen-binding fragment thereof (e.g., of Table 1) , described herein.
  • the disclosure provides insufflated administration of an antigen-binding protein, e.g., antibody or antigen-binding fragment thereof (e.g., of Table 1) , described herein.
  • an antibody of the disclosure e.g., antibody described herein (e.g., of Table 1) , is administered in association with one or more further therapeutic agents.
  • the anti-CoV-S antigen-binding protein e.g., antibody or antigen-binding fragment thereof of the present disclosure
  • another agent can be formulated into a single composition, e.g., for simultaneous delivery, or formulated separately into two or more compositions (e.g., a kit) .
  • Each component can be administered to a subject at a different time than when the other component is administered; for example, each administration may be given non-simultaneously (e.g., separately or sequentially) at intervals over a given period of time.
  • the separate components may be administered to a subject by the same or by a different route e.g., wherein an anti-CoV-S antibody or antigen-binding fragment thereof.
  • the present disclosure provides a vessel (e.g., a plastic or glass vial, e.g., with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising any of the anti-CoV-S antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof (e.g., of Table 1) , polypeptides (e.g., an HC, LC, VH or VL of Table 1) or polynucleotides or vectors set forth herein or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier.
  • a vessel e.g., a plastic or glass vial, e.g., with a cap or a chromatography column, hollow bore needle or a syringe cylinder
  • any of the anti-CoV-S antigen-binding proteins e.g., antibodies or antigen-binding fragments thereof (e.g., of Table 1)
  • polypeptides e.
  • the present disclosure also provides an injection device comprising one or more antigen-binding proteins (e.g., antibody or antigen-binding fragment) that bind specifically to CoV-S, e.g., those of Table 1, or a pharmaceutical composition thereof.
  • the injection device may be packaged into a kit.
  • An injection device is a device that introduces a substance into the body of a subject via a parenteral route, e.g., intramuscular, subcutaneous or intravenous.
  • an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., comprising the antibody or fragment or a pharmaceutical composition thereof) , a needle for piecing skin and/or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore.
  • an injection device that comprises an antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof, from a combination of the present disclosure, or a pharmaceutical composition thereof is an intravenous (IV) injection device.
  • IV intravenous
  • Such a device can include the antigen-binding protein or a pharmaceutical composition thereof in a cannula or trocar/needle which may be attached to a tube which may be attached to a bag or reservoir for holding fluid (e.g., saline) introduced into the body of the subject through the cannula or trocar/needle.
  • fluid e.g., saline
  • the antibody or fragment or a pharmaceutical composition thereof may, in some embodiments, be introduced into the device once the trocar and cannula are inserted into the vein of a subject and the trocar is removed from the inserted cannula.
  • the IV device may, for example, be inserted into a peripheral vein (e.g., in the hand or arm) ; the superior vena cava or inferior vena cava, or within the right atrium of the heart (e.g., a central IV) ; or into a subclavian, internal jugular, or a femoral vein and, for example, advanced toward the heart until it reaches the superior vena cava or right atrium (e.g., a central venous line) .
  • an injection device is an autoinjector; a jet injector or an external infusion pump.
  • a jet injector uses a high-pressure narrow jet of liquid which penetrate the epidermis to introduce the antibody or fragment or a pharmaceutical composition thereof to a subject’s body.
  • External infusion pumps are medical devices that deliver the antibody or fragment or a pharmaceutical composition thereof into a subject’s body in controlled amounts. External infusion pumps may be powered electrically or mechanically.
  • Different pumps operate in different ways, for example, a syringe pump holds fluid in the reservoir of a syringe, and a moveable piston controls fluid delivery, an elastomeric pump holds fluid in a stretchable balloon reservoir, and pressure from the elastic walls of the balloon drives fluid delivery.
  • a set of rollers pinches down on a length of flexible tubing, pushing fluid forward.
  • fluids can be delivered from multiple reservoirs at multiple rates.
  • the present disclosure provides methods for administering an anti-CoV-S antigen-binding protein of the present disclosure, e.g., those of Table 1, comprising introducing the antigen-binding protein into the body of a subject (e.g., a human) .
  • the method comprises the human subject taking the anti-CoV-S antigen-binding protein of the present disclosure via an intranasal, inhaled, or insufflated route.
  • an antibody described herein is intranasally administered to the human subject.
  • Antibody A or Antibody B or Antibody C or Antibody D or Antibody E or Antibody F, or a combination of any two, or three, or four, antibodies thereof, is intranasally administered to the human subject.
  • Antibody B is intranasally administered to the human subject.
  • the present disclosure provides methods for administering an anti-CoV-S antigen-binding protein, e.g., antibody or antigen-binding fragment thereof (e.g., of Table 1) , comprising introducing the protein into the body of a subject.
  • the method comprises piercing the body of the subject with a needle of a syringe and injecting the antigen-binding protein into the body of the subject, e.g., into the vein, artery, tumor, muscular tissue or subcutis of the subject.
  • the present disclosure provides methods for administering the pharmaceutical composition, or one or more antibodies or antigen binding fragments to a nasal cavity or a mucosa of a subject.
  • the pharmaceutical composition or the one or more antibodies or antigen binding fragment (s) is/are administrated to a mucosa location.
  • Mucosa is the soft tissue that lines the body’s canals and organs in the digestive, respiratory and reproductive systems.
  • the mucosa is located in the nose, mouth, throat, ear, genital, or anus.
  • the mucosa is located in the esophagus, lungs, stomach, intestine, bladder, or uterus.
  • the mucosa is located in the mouth, esophagus, or stomach. In some embodiments, the mucosa is located in the nose, mouth, pharynx, trachea, or lung. In some embodiments, the mucosa is nasal mucosa. In some embodiments, the pharmaceutical composition or the one or more antibodies or antigen binding fragment (s) is/are administrated intranasally.
  • the disclosure provides methods of preventing a disease in a subject in need thereof, comprising administering the pharmaceutical composition, the antibody or antigen binding fragment, or a combination thereof to the subject.
  • the disclosure provides methods of treating a disease in a subject in need thereof, comprising administering the pharmaceutical composition, the antibody or antigen binding fragment, or a combination thereof to the subject.
  • the pharmaceutical composition is administered to a mucosa of the subject.
  • the mucosa is located in the nose, mouth, throat, ear, genital, or anus.
  • the mucosa is located in the esophagus, lungs, stomach, intestine, bladder, or uterus.
  • the mucosa is located in the mouth, esophagus, or stomach.
  • the mucosa is located in the nose, mouth, pharynx, trachea, or lung.
  • the mucosa is nasal mucosa.
  • the administration is intranasal administration.
  • the administration starts prior to the onset of any symptom of the disease.
  • the pharmaceutical composition is a vaccine.
  • the vaccine comprises a protein antigen as an active ingredient.
  • the pharmaceutical composition comprises an active ingredient (e.g., an antibody) that neutralizes an infectious agent (e.g., a virus) .
  • an active ingredient e.g., an antibody
  • an infectious agent e.g., a virus
  • the administration starts after the onset of at least one symptom of the disease.
  • the symptom is a respiratory symptom.
  • the respiratory symptom is allergy, nasal congestion, nasal infection, or a combination thereof.
  • the disease is a respiratory infection caused by a virus, a bacterial, a fungus, or a combination thereof.
  • the disease is a viral infection.
  • the disease is a chronic viral infection.
  • the disease is an acute viral infection.
  • the disease is a coronavirus infection.
  • the disease is SARS-CoV-2 infection.
  • the present disclosure provides methods for treating or preventing viral infection (e.g., coronavirus infection) by administering a therapeutically effective amount of the active ingredient in the pharmaceutical composition (e.g., an antigen-binding protein, e.g., antibody or antigen-binding fragment, (e.g., of Table 1) ) to a subject (e.g., a human) in need of such treatment or prevention.
  • a therapeutically effective amount of the active ingredient in the pharmaceutical composition e.g., an antigen-binding protein, e.g., antibody or antigen-binding fragment, (e.g., of Table 1)
  • a subject e.g., a human
  • Coronavirus infection may be treated or prevented, in a subject, by administering an anti-CoV-S antigen-binding protein of the present disclosure to a subject.
  • an effective or therapeutically effective dose of the antigen-binding protein, e.g., antibody or antigen-binding fragment (e.g., of Table 1) , for treating or preventing a viral infection refers to the amount of the antibody or fragment sufficient to alleviate or prevent one or more signs and/or symptoms of the infection in the treated subject, whether by inducing the regression or elimination of such signs and/or symptoms or by inhibiting the progression of such signs and/or symptoms.
  • the dose amount may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like.
  • an effective or therapeutically effective dose of antibody or antigen-binding fragment thereof of the present disclosure, for treating or preventing viral infection e.g., in an adult human subject, is about 0.01 mg to about 200 mg per dose.
  • about 0.01 mg to about 200 mg of the antigen-binding protein is delivered to a nasal cavity of a human per administration.
  • the dose per administration is about 0.01 mg to about 0.05 mg. In some embodiments, the dose per administration is about 0.05 mg to about 0.1 mg. In some embodiments, the dose per administration is about 0.1 mg to about 0.2 mg. In some embodiments, the dose per administration is about 0.2 mg to about 0.3 mg. In some embodiments, the dose per administration is about 0.3 mg to about 0.5 mg. In some embodiments, the dose per administration is about 0.5 mg to about 0.7 mg. In some embodiments, the dose per administration is about 0.7 mg to about 1 mg. In some embodiments, the dose per administration is about 1 mg to about 2 mg.
  • the dose per administration is about 2 mg to about 5 mg. In some embodiments, the dose per administration is about 5 mg to about 10 mg. In some embodiments, the dose per administration is about 10 mg to about 30 mg. In some embodiments, the dose per administration is about 30 mg to about 100 mg. In some embodiments, the dose per administration is about 100 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 0.1 mg. In some embodiments, the dose per administration is about 0.05 mg to about 0.2 mg. In some embodiments, the dose per administration is about 0.1 mg to about 0.3 mg. In some embodiments, the dose per administration is about 0.2 mg to about 0.5 mg. In some embodiments, the dose per administration is about 0.3 mg to about 0.7 mg.
  • the dose per administration is about 0.5 mg to about 1 mg. In some embodiments, the dose per administration is about 0.7 mg to about 2 mg. In some embodiments, the dose per administration is about 1 mg to about 5 mg. In some embodiments, the dose per administration is about 2 mg to about 10 mg. In some embodiments, the dose per administration is about 5 mg to about 30 mg. In some embodiments, the dose per administration is about 10 mg to about 100 mg. In some embodiments, the dose per administration is about 30 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 0.2 mg. In some embodiments, the dose per administration is about 0.05 mg to about 0.3 mg. In some embodiments, the dose per administration is about 0.1 mg to about 0.5 mg.
  • the dose per administration is about 0.2 mg to about 0.7 mg. In some embodiments, the dose per administration is about 0.3 mg to about 1 mg. In some embodiments, the dose per administration is about 0.5 mg to about 2 mg. In some embodiments, the dose per administration is about 0.7 mg to about 5 mg. In some embodiments, the dose per administration is about 1 mg to about 10 mg. In some embodiments, the dose per administration is about 2 mg to about 30 mg. In some embodiments, the dose per administration is about 5 mg to about 100 mg. In some embodiments, the dose per administration is about 10 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 0.3 mg. In some embodiments, the dose per administration is about 0.05 mg to about 0.5 mg.
  • the dose per administration is about 0.1 mg to about 0.7 mg. In some embodiments, the dose per administration is about 0.2 mg to about 1 mg. In some embodiments, the dose per administration is about 0.3 mg to about 2 mg. In some embodiments, the dose per administration is about 0.5 mg to about 5 mg. In some embodiments, the dose per administration is about 0.7 mg to about 10 mg. In some embodiments, the dose per administration is about 1 mg to about 30 mg. In some embodiments, the dose per administration is about 2 mg to about 100 mg. In some embodiments, the dose per administration is about 5 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 0.5 mg. In some embodiments, the dose per administration is about 0.05 mg to about 0.7 mg.
  • the dose per administration is about 0.1 mg to about 1 mg. In some embodiments, the dose per administration is about 0.2 mg to about 2 mg. In some embodiments, the dose per administration is about 0.3 mg to about 5 mg. In some embodiments, the dose per administration is about 0.5 mg to about 10 mg. In some embodiments, the dose per administration is about 0.7 mg to about 30 mg. In some embodiments, the dose per administration is about 1 mg to about 100 mg. In some embodiments, the dose per administration is about 2 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 0.7 mg. In some embodiments, the dose per administration is about 0.05 mg to about 1 mg. In some embodiments, the dose per administration is about 0.1 mg to about 2 mg.
  • the dose per administration is about 0.2 mg to about 5 mg. In some embodiments, the dose per administration is about 0.3 mg to about 10 mg. In some embodiments, the dose per administration is about 0.5 mg to about 30 mg. In some embodiments, the dose per administration is about 0.7 mg to about 100 mg. In some embodiments, the dose per administration is about 1 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 1 mg. In some embodiments, the dose per administration is about 0.05 mg to about 2 mg. In some embodiments, the dose per administration is about 0.1 mg to about 5 mg. In some embodiments, the dose per administration is about 0.2 mg to about 10 mg. In some embodiments, the dose per administration is about 0.3 mg to about 30 mg.
  • the dose per administration is about 0.5 mg to about 100 mg. In some embodiments, the dose per administration is about 0.7 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 2 mg. In some embodiments, the dose per administration is about 0.05 mg to about 5 mg. In some embodiments, the dose per administration is about 0.1 mg to about 10 mg. In some embodiments, the dose per administration is about 0.2 mg to about 30 mg. In some embodiments, the dose per administration is about 0.3 mg to about 100 mg. In some embodiments, the dose per administration is about 0.5 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 5 mg. In some embodiments, the dose per administration is about 0.05 mg to about 10 mg.
  • the dose per administration is about 0.1 mg to about 30 mg. In some embodiments, the dose per administration is about 0.2 mg to about 100 mg. In some embodiments, the dose per administration is about 0.3 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 10 mg. In some embodiments, the dose per administration is about 0.05 mg to about 30 mg. In some embodiments, the dose per administration is about 0.1 mg to about 100 mg. In some embodiments, the dose per administration is about 0.2 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 30 mg. In some embodiments, the dose per administration is about 0.05 mg to about 100 mg. In some embodiments, the dose per administration is about 0.1 mg to about 200 mg.
  • the dose per administration is about 0.01 mg to about 100 mg. In some embodiments, the dose per administration is about 0.05 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg. In some embodiments, the dose per administration is about 0.02 mg. In some embodiments, the dose per administration is about 0.03 mg. In some embodiments, the dose per administration is about 0.04 mg. In some embodiments, the dose per administration is about 0.05 mg. In some embodiments, the dose per administration is about 0.06 mg. In some embodiments, the dose per administration is about 0.07 mg. In some embodiments, the dose per administration is about 0.08 mg. In some embodiments, the dose per administration is about 0.09 mg. In some embodiments, the dose per administration is about 0.1 mg.
  • the dose per administration is about 0.12 mg. In some embodiments, the dose per administration is about 0.14 mg. In some embodiments, the dose per administration is about 0.17 mg. In some embodiments, the dose per administration is about 0.2 mg. In some embodiments, the dose per administration is about 0.23 mg. In some embodiments, the dose per administration is about 0.27 mg. In some embodiments, the dose per administration is about 0.3 mg. In some embodiments, the dose per administration is about 0.35 mg. In some embodiments, the dose per administration is about 0.4 mg. In some embodiments, the dose per administration is about 0.45 mg. In some embodiments, the dose per administration is about 0.5 mg. In some embodiments, the dose per administration is about 0.6 mg.
  • the dose per administration is about 0.7 mg. In some embodiments, the dose per administration is about 0.8 mg. In some embodiments, the dose per administration is about 0.9 mg. In some embodiments, the dose per administration is about 1 mg. In some embodiments, the dose per administration is about 0.01 mg to about 5 mg. In some embodiments, the dose per administration is about 0.05 mg to about 2 mg. In some embodiments, the dose per administration is about 0.1 mg to about 1 mg. In some embodiments, the dose per administration is about 0.15 mg to about 0.75 mg. In some embodiments, the dose per administration is about 0.2 mg to about 0.6 mg. In some embodiments, the dose per administration is about 0.25 mg to about 0.5 mg. In some embodiments, the dose per administration is about 0.3 mg to about 0.4 mg. In some embodiments, the dose per administration is about 0.35 mg.
  • about 0.01-2 mL of the pharmaceutical composition is administered to a nasal cavity of a human; that is, the volume per administration is about 0.01 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.02 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.04 mL.In some embodiments, the volume per administration is about 0.04 mL to about 0.06 mL. In some embodiments, the volume per administration is about 0.06 mL to about 0.08 mL. In some embodiments, the volume per administration is about 0.08 mL to about 0.1 mL.
  • the volume per administration is about 0.1 mL to about 0.2 mL. In some embodiments, the volume per administration is about 0.2 mL to about 0.4 mL. In some embodiments, the volume per administration is about 0.4 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.6 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.8 mL to about 1 mL. In some embodiments, the volume per administration is about 1 mL to about 1.5 mL. In some embodiments, the volume per administration is about 1.5 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.04 mL.
  • the volume per administration is about 0.02 mL to about 0.06 mL. In some embodiments, the volume per administration is about 0.04 mL to about 0.08 mL. In some embodiments, the volume per administration is about 0.06 mL to about 0.1 mL. In some embodiments, the volume per administration is about 0.08 mL to about 0.2 mL. In some embodiments, the volume per administration is about 0.1 mL to about 0.4 mL. In some embodiments, the volume per administration is about 0.2 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.4 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.6 mL to about 1 mL.
  • the volume per administration is about 0.8 mL to about 1.5 mL. In some embodiments, the volume per administration is about 1 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.06 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.08 mL. In some embodiments, the volume per administration is about 0.04 mL to about 0.1 mL. In some embodiments, the volume per administration is about 0.06 mL to about 0.2 mL. In some embodiments, the volume per administration is about 0.08 mL to about 0.4 mL. In some embodiments, the volume per administration is about 0.1 mL to about 0.6 mL.
  • the volume per administration is about 0.2 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.4 mL to about 1 mL. In some embodiments, the volume per administration is about 0.6 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.8 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.08 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.1 mL. In some embodiments, the volume per administration is about 0.04 mL to about 0.2 mL. In some embodiments, the volume per administration is about 0.06 mL to about 0.4 mL.
  • the volume per administration is about 0.08 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.1 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.2 mL to about 1 mL. In some embodiments, the volume per administration is about 0.4 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.6 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.1 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.2 mL. In some embodiments, the volume per administration is about 0.04 mL to about 0.4 mL.
  • the volume per administration is about 0.06 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.08 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.1 mL to about 1 mL. In some embodiments, the volume per administration is about 0.2 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.4 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.2 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.4 mL. In some embodiments, the volume per administration is about 0.04 mL to about 0.6 mL.
  • the volume per administration is about 0.06 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.08 mL to about 1 mL. In some embodiments, the volume per administration is about 0.1 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.2 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.4 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.04 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.06 mL to about 1 mL.
  • the volume per administration is about 0.08 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.1 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.04 mL to about 1 mL. In some embodiments, the volume per administration is about 0.06 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.08 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.8 mL.
  • the volume per administration is about 0.02 mL to about 1 mL. In some embodiments, the volume per administration is about 0.04 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.06 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 1 mL. In some embodiments, the volume per administration is about 0.02 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.04 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.02 mL to about 2 mL.
  • the volume per administration is about 0.01 mL. In some embodiments, the volume per administration is about 0.05 mL. In some embodiments, the volume per administration is about 0.1 mL. In some embodiments, the volume per administration is about 0.15 mL. In some embodiments, the volume per administration is about 0.2 mL. In some embodiments, the volume per administration is about 0.25 mL. In some embodiments, the volume per administration is about 0.3 mL. In some embodiments, the volume per administration is about 0.4 mL. In some embodiments, the volume per administration is about 0.5 mL. In some embodiments, the volume per administration is about 0.6 mL. In some embodiments, the volume per administration is about 0.7 mL.
  • the volume per administration is about 0.8 mL. In some embodiments, the volume per administration is about 0.9 mL. In some embodiments, the volume per administration is about 1 mL. In some embodiments, the volume per administration is about 1.1 mL. In some embodiments, the volume per administration is about 1.2 mL. In some embodiments, the volume per administration is about 1.3 mL. In some embodiments, the volume per administration is about 1.4 mL. In some embodiments, the volume per administration is about 1.5 mL. In some embodiments, the volume per administration is about 1.6 mL. In some embodiments, the volume per administration is about 1.7 mL. In some embodiments, the volume per administration is about 1.8 mL.
  • the volume per administration is about 1.9 mL. In some embodiments, the volume per administration is about 2 mL. In some embodiments, the volume per administration is about 0.1 mL to about 2 mL. In some embodiments, the volume per administration is about 0.2 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.3 mL to about 1.2 mL. In some embodiments, the volume per administration is about 0.4 mL to about 1 mL. In some embodiments, the volume per administration is about 0.5 mL to about 0.9 mL. In some embodiments, the volume per administration is about 0.6 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.7 mL.
  • about 0.01-2 mL of the pharmaceutical composition is administered by nasal spray to the nasal cavity.
  • the frequency and the duration of the treatment can be adjusted.
  • the pharmaceutical composition or antigen-binding protein of the present disclosure can be administered at an initial dose, followed by one or more secondary doses, (e.g., once a day, twice a day, 3 times a day, 4 times a day, once every at least one day) .
  • the initial dose may be followed by administration of a second or a plurality of subsequent doses in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.
  • the pharmaceutical composition or antigen-binding protein is administered for about 1-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-2 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-3 days.
  • the pharmaceutical composition or antigen-binding protein is administered for about 3-4 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 4-5 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 5-7 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 7-10 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 10-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 14-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 20-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-3 days.
  • the pharmaceutical composition or antigen-binding protein is administered for about 2-4 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 3-5 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 4-7 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 5-10 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 7-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 10-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 14-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-4 days.
  • the pharmaceutical composition or antigen-binding protein is administered for about 2-5 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 3-7 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 4-10 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 5-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 7-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 10-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-5 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-7 days.
  • the pharmaceutical composition or antigen-binding protein is administered for about 3-10 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 4-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 5-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 7-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-7 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-10 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 3-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 4-20 days.
  • the pharmaceutical composition or antigen-binding protein is administered for about 5-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-10 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 3-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 4-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 3-30 days.
  • the pharmaceutical composition or antigen-binding protein is administered for about 1-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
  • the pharmaceutical composition or antigen-binding protein is administered for about 7-14 days.
  • the pharmaceutical composition or antigen-binding protein is administered to the subject about 1-10 times a day, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, times a day.
  • the pharmaceutical composition or antigen-binding protein is administered to the subject about 1-6 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 2-4 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 2-3 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 3-4 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about once a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about twice a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 3 times a day.
  • the pharmaceutical composition or antigen-binding protein is administered to the subject about 4 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 5 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 6 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject no more than 6, 5, 4, 3, 2, or 1 time (s) a day.
  • the term “subject” refers to a mammal (e.g., rat, mouse, cat, dog, cow, pig, sheep, horse, goat, rabbit) , preferably a human, for example, in need of prevention and/or treatment of a disease or disorder such as viral infection or cancer.
  • the subject may have a viral infection, or be predisposed to developing an infection.
  • Subjects predisposed to developing an infection, or subjects who may be at elevated risk for contracting an infection include subjects with compromised immune systems because of autoimmune disease, subjects receiving immunosuppressive therapy (for example, following organ transplant) , subjects afflicted with human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS) , subjects with forms of anemia that deplete or destroy white blood cells, subjects receiving radiation or chemotherapy, or subjects afflicted with an inflammatory disorder. Additionally, subjects of very young (e.g., 5 years of age or younger) or old age (e.g., 65 years of age or older) are at increased risk.
  • immunosuppressive therapy for example, following organ transplant
  • HIV human immunodeficiency syndrome
  • AIDS acquired immune deficiency syndrome
  • subjects with forms of anemia that deplete or destroy white blood cells subjects receiving radiation or chemotherapy
  • subjects of very young e.g., 5 years of age or younger
  • old age e.g., 65 years of age or older
  • a subject may be at risk of contracting a viral infection due to proximity to an outbreak of the disease, e.g., subject resides in a densely populated city or in close proximity to subjects having confirmed or suspected infections of a virus, or choice of employment, e.g. hospital worker, pharmaceutical researcher, traveler to infected area, or frequent flier.
  • Treat” or “treating” means to administer a composition comprising an active ingredient, e.g., antibody or antigen-binding fragment of the present disclosure (e.g., of Table 1) , to a subject having one or more signs or symptoms of a disease or infection, e.g., viral infection, for which the active ingredient is effective when administered to the subject at an effective or therapeutically effective amount or dose (as discussed herein) .
  • an active ingredient e.g., antibody or antigen-binding fragment of the present disclosure (e.g., of Table 1)
  • a subject having one or more signs or symptoms of a disease or infection e.g., viral infection
  • the present disclosure also encompasses prophylactically administering a composition comprising an active ingredient, e.g., antibody or antigen-binding fragment thereof of the present disclosure (e.g., of Table 1) , to a subject who is at risk of a disease (e.g., viral infection) so as to prevent such a disease.
  • a disease e.g., viral infection
  • Passive antibody-based immunoprophylaxis has proven an effective strategy for preventing subject from viral infection. See e.g., Berry et al., Passive broad-spectrum influenza immunoprophylaxis. Influenza Res Treat. 2014; 2014: 267594. Epub 2014 Sep 22; and Jianqiang et al., Passive immune neutralization strategies for prevention and control of influenza A infections, Immunotherapy.
  • Prevent means to administer a composition comprising an active ingredient, e.g., antibody or antigen-binding fragment of the present disclosure (e.g., of Table 1) , to a subject to inhibit the manifestation of a disease (e.g., viral infection) in the body of a subject, for which the active ingredient is effective when administered to the subject at an effective or therapeutically effective amount or dose (as discussed herein) .
  • an active ingredient e.g., antibody or antigen-binding fragment of the present disclosure (e.g., of Table 1)
  • a disease e.g., viral infection
  • a sign or symptom of a viral infection in a subject is survival or proliferation of virus in the body of the subject, e.g., as determined by viral titer assay (e.g., coronavirus propagation in embryonated chicken eggs or coronavirus spike protein assay) .
  • viral titer assay e.g., coronavirus propagation in embryonated chicken eggs or coronavirus spike protein assay.
  • Other signs and symptoms of viral infection are discussed herein.
  • the subject may be a non-human animal
  • the active ingredient or the antigen-binding proteins (e.g., antibodies and antigen-binding fragments) discussed herein may be used in a veterinary context to treat and/or prevent disease in the non-human animals (e.g., cats, dogs, pigs, cows, horses, goats, rabbits, sheep, and the like) .
  • the present disclosure provides a method for treating or preventing viral infection (e.g., coronavirus infection) or for inducing the regression or elimination or inhibiting the progression of at least one sign or symptom of viral infection such as: --fever or feeling feverish/chills; --cough; --sore throat; --runny or stuffy nose; --sneezing; --muscle or body aches; --headaches; --fatigue (tiredness) ; --vomiting; --diarrhea; --respiratory tract infection; --chest discomfort; --shortness of breath; --bronchitis; and/or --pneumonia, which sign or symptom is secondary to viral infection, in a subject in need thereof (e.g., a human) , by administering a therapeutically effective amount of the active ingredient (e.g., anti-CoV-S antigen-binding protein (e.g., of Table 1) ) to the subject, for example, by administering of the active ingredient into the body of the subject
  • the disclosure provides a method of treating a respiratory infection, said method comprising administering an active ingredient (e.g., an antibody) described herein intranasally to a mammal with the respiratory infection, thereby treating the respiratory infection.
  • an active ingredient e.g., an antibody
  • the disclosure provides a method of treating a SARS-CoV-2 infection, said method comprising administering active ingredient (e.g., an antibody) described herein intranasally to a mammal infected with SARS-CoV-2, thereby treating the SARS-CoV-2 infection.
  • the disclosure provides a method of treating a SARS-CoV-2 infection, said method comprising administering an active ingredient (e.g., an antibody) to a mammal infected with SARS-CoV-2, thereby treating the SARS-CoV-2 infection.
  • an active ingredient e.g., an antibody
  • the disclosure provides a method of delivering high local concentration of an active ingredient (e.g., an antibody) in the nasal cavity while having a low exposure in the blood circulation (systemic exposure) .
  • the active ingredient comprises an antibody or antigen binding fragment thereof.
  • the anti-CoV-S antigen-binding proteins may be used to detect and/or measure CoV-S in a sample.
  • Exemplary assays for CoV-S may include, e.g., contacting a sample with an anti-CoV-S antigen-binding protein of the disclosure, wherein the anti-CoV-S antigen-binding protein is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate CoV-S from samples.
  • the presence of an anti-CoV-S antigen-binding protein complexed with CoV-S indicates the presence of CoV-S in the sample.
  • an unlabeled anti-CoV-S antibody can be used in combination with a secondary antibody which is itself detectably labeled.
  • the detectable label or reporter molecule can be a radioisotope, such as 3 H, 14 C , 32 P, 35 S, or 125 I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, ⁇ -galactosidase, horseradish peroxidase, or luciferase.
  • the present disclosure includes a method for detecting the presence of spike protein polypeptide in a sample comprising contacting the sample with an anti-CoV-S antigen-binding protein and detecting the presence of a CoV-S/anti-CoV-S antigen-binding protein wherein the presence of the complex indicates the presence of CoV-S.
  • An anti-CoV-S antigen-binding protein of the disclosure (e.g., of Table 1) may be used in a Western blot or immune-protein blot procedure for detecting the presence of CoV-S or a fragment thereof in a sample.
  • Such a procedure forms part of the present disclosure and includes the steps of e.g.:
  • a membrane or other solid substrate comprising a sample to be tested for the presence of CoV-S, e.g., optionally including the step of transferring proteins from a sample to be tested for the presence of CoV-S (e.g., from a PAGE or SDS-PAGE electrophoretic separation of the proteins in the sample) onto a membrane or other solid substrate using a method known in the art (e.g., semi-dry blotting or tank blotting) ; and contacting the membrane or other solid substrate to be tested for the presence of CoV-S or a fragment thereof with an anti-CoV-S antigen-binding protein of the disclosure.
  • a method known in the art e.g., semi-dry blotting or tank blotting
  • Such a membrane may take the form, for example, of a nitrocellulose or vinyl-based (e.g., polyvinylidene fluoride (PVDF) ) membrane to which the proteins to be tested for the presence of CoV-S in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel have been transferred (e.g., following electrophoretic separation in the gel) .
  • PAGE polyacrylamide gel electrophoresis
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • Detection of the bound antigen-binding protein indicates that the CoV-S protein is present on the membrane or substrate and in the sample. Detection of the bound antigen-binding protein may be by binding the antigen-binding protein with a secondary antibody (an anti-immunoglobulin antibody) which is detectably labeled and, then, detecting the presence of the secondary antibody label.
  • a secondary antibody an anti-immunoglobulin antibody
  • anti-CoV-S antigen-binding proteins e.g., antibodies and antigen-binding fragments (e.g., of Table 1)
  • Such a method forms part of the present disclosure and comprises, e.g.,
  • the antigen-binding protein itself is detectably labeled, it can be detected directly.
  • the antigen-binding protein may be bound by a detectably labeled secondary antibody wherein the label is then detected.
  • kits comprising one or more components that include, but are not limited to, a pharmaceutical composition described herein suitable for intranasal administration, and a nasal administration device for accurate delivery of said intranasal pharmaceutical composition.
  • the nasal administration device is a nasal pump device.
  • the nasal administration device delivers about 0.01 mg/dose to about 20 mg/dose of an intranasal pharmaceutical composition described herein, by a single or multiple actuation (s) e.g., 0.1 mL per spray to each nostril, spray twice to each nostril (for a total of 0.4 mL) of antibody of 1 mg/mL, gives 0.4 mg per dose.
  • the kit comprises a pharmaceutical composition described herein suitable for intranasal administration, and a nasal administration device.
  • the nasal administration device is a nasal pump device.
  • the disclosure provides a kit comprising a pharmaceutical composition described herein suitable for intranasal administration, and a nasal administration device, and instructions for the use of the kit.
  • the intranasal compositions of this disclosure are most effective when proper product delivery design is utilized.
  • the preferred product design includes a composition of the disclosure contained within a delivery system, such as a bottle and a pump, for nasal delivery of the formulation in a mist of spray droplets to coat the mucosa of the nasal cavity upon administration.
  • Preferred pumps for use in such products of the disclosure are metered multi-dose pumps; however, single unit dose containers are also acceptable to deliver an intranasal pharmaceutical composition described herein to the nasal cavity.
  • the selection of the pump is based on the desired dose/spray volume and spray pattern appropriate for local delivery to the nasal mucosa.
  • compositions can be effectively contained in a package comprising a bottle fitted with a cap, and the cap can be screwed on or snapped on or sealed with a pump head, and are delivered by a metered-dose spray pump designed for intranasal application in volumes of about 0.02 to 0.2 ml per press.
  • Suitable compositions of the present disclosure include about 0.02 mg/dose to about 200 mg/dose of an intranasal pharmaceutical formulation described herein relative to the total weight of the composition.
  • kits comprising one or more components that include, but are not limited to, an anti-CoV-S antigen-binding protein, e.g., an antibody or antigen-binding fragment as discussed herein (e.g., of Table 1) , in association with one or more additional components including, optionally, a further therapeutic agent.
  • an anti-CoV-S antigen-binding protein e.g., an antibody or antigen-binding fragment as discussed herein (e.g., of Table 1)
  • additional components including, optionally, a further therapeutic agent.
  • the kit of the disclosure includes a pharmaceutical composition for parenteral administration to a subject and a device (e.g., an injection device) for performing such administration.
  • a device e.g., an injection device
  • the kit can include one or more hypodermic needles or other injection devices as discussed above containing the anti-CoV-S antigen-binding protein, e.g., antibody or antigen-binding fragment thereof of the present disclosure (e.g., of Table 1) .
  • the kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit.
  • information concerning the pharmaceutical compositions and dosage forms in the kit aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely.
  • the following information regarding a combination of the disclosure may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.
  • the sequence of the spike protein of wild-type SARS-CoV-2 is according to SEQ ID NO: 70:
  • the sequence of the RBD-domain of the spike protein of wild-type SARS-CoV-2 is according to SEQ ID NO: 71:
  • the sequence of the spike protein of alpha (B. 1.1.7) SARS-CoV-2 is according to SEQ ID NO: 72:
  • the sequence of the RBD-domain of the spike protein of alpha (B. 1.1.7) SARS-CoV-2 is according to SEQ ID NO: 73:
  • the sequence of the spike protein of beta (B. 1.351) SARS-CoV-2 is according to SEQ ID NO: 74:
  • the sequence of the RBD-domain of the spike protein of beta (B. 1.351) SARS-CoV-2 is according to SEQ ID NO: 75:
  • SARS-CoV-2 The sequence of the spike protein of gamma (P. 1) SARS-CoV-2 is according to SEQ ID NO: 76:
  • the sequence of the RBD-domain of the spike protein of gamma is according to SEQ ID NO: 77:
  • the sequence of the spike protein of delta (B. 1.617.2) SARS-CoV-2 is according to SEQ ID NO: 78:
  • the sequence of the RBD-domain of the spike protein of delta (B. 1.617.2) SARS-CoV-2 is according to SEQ ID NO: 79:
  • SARS-CoV-2 The sequence of the spike protein of omicron (BA. 1) SARS-CoV-2 is according to SEQ ID NO: 80:
  • the sequence of the RBD-domain of the spike protein of omicron (BA. 1) SARS-CoV-2 is according to SEQ ID NO: 81:
  • the active ingredient of Antibody B is IgG1 antibody, and the dosage form is nasal spray, with a specification of 5mg/mL and 70 ⁇ L per press.
  • Excipients are: disodium hydrogen phosphate, sodium dihydrogen phosphate, Tween 80, trehalose, glycerol and hydroxypropyl methylcellulose. The excipients used are all approved excipients with known safety. See Table 4A.
  • Antibody B in an aqueous solution at a concentration of about 5 mg/mL was nasally sprayed to Cynomolgus monkeys twice a day for 14 days, 0.38 mL per dose. Blood samples and nasal swab samples were collected. The concentration of Antibody B in the samples was determined ( Figure 1G) . The concentration of Antibody B in the nasal cavity was on average at least about 50 times higher than in the circulating blood within 12 hour of antibody administration. Administration of Antibody B via nasal spray provided a high local concentration of Antibody B in the nasal cavity that was about 1000 times higher than the Antibody B concentration needed for 50%neutralization (IC50) of SARS-CoV-2 viruses, while at the same time kept a low systemic exposure for improved safety.
  • IC50 50%neutralization
  • Vero (catalog no. CCL-81, ATCC) , Vero E6 (catalog no. CRL-1586 TM ) , 293T cells (catalog no. GDC187, CCTCC) , L-929 (catalog no. GDC034, CCTCC) , HEp-2 (catalog no. GDC004, CCTCC) , and Huh7 cells (catalog no. GDC134, CCTCC) were cultured in DMEM (Gibco) with 10%FBS (Gibco) , 100 units/ml penicillin, and 100 ⁇ g/ml streptomycin (Gibco) at 37°C and 5%CO 2 .
  • OVCAR-3 cells (catalog no.
  • TCHu228, CCTCC were cultured in RPMI Medium 1640 (Gibco) with 20%FBS at 37°C and 5%CO 2 .
  • HEK293F cells (Thermo) were cultured using Freestyle 293 expression medium (Invitrogen) in an incubator at 37°C and 8%CO 2 with shaking at 180 rpm/min.
  • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, strain: GWHBEBW01000000) was originally isolated from a swab sample of a 33 year old man travelling from Indonesia to Wuhan in June 2021. All processes in this study involving authentic SARS-CoV-2 were performed in a biosafety level 3 (BSL-3) facility.
  • Groups of 6 female Syrian golden hamsters were challenged intranasally with 10 4 PFU of the Delta and Omicron variants per animal in 100 ⁇ l of PBS after anesthetization with isoflurane.
  • For the prophylactic group (Group 1) hamsters were intranasally administered 100 ⁇ l of Antibody B (5 mg/kg for Delta challenge and 15 mg/kg for Omicron challenge) 30 min before challenge and given two more doses at 24 h and 48 h post challenge. The activity of Antibody B was also assessed in an in vivo therapeutic experiment.
  • hamsters were intranasally administered 100 ⁇ l of 5 mg/kg (Delta challenge) or 15 mg/kg (Omicron challenge) Antibody B (Group 2) or PBS (Group 3, this group was established only in the Delta challenge but not the omicron challenge) at 3 h post challenge, followed by two doses of 100 ⁇ l of 5mg/kg (Delta challenge) or 15 mg/kg (Omicron challenge) Antibody B at 24 h and 48 h post challenge.
  • the negative control group (Group 4; this group was named Group 3 in the Omicron challenge) was treated the same as Groups 2 and 3 but with the equivalent volume of PBS instead of antibody.
  • Viral RNA in the nasal washes, throat swabs, and tissue homogenates was quantified by one-step real-time RT–PCR as described previously (Feng, L. et al. An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques. Nature communications 11, 1-11 (2020) ) . Briefly, viral RNA was purified using a QIAamp Viral RNA Mini Kit (Qiagen) and quantified with a II One-Step qRT–PCR Green Kit (Vazyme Biotech Co., Ltd.
  • ORF1ab-F 5’-CCCTGTGGGTTTTACACTTAA-3’; SEQ ID NO: 82
  • ORF1ab-R 5’-ACGATTGTGCATCAGCTGA-3’; SEQ ID NO: 83
  • the amplification procedure was set up as follows: 50°C for 3 min, 95°C for 30 s, and 40 cycles consisting of 95°C for 10 s and 60°C for 30 s.
  • the expression level of cytokines in lung tissues of infected animals was determined similarly, with the primers shown in the supplementary material.
  • virus titer was determined with a plaque assay as previously described, with slight modification.
  • Zhang, Q. et al. A serological survey of SARS-CoV-2 in cat in Wuhan. Emerg Microbes Infect 9, 2013-2019, doi: 10.1080/22221751.2020.1817796 (2020) ) .
  • virus samples were serially 10-fold diluted with DMEM with 2.5%FBS and inoculated onto Vero E6 cells seeded overnight at 1.5 ⁇ 10 5 /well in 24-well plates; after incubation at 37°C for 1 h, the inoculum was replaced with DMEM containing 2.5%FBS and 0.9%carboxymethyl-cellulose.
  • the plates were fixed with 8%paraformaldehyde and stained with 0.5%crystal violet 3 days later.
  • Virus titer was calculated with the dilution gradient with 10 ⁇ 100 plaques.
  • Lung tissue was fixed with 10%neutral formaldehyde for H&E staining. Histologic lesion severity was scored per lung lobe according to a standardized scoring system evaluating the presence of interstitial pneumonia, type II pneumocyte hyperplasia, edema and fibrin deposition, and perivascular lymphoid cuffing.
  • Antibody B shows protective efficacy in vivo in hamster model through intranasal administration
  • Antibody B potently and broadly neutralizes different SARS-CoV-2 VOCs
  • Antibody B The neutralization activity of Antibody B was first measured with pseudotyped SARS-CoV-2 viruses, including SARS-CoV-2 WT, Delta and Omicron variants ( Figure 1B) .
  • Antibody B showed broadly neutralizing activities against all tested pseudoviruses, with IC 50 values ranging from 4.75 to 165 ng/ml.
  • IC 50 values ranging from 4.75 to 165 ng/ml.
  • Antibody B neutralized authentic SARS-CoV-2 WT, Delta, and Omicron with IC 50 values of 2.63 ng/ml, 3.38 ng/ml, and 108.6 ng/ml, respectively (Figure 1C) .
  • the IC 50 of Antibody B against omicron was lower than that of other tested antibodies (Cao Y, et al. Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature, (2021) ) .
  • Antibody B delivered via intranasal administration shows protective efficacy against Delta variant in vivo in a hamster model
  • Antibody B exhibited protective efficacy in hACE2-transgenic mice (K18 mice) challenged with SARS-CoV-2 WT and the Beta variant when administered intraperitoneally. While K18 mice served as a very useful animal model for evaluating vaccines and therapeutics in the early stage of the pandemic, they are much more expensive, have more limited supplies, and have recently been replaced/or partially replaced by golden (Syrian) hamsters. SARS-CoV-2 can cause lethality in K18 mice and severe damage to various organs, including the lung, liver, kidney, and even brain (McCray PB, Jr., et al. Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus.
  • the golden (Syrian) hamster can be naturally infected by SARS-CoV-2, which results in the presence of viral antigens in the nasal mucosa, bronchial epithelial cells and areas of lung consolidation after inoculation with SARS-CoV-2 (Sia SF, et al. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters. Nature 583, 834-838 (2020) ) . It has been widely accepted as an animal model by the field for the development of vaccines and therapies against SARS-CoV-2.
  • Antibody B reduces lung injury caused by Delta variant infection
  • Antibody B delivered via intranasal administration also shows protective efficacy against Omicron variant in vivo in a hamster model
  • Antibody B can efficiently neutralize authentic Delta and Omicron variants and prevent viral infection in vivo at a low dose when delivered through nasal administration.
  • Antibody B is a good clinical candidate for use against infections caused by newly emerging SARS-CoV-2 variants (e.g., Delta and Omicron) in humans.
  • SARS-CoV-2 variants e.g., Delta and Omicron
  • Antibody B could relieve patients’ burden due to the low dose used in animal studies and be more acceptable to different patients due to its convenient and safe nasal administration.
  • Antibody B is a broad nmAb that can inhibit SARS-CoV-2 and all currently recognized VOCs. Due to the conserved sites in the RBD recognized by Antibody B, it is very possible that Antibody B is capable of neutralizing additional SARS-CoV-2 variants that will appear in the future.
  • Antibody B is a fully humanized monoclonal antibody that is expressed in mammalian cells transfected with plasmids carrying its heavy chain and light chain genes. Antibody B was expressed by Chinese hamster ovarian cells CHOK1Q. The final cell bank virus safety test proved that there was no risk of contamination by exogenous factors.
  • CHOK1Q cells were resuscitated with CD02 medium and were cryopreserved after expanded culture to establish the primary cell bank.
  • Antibody B The heavy chain and light chain amino acid sequences of Antibody B are shown below. The corresponding DNA sequence are shown afterward each.
  • the plasmids carrying the target gene were transfected into the host cell by electric transfer.
  • Cells were cultured in BalanCD CHO GrowthA medium containing 4 mM Glutamine for 3 days, diluted and passaged once, and sampled and counted after 3 days of continuous nurturing.
  • the viable cell density is 7.01 ⁇ 10 5 cells/mL, and the cell viability is 97.82%.
  • Prepare for electric transfer Take 13 mL of host cell suspension and centrifuge at 200 g at room temperature for 5 min. Discard the supernatant, resuspend and mix with 500 ⁇ L EX-CELL CD CHO Fusion for later use.
  • Vero E6 cells (catalog no. CRL-1586 TM ) were cultured in DMEM (Gibco) with 10%FBS (Gibco) , 100 units/ml penicillin, and 100 ⁇ g/ml streptomycin (Gibco) at 37°C and with 5%CO 2 .
  • the SARS-CoV-2 WIV04 strain was originally isolated from a COVID-19 patient in 2019 (GISAID, accession no. EPI_ISL_402124) .
  • the SARS-CoV-2 Omicron virus was isolated from a throat swab of a patient from Hong Kong (CCPM-B-V-049-2112-18) . All processes in this study involving authentic SARS-CoV-2 were performed in a BSL-3 facility.
  • ELISA plates (corning 96well clear flat bottom, Cat no. 9018) were coated with 50 ⁇ l/well of SARS-CoV-2 RBD recombinant protein at 4 ⁇ g/ml in 0.05 M PBS, pH 9.6, 4°C overnight. Serially diluted Antibody B solution was added to each well and incubated at 37°C for 1h. Peroxidase-conjugated Mouse Anti-Human IgG Fc Antibody [HRP] , mAb (GenScript, Clone ID 50B4A9) was used as the detection antibody. The absorbance was measured at 450 nm using a microplate reader.
  • An authentic SARS-CoV-2 neutralization assay was performed to evaluate the inhibition of virus attachment by antibodies with a plaque assay in a BSL-3 laboratory (Zhao, S. et al. Identification of potent human neutralizing antibodies against SARS-CoV-2 implications for development of therapeutics and prophylactics. Nature communications 12, 4887, doi: 10.1038/s41467-021-25153-x (2021) ) .
  • Vero cells were seeded at 1.5 ⁇ 10 5 per well in 24-well culture plates overnight.
  • the antibody samples were serially diluted threefold in DMEM with 2.5%FBS.
  • An equal volume including 600 PFU/ml SARS-CoV-2 was added, and the antibody-virus mixture was incubated at 37°C for 1 h.
  • the mixture was added to a 24-well culture plate containing Vero cells.
  • Cells infected with 300 PFU/ml SARS-CoV-2 only and cells without virus were used as positive and uninfected controls, respectively.
  • the antibody-virus mixture was removed, and the medium was replenished with DMEM with 2.5%FBS plus 0.9%carboxymethyl cellulose.
  • the plates were fixed with 8%paraformaldehyde and stained with 0.5%crystal violet 3 days later.
  • Antibody B binds to conserved region in the SARS-CoV-2 S protein RBD
  • the SARS-CoV-2 S protein is the major antigenic protein and target for the development of neutralizing antibodies and vaccines (Walls et al. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281-292 e286 (2020) ) . It utilizes human angiotensin-converting enzyme II (hACE2) as its cellular receptor to enter the host through its receptor-binding domain (RBD) (Zhou P, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273 (2020) ) .
  • hACE2 human angiotensin-converting enzyme II
  • RBD receptor-binding domain
  • the region in the RBD that directly participates in the interaction between the RBD and ACE2 is termed the receptor-binding motif (RBM) (Lan J, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215-220 (2020) ; Shang J, et al. Structural basis of receptor recognition by SARS-CoV-2. Nature 581, 221-224 (2020) ; Wang Q, et al. Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. Cell 181, 894-904 e899 (2020) ) .
  • RBM receptor-binding motif
  • Antibody B retained its binding activity with EC 50 values of 22.5 ng/ml for the Delta RBD and 24.3 ng/ml for SARS-CoV-2 WT RBD, respectively, and still strongly bound to the Omicron RBD, with an EC 50 of 90.0 ng/ml ( Figure 4A) .
  • Antibody B recognizes two major regions in the RBD ( Figures 4D-4E) (Li T, et al. Potent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants. Nature communications 12, 6304 (2021) ) .
  • the nmAbs are powerful weapons to fight against infectious diseases.
  • a panel of neutralizing antibodies against SARS-CoV-2 from transgenic mice, convalescent patients and phage display have been identified and characterized.
  • these antibodies some of them have already been approved for treatment of COVID-19.
  • these mAbs had either lost or significantly decreased their protective efficacy against the two most dominant VOCs, delta and omicron (Cao Y, et al. Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature, (2021) ; Liu L, et al. Striking Antibody Evasion manifested by the Omicron Variant of SARS-CoV-2. Nature, (2021) ) .
  • S309 (VIR-7831) (Tortorici MA, et al. Science 370, 950-957 (2020) ) maintained its neutralizing activity against Delta and Omicron in a pseudovirus assay (Cao Y, et al. Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature, (2021) )
  • S309 (sotrovimab) is approved under an Emergency Use Authorization (EUA) for the treatment of mild-to-moderate COVID-19.
  • EUA Emergency Use Authorization
  • sotrovimab has not been observed in patients hospitalized due to COVID-19. Therefore, broader nmAbs against SARS-CoV-2 that yield better clinical outcomes are still highly desired.
  • Antibody B (B066) preparation was a clear, colorless to light yellow solution, which was contained in 6R vials and could be administered by nasal spraying for many times.
  • the solution in each vial was required to be administered at least 56 times (70 ⁇ L ⁇ 14 ⁇ L each time) .
  • the labeled amount was set to 6.0mL.
  • Table 5 For the composition of the preparation, see Table 5. There was no excessive feed in the preparation process.
  • hydroxypropyl methylcellulose (HPMC) hydroxypropyl methylcellulose
  • glycerin hydroxypropyl methylcellulose
  • benzalkonium chloride benzalkonium chloride
  • pH of 6.0 5 mg/ml of B066 antibody, 20mM of phosphate buffer (PB) , 4% (w/v) of trehalose (calculated by anhydrous trehalose) , 1.7% (w/v) of glycerin, 0.1% (w/v) of HPMC, 0.01% (w/v) of benzalkonium chloride, and 0.02% (w/v) of PS80, with pH of 6.0.
  • Golden hamster in-vivo challenge tests -Delta strain Antibody B coronavirus neutralizing antibodies were administered to golden hamsters (5 mg/mL, 100 ⁇ L/hamster) by nasal dripping. Delta strains (1 ⁇ 104 PFU, 100 ⁇ L in total, 50 ⁇ L/nostril) were used for animal challenge.
  • Administration was performed on Group 1 (G-1) at 30 min before challenge, and 1d and 2 d after challenge.
  • Administration was performed on Group 2 (G-2) at 3 h, 1 d and 2 d after challenge.
  • Administration was performed on Group 3 (G-3) at 1 d and 2 d after challenge.
  • Group 4 (G-4) was the control group.
  • the body weight data and the turbinate, trachea and lung tissue samples were collected.
  • the virus RNA level in the tissue samples was detected by qRT-PCR and the virus titer was determined by plaque assay.
  • Virus RNA level at 3 d after challenge, the virus RNA load Log (copies/g) in turbinate, trachea and left and right lung tissues of each group was as shown in Figure 6.
  • Virus titer at 3 d after challenge, the Log (virus titer PFU/g) in turbinate, trachea and left and right lung tissues of each group was as shown in Figure 7.
  • Golden hamster in-vivo challenge tests -Omicron strain Antibody B coronavirus neutralizing antibodies were administered to golden hamsters (100 ⁇ L/hamster) by nasal dripping. Omicron strains (1 ⁇ 104 PFU, 100 ⁇ L in total, 50 ⁇ L/nostril) were used for animal challenge. 10 mg/mL, 5 mg/mL and 2 mg/mL of Antibody B antibodies were respectively administered to G-1, G-2 and G-3 at 3 h before challenge and 1 d and 2 d after challenge. 10 mg/kg were administered to G-4 at 3 h before challenge by intraperitoneal injection. G-5 was the control group. The body weight data and the trachea and lung tissue samples were collected. The virus RNA level in the tissue samples was detected by qRT-PCR and the virus titer was determined by plaque assay.
  • Virus RNA level at 3 d after challenge, the virus RNA load Log (copies/g) in trachea and left and right lung tissues of each group is as shown in Figure 9.
  • Virus titer at 3 d after challenge, the Log (virus titer) in trachea and left and right lung tissues of each group is as shown in Figure 10.
  • Absorption The clinical route of Antibody B was nasal spraying. The mechanism of action was mainly through its retention in the nasal mucosa to neutralize the coronavirus that entered the human body through the nasal cavity.
  • Nasal dripping administration to mice Antibody B coronavirus neutralizing antibodies were administered to mice by nasal dripping.
  • 33 C57BL/6 male mice were divided into 5 groups, 6 mice/group, and the other 3 blank mice were used for the follow-up dilution of Antibody B standard curve samples without any treatment.
  • the concentration of Antibody B was 3 mg/mL, 1 mg/mL, 0.3 mg/mL, 0.1 mg/mL and 0 mg/mL respectively.
  • One dose was given by nasal dripping. Each dose included two times of nasal dripping for left and right nostrils (7.5 ⁇ L/drop each time) , 30 ⁇ L in total per dose.
  • the content of Antibody B in the nasal mucosa, blood, trachea, lung and liver tissues of mice was detected by enzyme-linked immunosorbent assay (ELISA) at 6 h and 24 h after administration.
  • ELISA enzyme-linked immunosorbent assay
  • the results show that the content of Antibody B in nasal mucosa of 3 mg/mL, 1 mg/mL, 0.3 mg/mL and 0.1 mg/mL dose groups was respectively 15.1 ⁇ g/mL, 8.26 ⁇ g/mL, 1.99 ⁇ g/mL, and 0.443 ⁇ g/mL at 6 h after administration, indicating that the content of Antibody B in nasal mucosa was positively correlated with the dose.
  • the content of Antibody B in trachea is respectively 2.7%, 4.5%, 4.5%and 15.5%of that in nasal mucosa.
  • the content of Antibody B in blood is respectively 0.28%, 0.20%, 0.36%and 0.85%of that in nasal mucosa. Only a trace amount of Antibody B is detected in 3 mg/mL and 1 mg/mL dose groups in lung and liver. It shows that Antibody B administered through nasal dripping is mainly distributed in the nasal mucosa.
  • SPECT/CT imaging of New Zealand rabbits The SPECT/CT imaging of experimental rabbits (supine position) was carried out by adopting chemically synthesized radiopharmaceuticals. The test was divided into a 131 I-antibody nasal dripping test group and a Na 131 I nasal dripping control group. Nasal dripping was carried out after intraperitoneal injection of chloral hydrate for anesthesia, respectively. The effects at 1 h, 3 h, 6 h, 12 h and 24 h after nasal dripping were collected.
  • Antibody B coronavirus neutralizing antibodies (5 mg/mL) were administered in a single dose to cynomolgus macaques by nasal spraying (3 females and 3 males) .
  • the content of Antibody B in the nasal swab collection solution of cynomolgus macaques after administration was detected by ELISA.
  • 3h-L represents the second collection of the left nostril after administration
  • 6h-L represents the third collection of the left nostril after administration
  • 6h-R represents the second collection of the right nostril after administration.
  • the drug concentration of 0 h nasal swab is the average of left and right nasal swabs.
  • the drug concentration of 6 h nasal swab is the data of the right nostril.
  • Antibody B neutralizing antibodies were administered through nasal cavities to study its pharmacokinetics and provide a reference for clinical dose, administration time and frequency. According to the understanding of different strains of coronaviruses, the transmissibility, infectivity and pathogenicity of wild-type, Delta and Omicron strains are different. Therefore, Antibody B is designed as a product with a target concentration in the nasal cavity that can achieve 100%inhibition rate against coronavirus wild type strain and Delta strain, and more than 90%inhibition rate against the Omicron strain (BA. 2.12.1) .
  • the content of Antibody B in the nasal mucosa is the highest and positively correlated with the dose.
  • the concentration in the nasal cavity can reach 15 ug/mL. This concentration can achieve 100%inhibition rate against the wild-type coronavirus strain and Delta strain, and more than 90%inhibition rate against the Omicron strain (BA. 2.12.1) .
  • the content of Antibody B in the nasal mucosa decreases to a level near the background value of the blank sample at 24 h after administration. This shows that the Antibody B antibody in the nasal mucosa of mice subjected to administration by nasal dripping within 6 h can maintain a predetermined target concentration (which can meet the setting of three doses in the daytime) .
  • Antibody B When Antibody B is administered by nasal spraying to cynomolgus macaques (5 mg/mL, 280 uL in a single dose) , the concentration reaches the highest concentration in the nasal cavity (35 ug/mL) at 15 min to 1 h after administration, then gradually decreases with time, and reaches 10 ug/mL at 3 h after administration. It is verified by the pseudovirus neutralization experiment that this concentration can achieve 100%inhibition rate against the coronavirus wild-type strain and Delta strain, and more than 95%inhibition rate against the Omicron strain (BA. 2.12.1) . At 6 h after administration, the concentration in the nasal cavity decreases to 3 ug/mL.
  • This concentration can achieve 100%inhibition rate against the coronavirus wild-type strain and Delta strain, but the inhibition rate against the Omicron strain (BA. 2.12.1) decreased to 80%. Since the goal of Antibody B is to achieve 100%inhibition against the coronavirus wild-type strain and Delta strain and 90%inhibition rate against the Omicron strain (BA. 2.12.1) , it can be preliminarily inferred from the above data that Antibody B can maintain the target concentration within 4 h after administration.
  • the SPECT/CT imaging study was carried out by using 131 I-labeled Antibody B ( 131 I-Antibody B) after nasal spraying to healthy adult rabbits.
  • the results are similar to the results of the above studies by nasal dripping to mice and nasal spraying to monkeys.
  • Antibody B is mainly distributed in the nasal cavity within 1-6h after nasal dripping administration, and a small amount is distributed in the pharynx. The concentration decreases rapidly from 6h to 24h, and then decreases to a level near the background level after 24h.
  • the distribution of the Antibody B neutralizing antibody in various tissues was studied by nasal dripping administration to mice.
  • the content of Antibody B in the trachea was 2.7%to 15.4%of the content in the nasal mucosa; the content of Antibody B in blood was 0.28%to 0.84%of the content in the nasal mucosa; in the lung and liver, only a small amount of Antibody B was detected in the high-dose group (near the lower limit of detection) . It shows that Antibody B administered through nasal dripping is mainly distributed in the nasal mucosa.
  • the content of Antibody B in the nasal mucosa and trachea decreases to a level near the background value of the blank sample, and the concentration of Antibody B in the blood, lung and liver remains at a low level without changing significantly.
  • the concentration of Antibody B in the blood is less than 1%of the nasal cavity concentration. From 1 h to 24 h after administration, the concentration of Antibody B in the blood remains at this low concentration and changes little.
  • the Antibody B neutralizing antibody is mainly distributed in the nasal mucosa, a small amount is distributed in the pharynx, and a low amount is distributed in other tissues such as liver, lung, and blood. Low-level system exposure provides a theoretical data support for the safety of Antibody B.
  • the pharmacokinetics of drug distribution in serum was studied by intravenous injection administration of Antibody B. After intravenous injection of 1 mg/kg Antibody B in mice, the half-life of serum was 16 d, and the 0-14 d AUC was 2579.2 ⁇ g ⁇ h/mL, AUC (0- ⁇ ) was 5655.7 ⁇ g ⁇ h/mL; the 0-14 d clearance CL was 0.177 mL/h/kg; the distribution volume Vd was 98 mL/kg.
  • This Investigator Initiated Trial (IIT) study was a randomized, double-blind, placebo-controlled clinical study on the safety and tolerance of the Antibody B neutralizing antibody nasal spray in 72 healthy subjects.
  • the main purpose of this study was to evaluate the safety and tolerance of the Antibody B neutralizing antibody nasal spray in healthy subjects, and the secondary purpose is to evaluate the drug concentration of the Antibody B neutralizing antibody nasal spray in serum and nasal and pharyngeal swabs of healthy subjects.
  • This trial was a randomized, double-blind, placebo-controlled clinical study. 72 healthy subjects (male and female) were included.
  • Administration dose and frequency were as follows: for the test group, Antibody B neutralizing antibody nasal spray; specification: 5 mg/mL, 5 mL/vial, 100 uL per spray; nasal administration, two times of spraying to each of left and right nostrils as one dose, 400 uL/dose; placebo group: only containing excipients; specification: 0 mg/mL, 5 mL/vial, 100 uL per spray; nasal administration, two times of spraying to each of left and right nostrils as one dose, 400 uL/dose. Only 1 dose was administered to Cohort 1; 2 doses were administered to Cohort 2 per day for 3 consecutive days; 2 doses were administered to Cohort 3 per day for 7 consecutive days.
  • the concentration of the Antibody B neutralizing antibody in the nasal cavity can be maintained at 5-11 ug/mL at 6 h after administration of the first dose. According to the in-vitro antiviral neutralizing activity study, the concentration in this range can achieve an inhibition rate of more than 99%against the coronavirus wild-type strain and Delta strain, and an inhibition rate of 80%against the Omicron strain BA. 2.
  • the second dose is administered at 6 h after administration of the first dose, and the concentration of the second dose can be maintained at 5-11 ug/mL at 6 h after administration of the second dose.
  • the concentration of the Antibody B neutralizing antibody in the nasal cavity decreases to the background level at 24 h after administration.
  • the antibody concentration in the pharynx of 72 subjects is not detected or the detected value is near the lower limit of quantitation. It is speculated that this product can be well attached to the surface of the nasal mucosa after nasal administration, and only a small amount may enter the pharynx and larynx.
  • Clinical pharmacological experiment design 72 healthy volunteers were selected for this IIT study, who were negative for coronavirus nucleic acid detection, aged 18 to 65 (critical value included) , with an appropriate gender ratio and body weight index in a range of 19.0-30.0 (critical value included) .
  • the selection of clinical dose was mainly based on the results of preclinical pharmacokinetic and pharmacodynamic studies of mice and cynomolgus macaques.
  • the Antibody B neutralizing antibody has a high binding ability to the S1 recombinant protein of the coronavirus wild-type strain S and mutant strains Alpha strain S, Beta strain S, Indian strain S, Delta strain S, Gamma strain S1, Lambda strain S1, and D614G strain.
  • Antibody B has strong pseudovirus neutralizing activity against these coronavirus strains, with IC50 of less than 10 ng/mL, and IC50 of only 1.18 ng/mL for the Delta strain. Antibody B also has super strong live virus neutralizing activity against the coronavirus wild-type strain and Beta strain live viruses, with IC50 of 6.03 ng/mL and 1.66 ng/mL respectively.
  • the live virus in-vivo challenge test shows that Antibody B has a highly effective preventive protection ability to golden hamsters infected with coronavirus Delta and Omicron strains. After nasal dripping of Antibody B with different concentrations to mice, the content of the antibody in the nasal mucosa of mice increased with the increase of the dose.
  • the nasal concentration of Antibody B in the 0.3 mg/mL 50 uL dose group at 6 h after administration was more than 1 ug/mL.
  • the concentration of the Antibody B neutralizing antibody in the nasal cavity was about 39 ⁇ g/mL. It is predicted that after nasal spraying of 140 ⁇ L of 5 mg/mL Antibody B to the human body, the concentration of Antibody B in the nasal cavity will remain much higher than the concentration required by the antibody to neutralize the coronavirus within 6 h.
  • Toxicity tests in ICR mice show that after two doses of 150 mg/kg Antibody B neutralizing antibody are injected intraperitoneally or 5 mg/mL Antibody B is administered by nasal dripping for 14 consecutive days with two doses per day and 25 ⁇ L per dose, there is no influence on general physical signs, blood routine, blood biochemistry and histopathology. After a single-dose intravenous injection of 10 mg/kg Antibody B, the blood biochemistry, blood routine and coagulation function of the cynomolgus macaques are normal.
  • the safety margin of Antibody B is respectively 300 and 20 times higher than that in mice and cynomolgus macaques. Therefore, there is sufficient safety margin for nasal spraying of 140 ⁇ L of 5mg/mL Antibody B for 14 consecutive days.
  • the mean concentration of the neutralizing antibody in the blood during the baseline period is 1503.0 ⁇ 2508.3 ng/mL
  • the mean concentration of the neutralizing antibody on the second day of the test is 1427.7 ⁇ 2433.2n g/mL
  • the mean concentration of the neutralizing antibody during the follow-up period is 1482.0 ⁇ 2015.7 ng/mL.
  • the data of the concentration of the antibody in nasal swabs after administration show a significant difference between the test group and the control group, and the concentration of the antibody in nasal swabs in the test group gradually decreases with time.
  • the data of the concentration of the neutralizing antibody in pharyngeal swabs in the test group and the control group are basically lower than the lower limit of quantitation.
  • the mean concentration of the neutralizing antibody in the blood during the baseline period is 1435.3 ⁇ 1954.5 ng/mL
  • the mean concentration of the neutralizing antibody on the fourth day of the test is 1355.9 ⁇ 1874.1 ng/mL
  • the mean concentration of the neutralizing antibody during the follow-up period is 1220.1 ⁇ 1713.3 ng/mL.
  • the mean concentration of the neutralizing antibody in nasal swabs during the baseline period is 4.28 ⁇ 1.79 ng/mL
  • the mean concentration of the neutralizing antibody in the left nose on the third day of the test period is 9323.1 ⁇ 12636 ng/mL
  • the mean of the placebo group is 5.59 ⁇ 0.97 ng/mL
  • the mean of the test group is 7940.7 ⁇ 15348 ng/mL
  • the mean concentration of the neutralizing antibody in the left and right noses of the test group is respectively 2780.5 ⁇ 5989.3 ng/mL and 6618.0 ⁇ 14222 ng/mL
  • the mean concentration of the neutralizing antibody in the left and right noses of the placebo group is respectively 4.86 ng/mL and 4.71 ⁇ 2.71 ng/mL.
  • the data of the concentration of the neutralizing antibody in pharyngeal swabs are basically lower than the lower limit of quantitation.
  • the mean concentration of the neutralization antibody in the blood during the baseline period is 2037.7 ⁇ 4676.6 ng/mL
  • the mean concentration of the neutralization antibody on the eighth day is 2025.0 ⁇ 4305.8 ng/mL
  • the mean concentration of the neutralization antibody on the tenth day is 1984.7 ⁇ 4223.9 ng/mL
  • the mean concentration of the neutralization antibody in the follow-up period is 1743.5 ⁇ 3436.0 ng/mL
  • the concentration of the antibody in blood is changed little and tends to be stable during the whole test.
  • the mean concentration of the nose swab neutralization antibody in the baseline period is 9.79 ⁇ 10.76 ng/mL; the mean concentration of the left nose neutralization antibody in the test period on the seventh day (on the day of the last administration) is 4713.5 ⁇ 6744.3 ng/mL, wherein the mean of the placebo group is 10.56 ⁇ 7.14 ng/mL, and the mean of the test group is 5418.9 ⁇ 6969.2 ng/mL; the mean concentration of the right nose neutralization antibody in the test period on the eighth day is 1272.5 ⁇ 2677.0 ng/mL, wherein the mean of the test group is 1304.9 ⁇ 2704.1 ng/mL, and the mean of the placebo group is 8.25 ng/mL; the mean concentration of the left nose neutralization antibody in the test period on the ninth day is 1496.7 ⁇ 8093.3 ng/mL, wherein the mean of the test group is 1634.5 ⁇ 8462.3 ng/mL, the mean of the placebo group is 27.54 ⁇ 38.31 ng/m
  • the nasal Antibody B concentration can reach 5-11 ug/mL.
  • the concentration in this range can reach an inhibition rate of over 99%on the coronavirus wild strain and Delta strain, and can also reach an inhibition rate of 80%on Omicron BA. 2.
  • Results of Cohort 2 and Cohort 3 show that in case of two doses of administration every day, the nasal concentration can be kept at concentration with a higher virus neutralization activity within 12 hours during administration. There is no significant drug aggregation in nasal cavities in case of continuous administration for days.
  • nose swab results show that: on the same day, the antibody concentration of the left nose is significantly lower than the antibody concentration of the right nose, and it is speculated that the difference may be caused by a case that a nose swab sample was collected from the left nose the day before the test. 14 days after the last administration of three cohorts, only a small amount of neutralization antibodies are detected from all the nose swabs, and for Cohort 3, after the last administration, it can be obviously seen that the antibody concentration in the nose is gradually decreased (72 h after the last administration, the antibody amount is basically close to the baseline level) . It can be seen that this study drug is mainly attached to the nasal cavity site and the antibody concentration is gradually reduced over time.
  • This embodiment illustrates a preclinical experimental result of an antibody B and antibody D mixture.
  • the mixture is named as A8G6; contained Antibody B and Antibody D, both obtained by sorting viral-specific memory B cells in a peripheral blood sample of a coronavirus patient in the convalescent period in a flow manner, and the sorted individual cells are subjected to antibody gene analysis; then an antibody gene sequence is cloned by a linear gene cassette; and finally, an antibody for nasal spraying is prepared by carrying out stable transfection expression on the antibody gene.
  • the Antibody B and the Antibody D are noncompetitively bound with different and complementary epitopes of a Receptor Binding Domain (RBD) of a SARS-Cov-2 spike protein, so that viruses are synergistically prevented from invading the cells through specific binding with an angiotensin-transferase 2 (ACE2) on human cells, and meanwhile, an immune complex formed by the viruses and a neutralization antibody is easier to phagocytose and eliminate by macrophage, thereby achieving the effect of inhibiting the viruses from infecting the cells.
  • RBD Receptor Binding Domain
  • ACE2 an angiotensin-transferase 2
  • the binding conditions of the Antibody D antibody with Spike proteins of a wild-type strain and multiple mutant strains are detected by Enzyme Linked Immunosorbent Assay (ELISA) : the experimental result can be found in Figure 14, the Antibody D has higher binding activities (EC50 ⁇ 500 ng/mL) with S1 recombinant proteins of multiple coronavirus mutant strains D614G and Gamma (P. 1) and S recombinant proteins of the wild-type strain, the Alpha strain (B. 1.1.7) , the Beta strain (B. 1.351) , the Kappa strain (B. 1.617) , the Delta strain (B. 1.617.2) , the Omicron BA.1 strain (BA. 1.1.529.1) , Omicron BA.
  • ELISA Enzyme Linked Immunosorbent Assay
  • BA. 1.1.529.2 BA. 4/5 (BA. 1.1.529.4 or BA.1.1.529.5)
  • the EC50 thereof respectively are 25.39 ng/mL, 19.45 ng/mL, 43.94 ng/mL, 17.82 ng/mL, 15.84 ng/mL, 254.5 ng/mL, 122.4 ng/mL, 102.5 ng/mL, 89.07 ng/mL, and 193.7 ng/mL.
  • IC50 respectively are 5.431 ng/mL, 5.574 ng/mL, 6.516 ng/mL, 6.06 ng/mL, 6.411 ng/mL, 6.246 ng/mL, 6.197 ng/mL, and 6.509 ng/mL.
  • the Antibody D has strong affinities for the spike proteins of the coronavirus wild-type strain, the Delta strain, the Omicron BA. 1 strain, the Omicron BA. 2 strain, and the Omicron BA. 4/BA. 5 strain (KD ⁇ 10 -8 M) .
  • the Antibody D partially competes with the ACE2 so as to blocking the binding of the ACE2 with the coronavirus spike (S) recombinant protein; the Antibody D does not block the binding of the ACE2 with the coronavirus Receptor Binding Domain (RBD) recombinant protein; the Antibody B completely competes with the ACE2 so as to blocking the binding of the ACE2 with the coronavirus RBD or S recombinant protein; and the A8G6 completely competes with the ACE2 so as to block the binding of the ACE2 with the coronavirus RBD or S recombinant protein.
  • S coronavirus spike
  • RBD coronavirus Receptor Binding Domain
  • Antibody B/Antibody D Fab a Antibody B/Antibody D antigen-binding fragment
  • S coronavirus mutant strain Omicron spike protein
  • RBDs receptor binding domains
  • the Antibody D and the Antibody B can be simultaneously bound to one “up” RBD
  • the Antibody D Fab identifies spatial epitopes of RBD proteins S345-352 and S440-450 by using antibody complementary determining regions (hereinafter referred to as CDRs) CDRH3, CDRL1 and CDRL3, and the Antibody B Fab identifies spatial epitopes of RBD proteins S475-480 AND s483-495 by using CDR
  • the Antibody B and the Antibody D can be simultaneously bound to the same Omicron S protein.
  • This test provides a molecular structure action mechanism and illuminates that the Antibody B/Antibody D antibody is bound with the S protein so as to block binding of the virus with the receptor ACE2 and produces the synergistic neutralization activity by different binding sites.
  • the study on the neutralization activities of the A8G6 for pseudoviruses of the coronavirus mutant strains Delta and Omicron (BA. 2, BA. 2.12.1, and BA. 5) is performed so as to determine the neutralization activities of the A8G6 coronavirus neutralization antibody against the coronavirus mutant strains Delta and Omicron (BA. 2, BA. 2.12.1, and BA. 5) .
  • the detection result shows that: the mean IC50 (mean ⁇ standard deviation) of the A8G6 coronavirus neutralization antibody for pseudoviruses of coronavirus mutant strains Delta and Omicron (B.1.1.529, BA. 2, BA. 2.12.1, and BA.
  • a Antibody B and Antibody D combined effect verification and pseudovirus neutralization experiment is performed: in order to determine the combined effect of the major active ingredients Antibody B and Antibody D in the coronavirus neutralization antibody nasal spray A8G6, a Antibody B and Antibody D combined BA. 2 coronavirus pseudovirus neutralization experiment is developed. Pseudovirus neutralization experimental data obtained after the Antibody B and the Antibody D are combined for use is analyzed by an analysis template MacSynergy II according to a Bliss independence model so as to determine a judgment standard of the combined effect in this experimental system, and the possible synergistic effect and antagonistic effect when the Antibody B and the Antibody D are combined for use are analyzed.
  • the Antibody B and Antibody D combined pseudovirus neutralization experiment result shows that the synergy volume is 263.0 ⁇ 31.1 ng/mL 2 %and is higher than a threshold (synergy volume >100 ng/mL 2 %) of the strong synergistic effect defined in MacSyerngy II, which indicates that combination of two antibodies has the strong synergistic effect; and the Antibody B and Antibody D combined pseudovirus neutralization experiment result shows that the the antagonism volume is -0.1 ⁇ 0.1 ng/mL 2 %and is within an interval range of an additive volume (-25 ng/mL 2 %to 25 ng/mL 2 %) defined by MacSyerngy II, which indicates that combination of the two antibodies has no antagonistic effect.
  • the CCK-8 cell activity detection result shows that in case of any concentration combination, the cell activity is about 100%, which indicates that when the antibodies are combined for use, no cell cytotoxicity is generated. From the above, it is shown that the combined use of both the Antibody B and Antibody D coronavirus neutralization antibodies has the strong synergistic effect without the antagonistic effect. Experimental results can be found in Table 7, Figure 21A and Figure 21B.
  • a neutralizing assay was performed to confirm the synergetic effect, using pseudoviruses and then authentic viruses.
  • Neutralization titers of antibodies were measured with a plaque reduction neutralization test (PRNT) using authentic SARS-CoV-2, Delta, and Omicron viruses. Briefly, Vero E6 cells (2.5 ⁇ 10 5 ) were seeded in each well of 24-well culture plates. The cells were incubated overnight at 37°C with 5%CO 2 . On the following day, each antibody was serially diluted 5-fold in the culture medium with the highest concentration being 100 ⁇ g/ml.
  • the diluted antibody was incubated with an equal volume including 600 PFU/ml SARS-CoV-2 at 37°C for 1 h, after which the antibody-virus mixtures were inoculated onto preseeded Vero E6 cell monolayers in 24-well plates. After 1 h of infection, the inoculum was removed, and 100 ⁇ l of overlay medium (DMEM supplemented with 0.8%methylcellulose, 2%FBS, and 1%P/S) was added to each well. After incubating the plates at 37°C for 96 h, the plates were fixed with 8%paraformaldehyde and stained with 0.5%crystal violet. The plaques in each well were counted and normalized to the non-antibody-treated controls to calculate relative infectivity. PRNT 50 values were calculated in GraphPad Prism 9.
  • the individual NAbs 55A8 (antibody D) and 58G6 (antibody B) could neutralize pseudotyped SARS-CoV-2 and the Delta, Omicron BA. 1 and BA. 2 variants, and Omicron BA.1+L452R ( Figure 22A) as well as other SARS-CoV-2 variants (data not shown) .
  • the combination of 58G6 and 55A8 showed clear synergetic effects against pseudotyped Omicron BA.1, BA. 1+L452R, and BA. 2 ( Figure 22A) .
  • the combination treatment showed synergetic effects against authentic Omicron BA. 1 ( Figure 22B) .
  • the IC50 for Omicron BA.1 (2.81 ng/mL) was much lower than the IC50 values for all other currently known NAbs.
  • the IC50 values of S30917 and LY-CoV140418 are 191.1 and 17.30 ng/mL, respectively. Therefore, the combination of 58G6 and 55A8 exhibited an enhanced neutralizing potency and breadth against SARS-CoV-2.
  • the Antibody B Antibody D ratio was set as 1: 4 by researching the pseudovirus neutralization activities of two antibodies of the A8G6 in case of different proportioning preparations for the Omicron BA. 4/5 and Delta strains, so that the neutralization activity for the current popular strain BA. 4/5 is continuously maintained, and the neutralization activity for the Delta-like strains can be maintained.
  • An A8G6 combined ratio determination-pseudovirus neutralization experiment is performed: in order to determine the combined ratio of two major active ingredients Antibody B and Antibody D in the A8G6 coronavirus neutralization antibody combined nasal spray, an Omicron (BA. 2, BA. 2.12.1, and BA. 5) and Delta coronavirus pseudovirus neutralization experiment is developed so as to determine the combined ratio of two antibodies in the A8G6.
  • the pseudovirus neutralization experiment results show that when the coronavirus neutralization antibodies Antibody D and Antibody B are in use separately, IC50 for the coronavirus mutant strain Omicron (BA. 2) pseudovirus respectively is 20.4 ng/mL and 1951 ng/mL, which indicates that the neutralization activity of the Antibody D for the BA.
  • the neutralization activities of the single antibodies and the combined antibodies are further researched, and the neutralization activity of the Antibody B for the BA.
  • 2.12.1 strain is lower than that of the Antibody D, so two combinations with high Antibody D proportions are selected for research, i.e., Antibody D+Antibody B (3: 2 and 4: 1) .
  • Results show that the IC50 for the BA.
  • 2.12.1 pseudovirus in cases of separate use of the Antibody D, separate use of the Antibody B and combined use of the Antibody D and the Antibody B (3: 2 and 4: 1) respectively is 49.9 ng/mL, 811.3 ng/mL, 49.5 ng/mL, and 43.6 ng/mL, which indicates that the combined antibody can maintain the similar neutralization activity for the BA.
  • 2.12.1 strain with that in case of separate use of the Antibody D (Table 9; Figure 23B) .
  • the BA. 5 strain has become a main epidemic strain, the neutralization activity of the combined antibody for the BA. 5 strain is studied, and results show that the mean IC50 (mean ⁇ standard difference) for the BA. 5 pseudovirus in cases of separate use of the Antibody D, separate use of the Antibody B and combined use of the Antibody D and the Antibody B (3: 2, 2: 1 and 4: 1) respectively is 221.8 ⁇ 42.1 ng/mL, > 100,000 ng/mL, 353.1 ⁇ 42.5 ng/mL, 408.5 ⁇ 92.3 ng/mL and 229.0 ⁇ 73.5 ng/mL (Table 10; Figure 23C) .
  • results show that the mean IC50 for the Delta pseudovirus in cases of separate use of the Antibody D, separate use of the Antibody B and combined use of the Antibody D and the Antibody B (3: 2, 2: 1 and 4: 1) respectively is 185.7 ⁇ 36.4 ng/mL, 0.89 ⁇ 0.06 ng/mL, 2.25 ⁇ 0.41 ng/mL, 2.14 ⁇ 0.79 ng/mL and 1.12 ⁇ 0.68 ng/mL (Table 11; Figure 23D) .
  • the Antibody D Antibody B ratio in the A8G6 preparation is determined as 4: 1.
  • In-vitro neutralization activities of the A8G6 antibody for coronavirus wild strain and Delta and Omicron strain live viruses by a plaque reduction neutralization test, in-vitro neutralization activities of the A8G6 and Antibody D for the wild-type, Delta and Omicron strain live viruses are detected. Results show that: IC50 of the Antibody D for the wild-type, Delta and Omicron live viruses respectively is 3.03 ng/mL, 26.4 ng/mL and 8.24 ng/mL; and IC50 of the A8G6 for the wild-type, Delta and Omicron live viruses respectively is 1.28 ng/mL, 4.37 ng/mL, and 2.81 ng/mL. respectively. It is proved that the A8G6 and the Antibody D have high neutralization activities for the wild-type, Delta and Omicron live viruses. Test results can be found in Figure 24.
  • Figure 25A-25C show the results of the protective efficacy of 55A8, 58G6 and 55A8/58G6 cocktails against Omicron in the hamster model.
  • Syrian golden hamsters challenged with 10 4 PFU of Omicron were treated with 58G6, 55A8 or the two-antibody cocktail at 1 h pre-infection and 24 and 48 h post-infection.
  • the turbinates, trachea and lungs were harvested on day 3 post-treatment and analyzed by viral titering (by PFU/g and qRT–PCR) . All the treatments led to robust viral clearance ( Figures 25B and 25C) .
  • the virus detected by measurement of the RNA copy numbers and PFU in the upper respiratory tract (turbinate and trachea) and lower respiratory tract (lungs) , was completely or almost completely inhibited in the combination treatment group, while viral RNA or live virus could be detected in the other two groups that received 58G6 or 55A8 monotherapy. Taken together, these results showed that the combination of 58G6 and 55A8 could efficiently suppress the Omicron variant in vivo through a synergetic effect.
  • hamsters treated with buffer had significant viral RNA copy numbers and viral titers in the turbinates and lungs.
  • Pretreatment with 55A8 or the 2-cocktail at 1 h pre-infection followed by two-dose administration lowered the Omicron viral RNA copy number in the lungs by 4–5 logs. More notably, in the turbinates, pretreatment with the 55A8 and 58G6 mixture 1 h pre-infection followed by two-dose administration (Group 3) reduced the Omicron viral RNA copy number by six orders of magnitude, producing a copy number that was significantly lower than that in Group 1 ( Figure 26B) .
  • Virus RNA level the virus RNA loads Log10 (copies/g) in the trachea and the left and right lung tissues of the golden hamster in the 10 mg/kg nasal dripping group respectively are 3.11 ⁇ 1.28, 2.10 ⁇ 0.78, and 2.24 ⁇ 0.62; the virus RNA loads Log10 (copies/g) in the trachea and the left and right lung tissues of the golden hamster in the 4 mg/kg nasal dripping group respectively are 3.67 ⁇ 1.43, 3.00 ⁇ 0.82, and 2.75 ⁇ 0.57; the virus RNA loads Log10 (copies/g) in the trachea and the left and right lung tissues of the golden hamster in the 2 mg/kg nasal dripping group respectively are is 4.55 ⁇ 1.35, 3.83 ⁇ 1.54, and 3.64 ⁇ 1.45; the virus RNA loads Log10 (copies/g) in the trachea and the left and right lung tissues of the golden hamster in the 1 mg/kg nasal dripping group respectively are 3.74 ⁇ 1.67, 3.02 ⁇ 0.45, and 2.47 ⁇ 0.84
  • the virus RNA loads in the lung tissues of the golden hamsters in the nasal dripping administration groups are reduced by 3.56-5.86 Log10 values and are significantly lower than that in the control group.
  • the virus RNA loads in the tracheae of the golden hamsters in the nasal dripping administration groups are reduced by 2.51-3.95 Log10 values relative to the control group. It is obtained by statistical analysis that the P values of the experimental groups and the control group are less than 0.01 and have significant differences.
  • the virus RNA loads in the trachea and the left and right lung tissues of the control group and the virus RNA loads in the tracheae and the left and right lung tissues of different dose administration groups are statistically analyzed; results show that data is in conformity with normal distribution and variance homogeneity analysis; and by using two-way analysis of variance (2-Way ANOVA) , the P values are less than 0.01 and have significant statistical differences. See Figure 27B.
  • Virus titer after 3 days of challenge, all the golden hamsters were dissected, the tracheae and the left and right lung tissues were respectively taken to perform virus titer measurement.
  • the arithmetic means of the Log10 (PFU/g) virus titers in the trachea and the left and right lung tissues of the golden hamster in the control group respectively are 4.96 ⁇ 0.79, 6.24 ⁇ 1.04, and 6.25 ⁇ 0.55;
  • the Log10 (PFU/g) virus titers in the tracheae and the left and right lung tissues in the 10 mg/kg, 4mg/kg and 1mg/kg nasal dripping groups are located at the detection limit (1.30) ;
  • the means of the Log10 (PFU/g) virus titers in the trachea and the left and right lung tissues in the 2 mg/kg nasal dripping group respectively are 1.78 ⁇ 1.18, 1.71 ⁇ 1.00, and 1.82 ⁇ 1.27; and compared to the control group, the virus
  • the virus titers in the trachea and the left and right lung tissues of the control group and the virus titers in the tracheae and the left and right lung tissues of different dose administration groups are statistically analyzed, and results show that data is in conformity with variance homogeneity analysis; and normal distribution of the virus titer data is detected by the Kolmogorov-Smirnov test method, and results show that data of the experimental groups is not in conformity with normal distribution near or below the detection limit, and data of the control group is in conformity with normal distribution.
  • 2-Way ANOVA two-way analysis of variance
  • Lung tissue pathology the lung pathological damage of the Omicron infected golden hamsters is not obvious, and the administration groups and the control group are compared and there is no significant differences.
  • Coronavirus mutant strain B. 1.1.7 and B. 1.351 were used as cell models for researching whether the Antibody D mediates the Antibody-Dependent Enhancement (ADE) effect of the coronavirus infection, and results indicate that the Antibody D has no ADE effect.
  • ADE Antibody-Dependent Enhancement
  • the Antibody B it was researched whether the Antibody B mediates the ADE effect of the coronavirus infection by using (i) coronavirus wild-type, D614G and D614G-based multiple mutant strain pseudoviruses and coronavirus mutant strains B.
  • Pharmacodynamics studies with respect to A8G6 neutralizing antibodies are mainly carried out with C57BL/6 mice and rhesus macaque for absorption and distribution tests.
  • studies on A8G6 in blood samples and tissue samples of mice and nasal swab samples of cynomolgus macaques are based on enzyme-linked immunosorbent assay (ELISA) .
  • the A8G6 coronavirus neutralizing antibody was administered by intravenous injection (1 mg/kg, 12 C57BL/6 mice, half male and half female) .
  • the level of the A8G6 antibody in the blood was tested by ELISA, and the results showed that at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 168 h (7 d) , 336 h (14 d) , 504 h (21 d) and 672 h (28 d) after administration, the means of the A8G6 antibody in the blood of mice were 26, 453 ⁇ 2, 730 ng/mL, 24, 100 ⁇ 3, 023 ng/mL, 19,814 ⁇ 2, 532 ng/mL, 15, 627 ⁇ 2, 245 ng/mL, 13, 133 ⁇ 831 ng/mL, 11, 573 ⁇ 1, 305 ng/mL, 9,098 ⁇ 1, 094 ng/mL, 8, 808
  • the level of A8G6 in nasal mucosa, blood, trachea, lung, and liver tissue of mice was tested by ELISA at 0.25 h, 1 h, 4 h, 8 h, and 24 h after the administration.
  • the results showed that at 0.25 h, 1 h, 4 h, 8 h, and 24 h after the administration, the geometric means of A8G6 in serum were 13.7 ng/mL, 15.2 ng/mL, 23.4 ng/mL, 37.7 ng/mL and 26.1 ng/mL, respectively, and the statistical AUC 0-24h in serum was 0.809 ⁇ g ⁇ h/mL; at 0.25 h, 1 h, 4 h, and 8 h after the administration, the levels of A8G6 in nasal mucosa were 55737 ng/ml, 43710 ng/ml, 3083 ng/ml, and 1239 ng/ml, respectively; at 24 h after the administration, the level of A
  • the levels of A8G6 in the blood was 0.02%, 0.03%, 0.76%, and 3.04%, respectively, and the levels of A8G6 in the trachea were 0.46%, 0.77%, 2.69%, and 7.07%of the antibody level in the nasal mucosa, respectively. It is indicated that by nasal dripping administration, the A8G6 antibody is predominantly distributed in the nasal mucosa and maintained at a high concentration for at least 8 h. Less A8G6 is distributed in the trachea, and the distribution of A8G6 in the lungs, liver and blood is extremely low. More details are shown in Figures 28A-28C.
  • the mean concentration was 50096 ⁇ 10263 ng/mL (36051-67343 ng/mL) and then slowly fluctuated down.
  • the nasal swab drug concentration was slightly reversed and repeated because the data collected at 2 h, 4 h, 6 h after the administration were from different groups treated on different dates.
  • the drug concentrations in nasal swabs was still maintained at a high level with a mean value of 7064 ⁇ 7625 ng/mL (357-20067 ng/mL) which decreased to about 14.1% (7064 vs 50096 ng/mL) of the mean value at 0.25 h after the administration.
  • the drug concentration was consistently maintained at a high level in the nasal swab samples after administration by nasal spray with three doses per day, with significant difference among animal individuals.
  • the drug concentration in nasal swabs reached the maximum with a mean value of 90870 ⁇ 37047 ng/mL (63052-160327 ng/mL) .
  • the nasal swab drug concentration was slightly reversed and repeated because the data collected at 2 h, 4 h, 6 h after the administration were from different groups treated on different dates.
  • the drug concentrations in nasal swabs was still maintained at a high level with a mean value of 17882 ⁇ 22343 ng/mL (4058-59077 ng/mL) which decreased to about 19.7% (17882 vs 90870 ng/mL) of the mean value at 0.25 h after the administration.
  • the peak drug concentrations Cmax at the administration site were 50096 and 90870 ng/mL, respectively, both reached the peaks at Tmax of 0.25 h; the half lives t 1/2 of the drug were 14.3 h and 12.4 h, respectively; AUC 0- 24h was 380994 ⁇ ng/mL and 872992 ⁇ ng/mL, respectively; AUC 0- ⁇ was 526542 and 1193712 ⁇ ng/mL, respectively; MRT 0-24h of the drug were 9.20 h and 8.59 h, respectively.
  • the Cmax, AUC 0-24h and AUC 0- ⁇ after the administration with three doses per day were 1.81, 2.29 and 2.27 times higher than those after the administration with one dose per day, respectively.
  • the administration with one dose and the administration with three doses were similar in terms of Tmax, t 1/2 and MRT 0-24h .
  • the PK feature of the drug is not changed after the administration with three doses per day.
  • the A8G6 neutralizing antibody is mainly distributed in the nasal mucosa, with low level in other tissues such as liver, lung, and blood. It is indicated that the concentration of the A8G6 antibody in nasal mucosa can maintain a predetermined target concentration which can meet the setting of the administration with three doses on the day, at 6-8 h after administration by nasal dripping.
  • the coronavirus can multiply in the lungs and other respiratory systems of golden hamsters; 2) infected golden hamsters lose weight, showing pathogenicity to the animals; and 3) the lungs of golden hamsters also show the same lesions seen in lungs of COVID-19 patients, so golden hamster was used as an animal model for in vivo live virus challenge.
  • the golden hamsters were administered with different doses of A8G6 by nasal dripping before/after inoculation with the Omicron strain, thereby evaluating the administration of A8G6 by nasal dripping in preventing and treating Omicron virus infection in golden hamsters.
  • Non-clinical pharmacodynamics studies found that A8G6 can effectively bind RBD proteins of different coronavirus strains, effectively neutralize different coronavirus pseudovirus strains Delta, Omicron (BA. 2, BA. 2.12.1, and BA. 5) , and live virus experiments also showed the superior virus neutralizing potency of A8G6.
  • the A8G6 coronavirus neutralizing antibody nasal spray was administered to rhesus macaques for 7 days on every other day, 0.7 mg/dose, where one dose was administered on D1, one dose was administered on D3, three doses were administered on D5 at an interval of 4 h ⁇ 5 min, and three doses were administered on D7 at an interval of 4 h ⁇ 5 min.
  • Nasal swab samples were taken during the administration period.
  • the peak drug concentrations Cmax at the administration site were 50096 and 90870 ng/mL, respectively, both reached the peaks at Tmax of 0.25 h; the half lives t 1/2 of the drug were 14.3 h and 12.4 h, respectively; AUC 0-24h was 380994 ⁇ ng/mL and 872992 ⁇ ng/mL, respectively; AUC 0- ⁇ was 526542 and 1193712 ng/mL, respectively; MRT 0-24h of the drug was 9.20 h and 8.59 h, respectively.
  • the Cmax, AUC 0-24h and AUC 0- ⁇ after the administration with three doses per day were 1.81, 2.29 and 2.27 times higher than those after the administration with one dose per day, respectively.
  • the administration with one dose and the administration with three doses were similar in terms of Tmax, t 1/2 and MRT 0-24h . It is indicated that the PK feature of the drug is not changed after the administration with three doses per day.
  • the concentration of A8G6 in the serum was extremely low, indicating that the AUC of A8G6 nasal spray administration system is low. It is therefore contemplated that the dosage of 5 mg/mL is selected as a clinically planned dosage, which can not only effectively prevent the current epidemic strains, but also greatly reduce the risk of losing neutralizing activity against future variants. In a toxicological experiment performed at this concentration, the animals in all groups did not exhibit abnormal alterations related to the drug.
  • Administration frequency the results of non-clinical pharmacodynamics and pharmacokinetics tests show that A8G6 coronavirus neutralizing antibody nasal spray can stay in the nasal cavity for a certain period of time after nasal spray administration, and maintain a high concentration. Therefore, clinically, 3 doses of nasal spray per day can be taken as needed.
  • the time of continuous administration can be considered to be not more than 14 days.
  • Antibody B and Antibody D as the two active components of the coronavirus neutralizing antibody A8G6, were screened from the peripheral blood lymphocytes of convalescent patients with coronavirus pneumonia and 209 coronavirus-specific antibodies were isolated from them.
  • the screened Antibody B is a super antibody that has a strong and effective neutralizing effect on the popular British strains, Indian strains, South African strains and Indian Delta strains, and has high affinity, high neutralizing potency, and broad-spectrum mutant activity against the coronavirus, and supports superior-activity antibody structure analysis.
  • Antibody D is a super antibody with high affinity and high neutralizing potency against the coronavirus Omicron (BA. 1 and BA. 2) strains.
  • the binding regions of Antibody B and Antibody D to RBD do not overlap with each other, and there is no competitive relationship between Antibody B and Antibody D.
  • Antibody B and Antibody D have a certain synergistic effect on each coronavirus strain.
  • the cocktail therapy with two Antibody B and Antibody D synergistic antibodies can be effective in broadly neutralizing SARS-CoV-2 and its variants, including Omicron strains BA. 1 and BA. 2.
  • the A8G6 antibody is one of the best in terms of affinity and neutralizing potency against the coronavirus.
  • the Antibody B neutralizing antibody and the Antibody D neutralizing antibody are the active components in A8G6 coronavirus neutralizing antibody nasal spray.
  • the Antibody B neutralizing antibody and the Antibody D neutralizing antibody were isolated and purified from the culture supernatant of stably expressing cell lines.
  • the clinical preparation of A8G6 contains a total antibody level of 5 mg/mL, where the mass ratio of the Antibody D neutralizing antibody to the Antibody B neutralizing antibody is 4: 1. Excipients in the clinical preparation can be found in Table 12 below:
  • Table 12 Overview of excipients in the A8G6 neutralizing antibody nasal spray
  • the label volume was set at 4.5 mL to ensure that the nasal spray can be sprayed at least 42 times in total.
  • Validation of biological analysis methods Two methods were developed to test the concentrations of A8G6 neutralizing antibody in human serum and nasal swabs.
  • the ELISA was performed to test the total anti-RBD antibody concentration. Since most of the subjects have been vaccinated against coronavirus, there are high and variable concentrations of anti-RBD baseline antibodies in the serum, so the first method is not suitable for testing the concentration of A8G6 in serum samples. Because the concentration of the anti-RBD baseline antibodies in the nasal cavity is very low, the first approach is more suitable for testing the concentration of A8G6 in the nasal cavity.
  • the second method is also developed and can specifically test the concentration of Antibody B in A8G6. This method is used to test the concentration of Antibody B in serum to estimate the concentration of A8G6.
  • the second Antibody B-specific assay was used to test serum samples which were samples mainly from Cohorts 3 and 4 after multiple-day administration, the results showed that the concentrations of Antibody B in all samples were below the lower limit of detection (0.5 ng/mL) , indicating that A8G6 enters the blood at an extremely low level.
  • a total of 1032 nasal swab samples were provided in this IIT study and the samples met the acceptance criteria.
  • the test results showed that the baseline antibody concentration in the nasal cavities of the subjects was less than 10 ng/mL, which was not affected by the vaccination status of the coronavirus vaccine. After administration of the A8G6 antibody by nasal spray, the antibody concentration in the nasal cavity increased significantly.
  • the results indicate that antibodies produced by vaccination itself have a low concentration in the nasal cavity, and the current analysis method can effectively test the concentration of A8G6 in the nasal cavity.
  • the IIT study is a randomized, double-blind, placebo-parallel controlled clinical study for safety, tolerance, and pharmacokinetics of the A8G6 neutralization antibody nasal spray in 108 healthy subjects (male or female) .
  • the primary objective thereof is to evaluate the safety and tolerance of the A8G6 neutralization antibody nasal spray in healthy subjects, and the secondary objective thereof is to evaluate drug concentrations in serums and by nasal swab testing of the A8G6 neutralization antibody nasal spray in healthy subjects.
  • Cohort 1 is subjected to a safety check at Day 2 and 3 ⁇ 1 day after final administration; for Cohort 2, at Day 4 and 3 ⁇ 1 day after final administration; for Cohort 3, at Day 8 and 3 ⁇ 1 day after final administration; and for Cohort 4, at Day17 and 7 ⁇ 2 day after final administration.
  • Administration dosages and frequencies are as following: a test group: the A8G6 neutralization antibody nasal spray; specification: 5 mg/mL, 6 mL/vial; intranasal administration, the nasal spray is sprayed once (or twice) in each of left and right nostrils as a single dosage, 140 ⁇ L (or 280 ⁇ L) /dosage; and a placebo group: only an excipient; specification: 0 mg/mL, 6 mL/vial, intranasal administration, the excipient is sprayed once (or twice) in each of left and right nostrils as a single dosage, 140 ⁇ L (or 280 ⁇ L) /dosage.
  • Dosages administered to different cohorts are as follows:
  • Cohort 1a (12 subjects) : intranasal administration, once for each of left and right nostrils as a single dosage, 140 ⁇ L/dosage, 1 dosage per day for a total of 1 day;
  • Cohort 1b (12 subjects) : intranasal administration, once for each of left and right nostrils as a single dosage, 140 ⁇ L/dosage, 2 dosage per day with a total of 1 day;
  • Cohort 1c (12 subjects) intranasal administration, once for each of left and right nostrils and repeating spaying once for each of left and right nostrils as a single dosage, 280 ⁇ L/dosage, 1 dosage per day with a total of 1 day;
  • Cohort 1d (12 subjects) : intranasal administration, once for each of left and right nostrils as a single dosage, 140 ⁇ L/dosage, 4 dosages per day at an interval of 4 h for a total of 1 day; and
  • Cohort 2 (12 subjects)
  • the A8G6 neutralization antibody concentration in the nasal cavity can be maintained above Omicron BA. 4/5 neutralization concentration IC90 (5 ug/mL) 4-8 h after administration of 1-2 dosages per day, but not completely above 5 ug/mL at 12 h after administration.
  • the Omicron BA.4/5 neutralization concentration IC90 (5 ug/mL) is maintained at 4-12 h after administration for 4 dosages per day.
  • the mean concentration after 12 h, 13-22 ug/mL is much higher than IC90, 5 ug/mL, which could be drug accumulation in the nasal cavity after multiple administration in a day. From the results, it can be seen that single-dosage administration can achieve a target concentration coverage for 8 h.
  • a BA. 4/5 pseudovirus neutralization experimental study is performed for nasal swab samples of A8G6 IIT. From the inhibition rates, nasal swab samples are collected at 4h after administration after 4 dosages per day, and could achieve an inhibition rate more than 90%for BA. 4/5 pseudovirus after 1: 10 dilution. The inhibition rates basically correspond to A8G6 concentrations in the nasal cavity. Inhibition rates for BA. 4/5 pseudovirus of some samples are above 80%at 8 h, 12 h and 24 h after nasal spray. The pseudovirus neutralization experiment confirms that A8G6 can maintain the useful effect against coronavirus variant BA. 4/5 in the nasal cavity for a longer time.
  • Blood drug concentrations of all 108 subjects in the study are not increased significantly after administration, while the Antibody B coronavirus antibody concentrations in blood in subjects in Cohorts 3 and 4 are basically below the lower detection limit before and after administration.
  • the administration route for test drugs is nasal spray, it is presumed that a main component thereof, the A8G6 coronavirus neutralization antibody, and components thereof rarely enters or do not enter the bloodstream.
  • the pharmacokinetic study after nasal administration provides a basis for further optimizing a nasal administration frequency and an dosing interval, safety data in human bodies and next phase I clinical trials.
  • Clinical pharmacology experimental design In the IIT study, 108 healthy volunteers, aged 18-65 years (including critical values) , were selected as healthy subjects, with a mean height of 164.16 ⁇ 7.89 cm, a mean weight of 58.10 ⁇ 11.13 kg and a mean BMI of 21.42 ⁇ 2.76 kg/m2. The sex ratio is appropriate. Clinical dosages are selected based primarily on results of pharmacogenetic and pharmacodynamic studies in the previous example on preclinical mice, cynomolgus macaques and rhesus macaque.
  • the combination nasal spray of the A8G6 coronavirus neutralization antibody has 2 active ingredients, a Antibody B neutralization antibody and a Antibody D neutralization antibody.
  • Antibody B and Antibody D have high binding and neutralizing abilities with wild strain S and mutant strains Alpha, Beta, Indian, Delta, Gamma, Kappa, Lambda, and D614G of the coronavirus.
  • the pseudo-viral neutralization experimental data after combination administration of Antibody B and Antibody D are analyzed by analyzing template MacSynergy II, to determine a standard of a combined effect thereof under the experimental system according to a Bliss independence model, and possible synergistic and antagonistic effects of the combination of the two drugs are analyzed.
  • Results show that combination of two coronavirus neutralizing antibodies, Antibody B and Antibody D, has a strong synergistic effect without antagonistic effect.
  • the mean IC50 values (a mean ⁇ standard deviation) of coronavirus neutralization antibody, A8G6, against coronavirus mutant strains Delta, Omicron (B. 1.1.529, BA. 2, BA. 2.12.1 and BA.
  • pseudoviruses are 6.4 ⁇ 1.0 ng/mL, 13.3 ⁇ 0.4 ng/mL, 13.2 ⁇ 4.9 ng/mL, 36.3 ⁇ 6.1 ng/mL and 407.6 ⁇ 92.1 ng/mL, respectively, indicating that A8G6 can effectively neutralize the above coronavirus mutant strains.
  • IC50 values of A8G6 against wild-type, Delta and Omicron (BA. 1) live viruses are 1.28 ng/mL, 4.37 ng/mL and 2.81 ng/mL, respectively.
  • the antibody combination shows very good synergistic effects.
  • the pharmacokinetics in rhesus macaque after intranasal administration was studied by using a higher one of the two candidate concentrations, 5 mg/mL, to provide a basis for clinical administration concentrations and dosages.
  • the rhesus macaque was administered with A8G6 by nasal spray, 1 dosage of 140 uL, 5 mg/mL (0.7 mg/dosage) , and the nasal swab testing concentration is 11457 ⁇ 14054 ng/ml 8-24 h after administration, twice as high as the antibody concentration required to neutralize the coronavirus Omicron BA. 4/5 IC90.
  • the pharmacokinetic study result further confirms that 5 mg/mL is a feasible clinical dosing concentration.
  • the toxicological test result shows that there is no toxic reaction and death after administration to mice via nasal dripping for 2 weeks in each administration group with 5 mg/mL Antibody D, 10 mg/mL Antibody D, 20 mg/mL Antibody D and 5 mg/mL A8G6 and 10 mg/mL A8G6; there is no toxic reaction and death for cynomolgus macaques administered with Antibody D via intravenous injection at 1 mg/kg; there is no toxic reaction and death for cynomolgus macaques administered via nasal spray with A8G6 ( (2.8+2.8) mg/dosage) and Antibody D (2.8 mg/dosage) groups for 2 weeks; and there is no toxic reaction and death for rhesus macaque in the Antibody D group (24 mg/mL) , combined low-dose group (2 mg/mL) , combined medium-dose group (10 mg/m
  • the final total antibody concentration of the combined formulation is determined as 5 mg/ml.
  • an A8G6 content is 0.7 mg/dosage (140 ⁇ L, 5 mg/mL A8G6) , and the highest dosage is four dosages per day for 14 days, a cumulative total of 39.2 mg A8G6 (7.84 mg Antibody B + 31.36 mg Antibody D) , or about 0.65 mg/kg (with Antibody B 0.16 mg/kg, and Antibody D 0.49 mg/mL) .
  • the amount of the Antibody B antibody entering the bloodstream is less than 1/100.
  • the mean blood neutralization antibody concentration is 2075.9 ⁇ 3048.7 ng/mL during the baseline period, 2406.4 ⁇ 3740.5 ng/mL at the second day of the trial, and 2436.3 ⁇ 3656.4 ng/mL during the follow-up period.
  • antibody concentrations are varied widely among subjects, throughout the trial, the blood antibody concentrations are not varied much and tended to be stable.
  • the antibody concentrations by nasal swab testing after administration are significantly different between the test groups and the control group, and the antibody concentrations by nasal swab testing of the test group is decreased gradually over time after administration.
  • the mean value of the neutralization antibody concentrations by nasal swab testing is 3.54 ⁇ 1.15 ng/mL during the baseline period; the mean neutralization antibody concentration in left nostril is 54534 ⁇ 22696 ng/mL at 15 min after administration on D1; the mean neutralization antibody concentration in left nostril is 65062 ⁇ 36064 ng/mL at 1 h after administration on D1; the mean neutralization antibody concentration in right nostril is 29131 ⁇ 21036 ng/mL at 4 h after administration on D1; the mean neutralization antibody concentration in right nostril is 14227 ⁇ 8486.2 ng/mL at 8 h after administration on D1; the mean neutralization antibody concentrations in left and right nostrils are 984.55 ⁇ 1539.9 ng/mL and 1734.5 ⁇ 2596.3, respectively at 24 h after administration on D1; and during the follow-up period, the mean neutralization antibody concentrations in left and right nostrils are 138.52 ⁇ 178.43 ng/mL and 768.51 ⁇ 18
  • the mean blood neutralization antibody concentration is 833.90 ⁇ 1283.6 ng/mL during the baseline period, 925.47 ⁇ 1440.7 ng/mL at the second day of the trial, and 970.30 ⁇ 1584.5 ng/mL during the follow-up period.
  • antibody concentrations are varied widely among subjects, throughout the trial, the antibody concentrations in the blood are not varied much and tended to be stable.
  • the mean value of the neutralization antibody concentrations by nasal swab testing is 23.12 ng/mL during the baseline period; during the trial period, the mean neutralization antibody concentration in left nostril is 29008 ⁇ 24144 ng/mL at 15 min before administration of a second dosage on D1; the mean neutralization antibody concentration in left nostril is 149822 ⁇ 97301 ng/mL at 1 h after administration of the second dosage on D1; the mean neutralization antibody concentration in right nostril is 29823 ⁇ 25443 ng/mL at 4 h after administration of the second dosage on D1; the mean neutralization antibody concentration in right nostril is 40142 ⁇ 70821 ng/mL at 8 h after administration of the second dosage on D1; the mean neutralization antibody concentrations in left and right nostrils are 1282.5 ⁇ 2326.4 ng/mL and 524.89 ⁇ 766.92, respectively at 24 h after administration of the first dosage; and during the follow-up period, the mean neutralization antibody concentrations in left and right nostrils are
  • the mean blood neutralization antibody concentration is 642.78 ⁇ 745.23 ng/mL during the baseline period, 594.53 ⁇ 737.58 ng/mL at the second day of the trial, and 574.35 ⁇ 642.12 ng/mL during the follow-up period.
  • antibody concentrations are varied widely among subjects, throughout the trial, the antibody concentrations in the blood are not varied much and tended to be stable.
  • the mean value of the neutralization antibody concentrations by nasal swab testing is 7.34 ng/mL during the baseline period; during the trial period, the mean neutralization antibody concentration in left nostril is 9388.5 ⁇ 5591.2 ng/mL at 15 min after administration on D1; the mean neutralization antibody concentration in left nostril is 5857.5 ⁇ 4232.6 ng/mL at 1 h after administration on D1; the mean neutralization antibody concentration in right nostril is 5578.1 ⁇ 3683.3 ng/mL at 4 h after administration on D1; the mean neutralization antibody concentration in right nostril is 2763.2 ⁇ 2986.2 ng/mL at 8 h after administration on D1; the mean neutralization antibody concentrations in left and right nostrils are 355.59 ⁇ 501.31 ng/mL and 373.58 ⁇ 682.39 ng/mL, respectively at 24 h after administration; and during the follow-up period, the mean neutralization antibody concentrations in left and right nostrils are 4.94 ⁇ 4.58 ng/mL and 7.81 ⁇ 7.1
  • the mean blood neutralization antibody concentration is 790.09 ⁇ 852.05 ng/mL during the baseline period, 681.38 ⁇ 751.58 ng/mL at the second day of the trial, and 669.56 ⁇ 723.34 ng/mL during the follow-up period.
  • antibody concentrations are varied widely among subjects, throughout the trial, the antibody concentrations in the blood are not varied much and tended to be stable.
  • the mean value of the neutralization antibody concentrations by nasal swab testing is 4.63 ⁇ 4.01 ng/mL during the baseline period; the mean neutralization antibody concentration in left nostril is 41901 ⁇ 42219 ng/mL at 15 min before administration of a fourth dosage on D1; the mean neutralization antibody concentration in left nostril is 85004 ⁇ 49356 ng/mL at 1 h after administration of a fourth dosage on D1; the mean neutralization antibody concentration in right nostril is 115448 ⁇ 39702 ng/mL at 4 h after administration of a fourth dosage on D1; the mean neutralization antibody concentration in right nostril is 22184 ⁇ 19545 ng/mL at 8 h after administration of a fourth dosage on D1; the mean neutralization antibody concentrations in left and right nostrils are 22894 ⁇ 29619 ng/mL and 13242 ⁇ 13701 ng/mL, respectively at 24 h after administration of a fourth dosage on D1; and during the follow-up period, the mean neutralization antibody concentrations
  • the mean blood neutralization antibody concentration is 492.48 ⁇ 725.80 ng/mL during the baseline period, 479.64 ⁇ 696.08 ng/mL at the fourth day of the trial, and 500.21 ⁇ 742.42 ng/mL during the follow-up period.
  • antibody concentrations are varied widely among subjects, throughout the trial, the antibody concentrations in the blood are not varied much and tended to be stable.
  • the mean value of the neutralization antibody concentrations by nasal swab testing is below the lower detection limit during the baseline period;
  • the mean neutralization antibody concentration in left nostril is 32159 ⁇ 15305 ng/mL at 0.5 h before administration of a second dosage on D1;
  • the mean neutralization antibody concentration in right nostril is 62697 ⁇ 47550 ng/mL at 0.5 h before administration of a fourth dosage on D3;
  • the mean neutralization antibody concentration in left nostril is 20455 ⁇ 39514 ng/mL at 12 h after final administration on D4;
  • the mean neutralization antibody concentration in right nostril is 17307 ⁇ 26128 ng/mL at 24 h after final administration on D4;
  • the mean neutralization antibody concentrations in left and right nostrils are 189.22 ⁇ 416.92 ng/mL and 1174.7 ⁇ 1669.2 ng/mL during the follow-up period.
  • the mean blood neutralization antibody concentration is 633.75 ⁇ 807.66 ng/mL during the baseline period, 591.95 ⁇ 729.15 ng/mL at 12 h after final administration during the trial, and 588.58 ⁇ 670.01 ng/mL during the follow-up period.
  • antibody concentrations are varied widely among subjects, throughout the trial, the antibody concentrations in the blood are not varied much and tended to be stable.
  • the mean value of the neutralization antibody concentrations by nasal swab testing is 7.91 ng/mL during the baseline period; the mean neutralization antibody concentration in left nostril is 30805 ⁇ 18892 ng/mL 0.5 h after administration of a second dosage on D1; the mean neutralization antibody concentration in right nostril is 19444 ⁇ 12761 ng/mL at 0.5 h before administration of a fourth dosage on D7; the mean neutralization antibody concentration in left nostril is 13507 ⁇ 7636.7 ng/mL at 12 h after final administration; the mean neutralization antibody concentration in right nostril is 5715.1 ⁇ 5630.7 ng/mL at 24 h after final administration; and the mean neutralization antibody concentrations in left and right nostrils are 474.62 ⁇ 624.14 ng/mL and 233.01 ⁇ 368.99 ng/mL during the follow-up period.
  • the mean blood neutralization antibody concentration is 1385.7 ⁇ 2775.5 ng/mL during the baseline period, 1255.8 ⁇ 2517.3 ng/mL at 12 h after final administration during the trial, 1278.9 ⁇ 2620.9 ng/mL in 3 days after final administration and 1276.2 ⁇ 2650.5 ng/mL during the follow-up period.
  • antibody concentrations are varied widely among subjects, throughout the trial, the antibody concentrations in the blood are not varied much and tended to be stable.
  • the mean value of the neutralization antibody concentration by nasal swab testing is 3.42 ng/mL during the baseline period; the mean neutralization antibody concentration in left nostril is 36138 ⁇ 23212 ng/mL at 0.5 h before administration of the second dosage on D1; the mean neutralization antibody concentration in left nostril is 53615 ⁇ 81154 ng/mL at 0.5 h before administration of the second dose on D8; the mean neutralization antibody concentration in left nostril is 54037 ⁇ 53645 ng/mL at 15 min before administration of the fourth dosage on D14; the mean neutralization antibody concentration in left nostril is 72717 ⁇ 80364 ng/mL at 1 h after administration of the fourth dosage on D14; the means neutralization antibody concentration in right nostril is 28702 ⁇ 25733 ng/mL 4 h after administration of the fourth dosage on D14; the means neutralization antibody concentration in right nostril is 30625 ⁇ 73799 ng/mL at 12 h after final administration; the mean neutralization antibody concentration in left nostril is 120
  • a large amount of neutralization antibodies could be detected by nasal swab testing after the subjects are administered, presuming that coronavirus neutralization antibody A8G6, a main component of the test drug, could be effectively adhered to the nasal cavity.
  • the drug concentration by nasal swab testing is decreased gradually over time after subjects stop receiving drugs; the drug concentration by nasal swab testing is decreased significantly at 24 h after final administration, the drug concentration by nasal swab testing in most subjects is below a detection limit in 3 ⁇ 1 days after final administration, and in some subjects in Cohort 4, the drug concentrations by nasal swab testing are below the detection limit at 48 h after administration.
  • Tm melting temperature
  • Tonset temperature of the onset of heat induced denaturing
  • Tg temperature of the onset of the aggregation
  • the intranasal formulation of Antibody B was determined to be: 5mg/ml Antibody B antibody, 20mM phosphate buffer, 4% (w/v) trehalose, 1.7% (w/v) glycerin, 0.1% (w/v) hydroxypropyl methylcellulose (HPMC) , 0.01% (w/v) benzalkonium chloride, 0.02% (w/v) PS80, pH 6.0.
  • basal formulation comprising 20mM PB (pH 6.0) , 8%trehalose, 0.02%PS80 to study the effect of eight additives for rat intranasal administration; and used the same basal formulation to study the effect of four additives for mouse intranasal administration.
  • Each rat was intranasally administered 25 ⁇ L/nasal cavity (total 50 ⁇ L) , and the nasal mucosal samples were collected 4 hours after administration and the amounts of antibodies in the samples were determined by ELISA.
  • the different formulations were shown in Table 16. As shown in Figure 30A, the results demonstrate that the formulations containing glycerin or HPMC retained more antibodies in the nasal cavity as compared to other formulations.
  • HPMC controlled release agent
  • glycerin a humectant
  • the concentration of trehalose as the cryoprotectant/osmotic pressure regulator. As shown in Table 18, the Tm, Tonset, and Tagg of the antibody were very similar at both formulations comprising 4%or 8% (w/v) of trehalose. Because the higher 8%concentration of trehalose resulted in higher overall osmotic pressure of the formulation (520 mOsm/kg) , which is more likely to cause discomfort in the nasal cavity, the trehalose concentration was set as 4%in the formulation, resulting in an overall osmotic pressure of 391 mOsm/kg.

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Abstract

The present disclosure relates to (i) intranasal and/or mucosal formulations for delivery of proteins, such as antibodies; and (ii) antibodies which bind to the spike protein of wild-type SARS-CoV-2, and variants thereof, such as alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and omicron (BA.1 or BA.2).

Description

INTRANASAL FORMULATIONS AND ANTI-SARS-COV-2-SPIKE PROTEIN ANTIBODIES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the priority to, and the benefit of, Chinese Application No. 202210151716.6, filed February 18, 2022, and Chinese Application No. 202210151970.6, filed February 18, 2022, all of which are herein incorporated by reference in their entireties.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
The contents of the electronic sequence listing (CMHB_001_02WO_SeqList_ST26. xml; Size: 88,220 bytes; and Date of Creation: February 17, 2023) are herein incorporated by reference in their entireties.
FIELD
The present disclosure relates to (i) intranasal formulations and application of proteins, such as antibodies; and (ii) antibodies capable of binding to the spike protein of multiple variants of SARS-CoV-2.
BACKGROUND
There is a need in the art for protein formulations which can be administered to the human subject through the nasal passages. There is also a need in the art for RBD-domain spike protein SARS-CoV-2 neutralizing antibodies that would treat or prevent SARS-CoV-2 infection.
SUMMARY
In one aspect, the disclosure provides a pharmaceutical composition comprising: a protein, a cryoprotectant, a humectant, an emulsifier, a controlled release agent, and a buffer.
In one aspect, the disclosure provides a pharmaceutical composition, comprising: an active ingredient or a precursor thereof; no more than about 40% (w/v) of a cryoprotectant; no more than about 30% (w/v) of a humectant; no more than about 5% (w/v) of an emulsifier; no more than about 10% (w/v) of a controlled release agent; and a buffer.
In some embodiments, the pharmaceutical composition is suitable for mucosal administration to a human. In some embodiments, the pharmaceutical composition is suitable for intranasal administration to a human.
In some embodiments, the cryoprotectant is selected from the group consisting of trehalose, glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol, and sucrose, or a combination thereof. In some embodiments, the cryoprotectant in the pharmaceutical composition is:
no more than 40% (w/v) trehalose;
no more than 40% (w/v) glycerol;
no more than 10% (w/v) dimethyl sulfoxide;
no more than 40% (w/v) ethylene glycol;
no more than 20% (w/v) polyethylene glycol; or
no more than 0.5 M sucrose.
In some embodiments, the cryoprotectant in the pharmaceutical composition is:
about 2%-about 8% (w/v) trehalose;
about 1%-about 20% (w/v) glycerol;
about 1%-about 10% (w/v) dimethyl sulfoxide;
about 4%-about 30% (w/v) ethylene glycol;
about 5%-about 15% (w/v) polyethylene glycol; or
about 0.1 M -about 0.4 M sucrose.
In some embodiments, the cryoprotectant is trehalose.
In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 12% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 8% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 4% (w/v) of the cryoprotectant.
In some embodiments, the humectant is selected from the group consisting of glycerin, mannitol, polyethylene glycol 400, polyethylene glycol 4000, D-sorbitol, chitosan, xylitol, and sodium hyaluronate, or a combination thereof.
In some embodiments, the humectant in the pharmaceutical composition is:
no more than 30% (w/v) glycerin;
no more than 7% (w/v) mannitol;
no more than 20% (w/v) polyethylene glycol 400;
about 5%-about 15% (w/v) polyethylene glycol 4000;
about 3%-about 15% (w/v) D-sorbitol;
about 0.01%-about 3% (w/v) chitosan;
no more than 10% (w/v) xylitol; or
about 0.1%-about 2.0% (w/v) sodium hyaluronate.
In some embodiments, the humectant in the pharmaceutical composition is:
about 2%-about 6% (w/v) glycerin;
about 3%-about 5% (w/v) mannitol;
about 10%-about 18% (w/v) polyethylene glycol 400;
about 5%-about 10% (w/v) polyethylene glycol 4000;
about 3%-about 6% (w/v) D-sorbitol;
about 0.01%-about 1% (w/v) chitosan;
about 1%-about 10% (w/v) xylitol; or
about 0.1%-about 1.0% (w/v) sodium hyaluronate.
In some embodiments, the humectant is glycerin.
In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 4% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.7% (w/v) to about 2% (w/v) of the humectant.
In some embodiments, the emulsifier is selected from the group consisting of TWEEN 80, polysorbate 20, lecithin, sorbitan esters, mono-and/or diglycerides, and sodium stearoyl lactylate, or a combination thereof. In some embodiments, the emulsifier is TWEEN 80.
In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.04% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.02% (w/v) of the emulsifier.
In some embodiments, the controlled release agent is selected from the group consisting of hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone K30, or a combination thereof. In some embodiments, the controlled release agent is hydroxypropyl methylcellulose (HPMC) .
In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 5% (w/v) , or about 0.01% (w/v) to about 1% (w/v) , of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.004% (w/v) to about 0.4% (w/v)  of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) , or about 0.1% (w/v) , of the controlled release agent.
In some embodiments, the controlled release agent is polyvinylpyrrolidone K30.
In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 10% (w/v) , or about 3% (w/v) to about 6% (w/v) , of the controlled release agent.
In some embodiments, the buffer is selected from the group consisting of a phosphate buffer, a tris buffer, and a glycine buffer.
In some embodiments, the buffer in the pharmaceutical composition is:
about 10 mM -about 100 mM phosphate;
about 10 mM -about 100 mM tris; or
about 0.1 M -about 0.5 M glycine.
In some embodiments, the buffer in the pharmaceutical composition is:
about 10 mM -about 40 mM phosphate;
about 10 mM -about 40 mM tris; or
about 0.1 M -about 0.2 M glycine.
In some embodiments, the buffer is a phosphate buffer. In some embodiments, the buffer in the pharmaceutical composition is about 20 mM phosphate or tris.
In some embodiments, the buffer has a pH of about 4-8, or about 5-7. In some embodiments, the buffer has a pH of about 5.5-6.5. In some embodiments, the buffer has a pH of about 6.0.
In some embodiments, the pharmaceutical composition comprises an antibiotic. In some embodiments, the antibiotic comprises benzalkonium chloride, benzyl alcohol, chlorobutanol, or a combination thereof.
In some embodiments, the antibiotic in the pharmaceutical composition is:
about 0.002%-about 0.02% (w/v) benzalkonium chloride;
no more than 3.0% (v/v) benzyl alcohol; or
about 0.5%-about 2.0% (w/v) chlorobutanol.
In some embodiments, the antibiotic in the pharmaceutical composition is:
about 0.005%-about 0.02% (w/v) benzalkonium chloride;
about 1.0%-about 3.0% (v/v) benzyl alcohol; or
about 1.0%-2.0% (w/v) chlorobutanol.
In some embodiments, the antibiotic is benzalkonium chloride.
In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.02% (w/v) , or about 0.01% (w/v) , of the antibiotic.
In some embodiments, the mean retention time of the active ingredient or a precursor thereof in a nasal cavity of a primate is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, or at least 12 hours, after intranasal administration of the pharmaceutical composition to the primate. In some embodiments, the primate is a rhesus macaque.
In some embodiments, the pharmaceutical composition comprises:
about 0.3% (w/v) to about 12% (w/v) of a cryoprotectant;
about 0.2% (w/v) to about 10% (w/v) of a humectant;
about 0.002% (w/v) to about 0.1% (w/v) of an emulsifier; and
about 0.004% (w/v) to about 0.4% (w/v) of a controlled release agent.
In some embodiments, the pharmaceutical composition comprises:
about 2%-about 8% (w/v) of the cryoprotectant;
about 1%–about 4% (w/v) of the humectant;
about 0.005%-about 0.04% (w/v) of the emulsifier;
about 0.01%-about 0.2% (w/v) of the controlled release agent; and
the buffer has a pH of about 5.5-6.5.
In some embodiments, the pharmaceutical composition comprises:
about 4% (w/v) of the cryoprotectant;
about 1.7%–about 2% (w/v) of the humectant;
about 0.01%-about 0.02% (w/v) of the emulsifier;
about 0.02%-about 0.1% (w/v) of the controlled release agent; and
the buffer has a pH of about 6.0.
In some embodiments, the pharmaceutical composition comprises:
about 4% (w/v) of the cryoprotectant;
about 2% (w/v) of the humectant;
about 0.01% (w/v) of the emulsifier;
about 0.02% (w/v) of the controlled release agent; and
the buffer has a pH of about 6.0.
In some embodiments, the pharmaceutical composition comprises:
about 4% (w/v) of the cryoprotectant;
about 2% (w/v) of the humectant;
about 0.02% (w/v) of the emulsifier;
about 0.1% (w/v) of the controlled release agent; and
the buffer has a pH of about 6.0.
In some embodiments, the pharmaceutical composition comprises:
about 4% (w/v) of the cryoprotectant;
about 1.7% (w/v) of the humectant;
about 0.02% (w/v) of the emulsifier;
about 0.1% (w/v) of the controlled release agent; and
the buffer has a pH of about 6.0.
In some embodiments, the buffer comprises about 20 mM phosphate salt. In some embodiments, the phosphate salt is a sodium phosphate salt.
In some embodiments, the cryoprotectant is trehalose, the humectant is glycerin, the emulsifier is TWEEN 80, the controlled release agent is HPMC.
In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) benzalkonium chloride.
In some embodiments, the active ingredient comprises a polypeptide. In some embodiments, the active ingredient comprises an antibody or an antigen binding fragment thereof. In some embodiments, the active ingredient has a concentration of about 0.1-100 mg/ml, about  0.5-50 mg/ml, or about 1-30 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 mg/ml. In some embodiments, the antibody or antigen fragment thereof binds to a coronavirus. In some embodiments, the antibody or antigen fragment thereof binds to SARS-CoV-2.
In some embodiments, the pharmaceutical composition comprises one or more of:
an antibody A or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 13, a HCDR2 comprising SEQ ID NO: 14, and a HCDR3 comprising SEQ ID NO: 15 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 10, a LCDR2 comprising SEQ ID NO: 11, and a LCDR3 comprising SEQ ID NO: 12;
an antibody A-1 or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 4, a HCDR2 comprising SEQ ID NO: 5, and a HCDR3 comprising SEQ ID NO: 6 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 1, a LCDR2 comprising SEQ ID NO: 2, and a LCDR3 comprising SEQ ID NO: 3;
an antibody B or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 23, a HCDR2 comprising SEQ ID NO: 24, and a HCDR3 comprising SEQ ID NO: 25 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, and a LCDR3 comprising SEQ ID NO: 22;
an antibody C or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 33, a HCDR2 comprising SEQ ID NO: 34, and a HCDR3 comprising SEQ ID NO: 35 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 30, a LCDR2 comprising SEQ ID NO: 31, and a LCDR3 comprising SEQ ID NO: 32;
an antibody D/E or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 43, a HCDR2 comprising SEQ ID NO: 44, and a HCDR3 comprising SEQ ID NO: 45 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 40, a LCDR2 comprising SEQ ID NO: 41, and a LCDR3 comprising SEQ ID NO: 42; and
an antibody F or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 63, a HCDR2 comprising SEQ ID NO: 64, and a HCDR3 comprising SEQ ID NO: 65 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62.
In some embodiments:
the antibody A or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 18 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 16 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
the antibody A-1 or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 8 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 7 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
the antibody B or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 28 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 26 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
the antibody C or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 38 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 36 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
the antibody D/E or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 48 or 52 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 46 or 50 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; and/or
the antibody F or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 68 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 66 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
In some embodiments, (e) the antibody D/E or antigen binding fragment comprises:
a heavy chain constant region (HCR) comprising or consisting of SEQ ID NO: 49 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a light chain constant region (LCR) comprising or consisting of SEQ ID NO: 47 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or
a HCR comprising or consisting of SEQ ID NO: 53 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a LCR comprising or consisting of SEQ ID NO: 51 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or
a combination thereof.
In some embodiments, the pharmaceutical composition comprises at least two of the antibodies or antigen binding fragments. In some embodiments, the pharmaceutical composition comprises (c) the antibody B or antigen binding fragment thereof and (e) the antibody D/E or antigen binding fragment thereof. In some embodiments, the ratio of (c) the antibody B or antigen binding fragment thereof to (e) the antibody D/E or antigen binding fragment thereof is between about 1: 2 and about 1: 8. In some embodiments, the ratio of (c) the antibody B or antigen binding fragment thereof to (e) the antibody D/E or antigen binding fragment thereof is about 1: 4.
In some embodiments, the pharmaceutical composition comprises (d) the antibody C or antigen binding fragment thereof.
In some embodiments, the pharmaceutical composition comprising (f) the antibody F or antigen binding fragment thereof.
In some embodiments, the antibody is an IgG, an IgM, or an IgA.
In some embodiments, the pharmaceutical composition is administered via a nasal administration device.
In one aspect, the disclosure provides a nasal or mucosal administration device comprising the pharmaceutical composition of the disclosure.
In one aspect, the disclosure provides a kit comprising the pharmaceutical composition of the disclosure and a nasal or mucosal administration device.
In some embodiments, the nasal administration device is a metered dose nasal pump device or a unit dose nasal pump device.
In one aspect, the disclosure provides an antibody or antigen binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) , a heavy chain complementarity determining region 2 (HCDR2) and a heavy chain complementarity determining region 3 (HCDR3) , wherein: the HCDR1 comprises the amino acid sequence of GFTFSGSA (SEQ ID NO: 13) ; the HCDR2 comprises the amino acid sequence of IVVGSGNT (SEQ ID NO: 14) ; and the HCDR3 comprises the amino acid sequence of AAPYCSSTSCRDGFDI (SEQ ID NO: 15) . In some embodiments, the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 18, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto. In some embodiments, the antibody or antigen binding fragment comprises a light chain variable domain (VL) comprising a light chain complementarity determining region 1 (LCDR1) , a light chain complementarity determining region 2 (LCDR2) and a light chain complementarity determining region 3 (LCDR3) , wherein: the LCDR1 comprises the amino acid sequence of QSVRSSY (SEQ ID NO: 10) ; the LCDR2 comprises the amino acid sequence of GAS (SEQ ID NO: 11) ; and the LCDR3 comprises the amino acid sequence of QQYGRSPWT (SEQ ID NO: 12) . In some embodiments, wherein the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
In one aspect, the disclosure provides an antibody or antigen binding fragment thereof, comprising:
a VH comprising a HCDR1 comprising SEQ ID NO: 23, a HCDR2 comprising SEQ ID NO: 24, and a HCDR3 comprising SEQ ID NO: 25 and a VL comprising a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, and a LCDR3 comprising SEQ ID NO: 22;
a VH comprising a HCDR1 comprising SEQ ID NO: 33, a HCDR2 comprising SEQ ID NO: 34, and a HCDR3 comprising SEQ ID NO: 35 and a VL comprising a LCDR1 comprising SEQ ID NO: 30, a LCDR2 comprising SEQ ID NO: 31, and a LCDR3 comprising SEQ ID NO: 32;
a VH comprising a HCDR1 comprising SEQ ID NO: 43, a HCDR2 comprising SEQ ID NO: 44, and a HCDR3 comprising SEQ ID NO: 45 and a VL comprising a LCDR1 comprising SEQ ID NO: 40, a LCDR2 comprising SEQ ID NO: 41, and a LCDR3 comprising SEQ ID NO: 42; or
a VH comprising a HCDR1 comprising SEQ ID NO: 63, a HCDR2 comprising SEQ ID NO: 64, and a HCDR3 comprising SEQ ID NO: 65 and a VL comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62; .
In some embodiments, the antibody or antigen binding fragment is for intranasal administration.
In some embodiments, the antibody or antigen binding fragment is capable of binding to a SARS-CoV-2 spike protein, optionally wherein the SARS-CoV-2 spike proteins comprises the amino acid sequence set forth in SEQ ID NO: 106, SEQ ID NO: 108, or SEQ ID NO: 110.
In some embodiments, the antibody is an IgG, an IgM, or an IgA.
In one aspect, the disclosure provides a polynucleotide encoding the antibody or antigen binding fragment of the disclosure.
In one aspect, the disclosure provides a vector comprising the polynucleotide of the disclosure.
In one aspect, the disclosure provides a composition comprising the antibody or antigen binding fragment, the polynucleotide, or the vector of the disclosure.
In one aspect, the disclosure provides a cell comprising the polynucleotide or the vector of the disclosure.
In one aspect, the disclosure provides a method of producing the antibody or antigen binding fragment of the disclosure, comprising culturing a cell comprising a vector encoding the antibody or antigen binding fragment thereof and harvesting the antibody or antigen binding fragment from the culture medium of the cell.
In one aspect, the disclosure provides a method of treating or preventing a SARS-CoV-2 infection in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen binding fragment of the disclosure.
In some embodiments, the antibody or antigen binding fragment is administered intranasally, subcutaneously, intravenously, or intramuscularly. In some embodiments, the antibody or antigen binding fragment is administered intranasally.
In one aspect, the disclosure provides a combination of antibodies or antigen binding fragments thereof, comprising at least two of the following antibodies or antigen binding fragments thereof:
an antibody A or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 13, a HCDR2 comprising SEQ ID NO: 14, and a HCDR3 comprising SEQ ID NO: 15 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 10, a LCDR2 comprising SEQ ID NO: 11, and a LCDR3 comprising SEQ ID NO: 12;
an antibody A-1 or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 4, a HCDR2 comprising SEQ ID NO: 5, and a HCDR3 comprising SEQ ID NO: 6 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 1, a LCDR2 comprising SEQ ID NO: 2, and a LCDR3 comprising SEQ ID NO: 3;
an antibody B or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 23, a HCDR2 comprising SEQ ID NO: 24, and a HCDR3 comprising SEQ ID NO: 25 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, and a LCDR3 comprising SEQ ID NO: 22;
an antibody C or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 33, a HCDR2 comprising SEQ ID NO: 34, and a HCDR3 comprising SEQ ID NO: 35 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 30, a LCDR2 comprising SEQ ID NO: 31, and a LCDR3 comprising SEQ ID NO: 32;
an antibody D/E or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 43, a HCDR2 comprising SEQ ID NO: 44, and a HCDR3 comprising SEQ ID NO: 45 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 40, a LCDR2 comprising SEQ ID NO: 41, and a LCDR3 comprising SEQ ID NO: 42; and
an antibody F or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 63, a HCDR2 comprising SEQ ID NO: 64, and a HCDR3 comprising SEQ ID NO: 65 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62.
In some embodiments:
the antibody A or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 18 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 16 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
the antibody A-1 or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 8 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 7 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
the antibody B or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 28 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 26 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
the antibody C or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 38 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 36 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
the antibody D/E or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 48 or 52 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 46 or 50 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; and/or
the antibody F or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 68 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 66 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
In some embodiments, the antibody D/E or antigen binding fragment comprises: a heavy chain constant region (HCR) comprising or consisting of SEQ ID NO: 49 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a light chain constant region (LCR) comprising or consisting of SEQ ID NO: 47 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or a HCR comprising or consisting of SEQ ID NO: 53 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a LCR comprising or consisting of SEQ ID NO: 51 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or a combination thereof.
In some embodiments, the combination of antibodies or antigen binding fragments comprises: the antibody B or antigen binding fragment thereof, and the antibody D/E or antigen binding fragment thereof.
In some embodiments, the ratio of the antibody B or antigen binding fragment thereof to the antibody D/E or antigen binding fragment thereof is between about 1: 2 and about 1: 8. In some embodiments, the ratio of the antibody B or antigen binding fragment thereof to the antibody D/E or antigen binding fragment thereof is about 1: 4.
In some embodiments, the combination of antibodies or antigen binding fragments comprises the antibody C or antigen binding fragment thereof. In some embodiments, the combination of antibodies or antigen binding fragments comprises the antibody F or antigen binding fragment thereof.
In some embodiments, the combination displays a synergistic effect against COVID-19 Omicron BA. 2, BA. 2.12.1, or BA. 5 strain.
In one aspect, the disclosure provides a method of delivering an active ingredient or a precursor thereof to a nasal cavity or a mucosa of a subject, comprising administering to the subject the pharmaceutical composition of the disclosure.
In one aspect, the disclosure provides a method of preventing a disease in a subject in need thereof, comprising administering the pharmaceutical composition, the antibody or antigen binding fragment, or the combination, to the subject.
In one aspect, the disclosure provides a method of treating a disease in a subject in need thereof, comprising administering the pharmaceutical composition, the antibody or antigen binding fragment, or the combination, to the subject.
In some embodiments, the pharmaceutical composition, or the antibody or antigen binding fragment or the combination, is administered to a mucosa of the subject.
In some embodiments, the pharmaceutical composition, or the antibody or antigen binding fragment or the combination, is administered intranasally to the subject.
In some embodiments, about 30-150 μl per administration of the pharmaceutical composition is delivered to a nasal cavity of the subject. In some embodiments, about 70-100 μl per administration of the pharmaceutical composition is delivered to the nasal cavity of the subject.
In some embodiments, about 0.15 mg to about 0.75 mg of the active ingredient or a precursor thereof is delivered to the nasal cavity per administration.
In some embodiments, the antibody or antigen binding fragment, or the combination, is delivered to the subject about 1-6 times per day. In some embodiments, the pharmaceutical  composition, the antibody or antigen binding fragment, or the combination, is delivered to the subject about 2-3 times per day.
In some embodiments, the pharmaceutical composition, the antibody or antigen binding fragment thereof, or the combination, is delivered to the subject for about 7-14 days.
In some embodiments, administering the pharmaceutical composition, the antibody or antigen binding fragment thereof, or the combination, starts prior to the onset of any symptom of the disease. In some embodiments, administering the pharmaceutical composition, the antibody or antigen binding fragment thereof, or the combination, starts after the onset of at least one symptom of the disease.
In some embodiments, the symptom is a respiratory symptom. In some embodiments, the disease comprises a respiratory symptom. In some embodiments, the respiratory symptom is allergy, nasal congestion, nasal infection, or a combination thereof.
In some embodiments, the disease is a respiratory infection caused by a virus, a bacterial, a fungus, or a combination thereof. In some embodiments, the disease is a viral infection. In some embodiments, the disease is a chronic viral infection. In some embodiments, the disease is an acute viral infection. In some embodiments, the disease is a coronavirus infection. In some embodiments, the disease is SARS-CoV-2 infection.
In some embodiments, the concentration of the antibody or antigen binding fragment thereof in a nasal cavity of the subject is at least 100-fold higher than the concentration of the antibody or antigen binding fragment thereof in the blood sample of the subject within 24 hours after the administration as measured by ELISA.
In various embodiments, any of the features or components of embodiments discussed above or herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value discussed above or herein may be combined with another related value discussed above or herein to recite a range with the values representing the upper and lower ends of the range, and such ranges are encompassed within the scope of the present disclosure.
BRIEF DESCRIPTION OF THE FIGURES
Figures 1A-1F show protective efficacy of Antibody B against the Delta variant of the SARS-Cov-2 virus (Delta variant) in a hamster model. Figure 1A: Animal experimental scheme. Figure 1B: Body weight changes. Figure 1C: Viral RNA (log10 (RNA copies per ml) ) detected  in the nasal washes of hamsters challenged with the Delta variant at 3 days post infection (dpi) . Figure 1D: Viral RNA (log10 (RNA copies per g) ) detected in the respiratory tracts of hamsters challenged with the Delta variant at 3 dpi. Figure 1E: Viral RNA (log10 (RNA copies per ml) ) detected in the throat swabs of hamsters challenged with the Delta variant at 3 dpi. Figure 1F: The number of infectious viruses (PFU) was measured in the respiratory tract at 3 dpi by a viral plaque assay in Vero E6 cells. Figure 1G shows the concentration of antibody B in the nasal cavity or in the circulating blood in a Cynomolgus monkey study.
Figures 2A-2B show histopathological changes in the lung at 3 dpi in hamsters challenged with Delta variant. Figure 2A: Representative images of the H&E-stained lung tissues of Group 1, 2, 3, and 4 hamsters at 3 days after Delta variant challenge. There was no obvious abnormality in the structure of the bronchus at any level. The alveolar wall was composed of a single layer of epithelium with a clear structure. No obvious inflammatory changes were observed (Group 1, prophylactic group) . Lung tissue showed small foci of alveolar wall thickening with a small amount of neutrophil infiltration (black arrows) ; vascular congestion and dilation were common (green arrows) (Group 2, 3-dose antibody treatment group) . Lung tissue showed mild alveolar wall thickening with neutrophil infiltration (black arrows) ; occasionally, there were a few necrotic epithelial cells, lymphocytes, and neutrophils in the bronchi (purple arrows) (Group 3, 2-dose antibody treatment group) . Diffuse hemorrhage in the alveolar wall (red arrow) was seen in the lung tissue; a large number of blood cells in the bronchi were often seen (green arrow) ; multifocal thickening of the alveolar wall with neutrophil infiltration (black arrow) was seen (Group 4, control group without antibody) . Figure 2B: Sections were comprehensively evaluated for histological changes and inflammation progression.
Figures 3A-3E show protective efficacy of Antibody B against omicron in the hamster model. Figure 3A: Animal experimental scheme. Figure 3B: Viral RNA (log10 (RNA copies per ml) ) detected in the nasal washes of hamsters challenged with Omicron variant at 3 dpi. Figure 3C: Viral RNA (log10 (RNA copies per g) ) detected in respiratory tracts of hamsters challenged with Omicron variant at 3 dpi. Figure 3D: Viral RNA (log10 (RNA copies per ml) ) detected in the throat swabs of hamsters challenged with Omicron at 3 dpi. Figure 3E: The number of infectious virus (PFU) was measured in the respiratory tract at 3 dpi by the viral plaque assay in Vero E6 cells.
Figures 4A-4E show activities of Antibody B. Figure 4A: Binding of Antibody B to different RBDs, as measured by an ELISA. Figure 4B: Neutralization of pseudotyped SARS-CoV-2 wildtype (WT) and different variants by Antibody B. Inhibitory rates of infection were  calculated by scanning fluorescent plaques. Figure 4C: Evaluation of the neutralization activity of Antibody B against live SARS-CoV-2 WT and the Delta and Omicron variants. Figure 4D: Binding regions of Antibody B. RBDs from WT SARS-CoV-2 and the Alpha, Beta, Gamma, Delta and Omicron variants are aligned. The lines indicate the binding regions of Antibody B. Figure 4E: Structure of the complex of Antibody B Fab bound onto the SARS-CoV-2 RBD. On the left, the secondary structure elements of the RBD and heavy and light chains of Antibody B Fab are colored green, cyan and violet, respectively. The surface of the RBD is colored green. The mutated residues of the Delta variant are colored blue (middle) , while those from the Omicron are colored red (right) .
Figure 5 shows the weight change of golden hamsters after challenge (*p<0.05; **p<0.01; ***p<0.001) .
Figure 6 shows viral RNA level in the indicated tissue at 3-day post infection (dpi) . L.O.D = limit of detection is 1.649.
Figure 7 shows viral titer in the indicated tissue at 3-day post infection (dpi) . (**p<0.01; ****p<0.0001) . L.O.D = limit of detection is 1.3.
Figure 8 shows the weight change of golden hamsters after challenge.
Figure 9 shows the viral load of tissues of golden hamsters in each group after challenge. (****p<0.0001) . L.O.D = limit of detection is 1.649.
Figure 10 shows the virus titer of tissues of golden hamsters in each group after challenge. (****p<0.0001)
Figure 11A shows the amount of Antibody B antibody in mice nasal mucosa. Figure 11B shows the amount of Antibody B antibody in mice blood. Figure 11C shows the amount of Antibody B antibody in mice trachea.
Figure 12A shows the radioactive retention in the nasal cavities of healthy adult rabbits within 24h after nasal dripping of 131I-Antibody B antibody and Na131I. For each time point, the bar on the left represents the value for 131I-Antibody B and the bar on the right represents the value for Na131I. Figure 12B shows the downward trend of radioactive retention in the nasal cavities of healthy adult rabbits within 24h after nasal dripping of 131I-Antibody B antibody and Na131I. Figure 12C shows the radioactive retention in the nasal cavities and pharynges of healthy adult rabbits within 24h after nasal dripping of 131I-Antibody B antibody and Na131I. For each time point, the bar on the left represents the value for 131I-Antibody B and the bar on the right represents the value for Na131I. Figure 12D shows the downward trend of the radioactive retention  in the nasal cavities and pharynges of healthy adult rabbits within 24h after nasal dripping of 131I-Antibody B antibody and Na131I.
Figure 13 shows the drug content in nasal swabs of cynomolgus macaques after nasal spraying.
Figure 14 shows the binding ability of Antibody D to recombinant proteins of wild-type and various mutant strains of coronaviruses.
Figure 15 shows the binding ability of Antibody B to recombinant proteins of wild-type and various mutant strains of coronaviruses.
Figure 16 shows affinity results between Antibody D antibody and wild-type, Delta, Omicron BA. 1, Omicron BA. 2 and Omicron BA. 4/5) of coronaviruses.
Figure 17 shows affinity results between Antibody B antibody and coronaviruses Delta and Omicron (BA. 1 and BA. 2) .
Figure 18 shows blocking of binding of Omicron RBD/Sto ACE2 through Antibody D, Antibody B and A8G6 antibodies. The upper figures show that the antibodies compete with Omicron RBD for binding to ACE2 recombinant protein. The lower figures show that the antibodies compete with Omicron S for binding to ACE2 recombinant protein. The figures from left to right sequentially represent Antibody B, Antibody D and A8G6. The black curve represents the control group. When no antibody is added, ACE2 can bind to RBD or S coated on the chip, and the response value increases with time, the curve shows an upward trend (blue curve) ; the red curve shows that after the antibody is added in advance to bind to RBD or S on the chip, ACE2 can no longer bind to RBD or S on the chip, and the response value does not increase with time. This proves the follows: 1. Antibody D partially competes with ACE2 to block its binding to the recombinant protein of spike protein (S) of a coronavirus; Antibody D does not compete with ACE2 and cannot block its binding to the recombinant protein of Receptor Binding Domain (RBD) of a coronavirus. 2. Antibody B completely competes with ACE2, to block its binding to a coronavirus RBD or S recombinant protein. 3. A8G6 completely competes with ACE2 to block its binding to a coronavirus RBD or S recombinant protein.
Figure 19A shows pseudovirus neutralization ability tests of Antibody B against wild-type and various mutant strains of a coronavirus. Figure 19B shows pseudovirus neutralization ability tests of Antibody B against wild-type and various mutant strains of coronaviruses.
Figure 20 shows pseudovirus neutralization test results of A8G6 coronavirus neutralizing antibodies.
Figure 21A shows 3D Synergy 95%pattern-20220811 of BA. 2 pseudovirus neutralization tests under joint use of Antibody B and Antibody D. Figure 21B shows 3D Synergy 95%pattern-20220816 of BA. 2 pseudovirus neutralization tests under joint use of Antibody B and Antibody D.
Figure 22A: The neutralizing potencies of 58G6 alone, 55A8 alone or 58G6 and 55A8 in combination against SARS-CoV-2 and the Delta, Omicron BA. 1, Omicron BA. 1 +L452R, and Omicron BA. 2 variants were measured with a pseudovirus neutralization assay. The dashed line indicates a 0%or 50%reduction in viral neutralization. Data are presented as the mean values. Figure 22B: Neutralization against authentic SARS-CoV-2, Delta and Omicron BA. 1 viruses.
Figure 23A shows BA. 2 pseudovirus neutralization test results under joint use of Antibody B and Antibody D at different ratios. Figure 23B shows BA. 2.12.1 pseudovirus neutralization test results under joint use of Antibody B and Antibody D at different ratios. Figure 23C shows BA. 5 pseudovirus neutralization test results under joint use of Antibody B and Antibody D at different ratios. Figure 23D shows Delta pseudovirus neutralization test results under joint use of Antibody B and Antibody D at different ratios.
Figure 24 shows neutralization activities of A8G6 and Antibody D against live viruses of wild-type, Delta and Omicron strains.
Figure 25A shows an animal experimental scheme. Figure 25B shows the viral RNA (log10 (RNA copies per g) ) detected in the respiratory tract of hamsters challenged with the Omicron variant at 3 dpi. For the bar chart of each location, the order is Group 1, Group 2, Group 3, Group 4, from left to right. Figure 25C shows the number of infectious viruses (PFU) in the respiratory tract measured at 3 dpi with a viral plaque assay performed with Vero E6 cells. For the bar chart of each location, the order is Group 1, Group 2, Group 3, Group 4, from left to right.
Figures 26A-26D show the results of the protective efficacy of 55A8/58G6 cocktails against Omicron in a hamster model. Figure 26A shows an animal experimental scheme. Figure 26B shows the viral RNA (log10 (RNA copies per g) ) detected in the respiratory tract of hamsters challenged with the Omicron variant at 3 dpi. For the bar chart of each location, the order is Group 1, Group 2, Group 3, Group 4, Group 5, from left to right. Figure 26C shows the number of infectious viruses (PFU) in the respiratory tract measured at 3 dpi with a viral plaque assay performed with Vero E6 cells. For the bar chart of each location, the order is Group 1, Group 2, Group 3, Group 4, Group 5, from left to right. Figure 26D shows the weight changes in the hamster model treated with 2-cocktail as a measure of the protective efficacy against Omicron.
Figures 27A-27D shows the results of the prophylactic dose of 55A8/58G6 cocktails against Omicron in the hamster model. Figure 27A shows an animal experimental scheme. Figure 27B shows the viral RNA (log10 (RNA copies per g) ) detected in the respiratory tract of hamsters challenged with the Omicron variant at 3 dpi (**p<0.01; ****p<0.0001; L.O.D (limit of detection) is 1.30) . For the bar chart of each location, the order is Group 1, Group 2, Group 3, Group 4, Group 5, from left to right. Figure 27C shows the number of infectious viruses (PFU) in the respiratory tract measured at 3 dpi with a viral plaque assay performed with Vero E6 cells (****p<0.0001; L.O.D (limit of detection) is 1.30) . For the bar chart of each location, the order is Group 1, Group 2, Group 3, Group 4, Group 5, from left to right. Figure 27D shows weight changes in the hamster model after treatment with 2-cocktails as a measure to define the prophylactic dose effective against Omicron.
Figure 28A shows the content of A8G6 antibodies in nasal mucosa of mice. Figure 28B shows the content of A8G6 antibodies in blood of mice. Figure 28C shows the content of A8G6 antibodies in tracheas of mice.
Figure 29 shows mean drug concentration-time curves of nasal swabs after different times of A8G6 coronavirus neutralizing antibody nasal spray to rhesus monkeys every day.
Figure 30A shows Antibody B antibody concentration in rat mucosal samples 4-hours post administration. LOB is the maximal value of the blank control (Group I) . Figure 30B shows Antibody B antibody concentration in mouse mucosal samples 6-hours post administration. Two animals in the glycerin group and one animal in the HPMC group has antibody level that is higher than the upper detection limit. LOB is the maximal value of the blank control (Group F) .
DETAILED DESCRIPTION
Definitions
In order that the application may be more completely understood, several definitions are set forth below. Such definitions are meant to encompass grammatical equivalents.
The term “about” in relation to a reference numerical value can include the numerical value itself and a range of values plus or minus 10%from that numerical value. For example, the amount “about 10” includes 10 and any amounts from 9 to 11.
Specific embodiments disclosed herein can be further limited in the claims using “consisting of” or “consisting essentially of” language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or  ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic (s) . Embodiments of the disclosure so claimed are inherently or expressly described and enabled herein.
The terms “a, ” “an, ” “the” and similar referents used in the context of describing the disclosure (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.
Before the present methods are described, it is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.
The term “coronavirus” or “CoV” refers to any virus of the coronavirus family, including but not limited to SARS-CoV-2, MERS-CoV, and SARS-CoV. SARS-CoV-2 refers to the recently emerged coronavirus. SARS-CoV-2 has also been known as 2019-nCoV and Wuhan coronavirus. Without being held to theory or mechanism, it binds via the viral spike protein to human host cell receptor angiotensin-converting enzyme 2 (ACE2) . The spike protein also binds to and is cleaved by TMPRSS2, which activates the spike protein for membrane fusion of the virus.
The term “CoV-S” , also called “S” or “S protein” refers to the spike protein of a coronavirus, and can refer to specific S proteins such as SARS-CoV-2-S, MERS-CoV S, and SARS-CoV S. The wild-type SARS-CoV-2-Spike protein is a 1273 amino acid type I membrane glycoprotein which assembles into trimers that constitute the spikes or peplomers on the surface of the enveloped coronavirus particle. The protein has two essential functions, host receptor binding and membrane fusion, which are attributed to the N-terminal (S1) and C-terminal (S2) halves of the S protein. CoV-S binds to its cognate receptor via a receptor binding domain (RBD) present in the S1 subunit. The amino acid sequence of wild-type SARS-CoV-2 spike protein is  exemplified by SEQ ID NO: 70. The amino acid sequence of alpha (B. 1.1.7) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 72. The amino acid sequence of beta (B. 1.351) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 74. The amino acid sequence of gamma (P. 1) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 76. The amino acid sequence of delta (B.1.617.2) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 78. The amino acid sequence of omicron (BA. 1) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 80. Unless further specified, the term “CoV-S” includes protein variants of CoV spike protein isolated from different CoV isolates as well as recombinant CoV spike protein or a fragment thereof. The term “alpha CoV-S” or “alpha (B. 1.1.7) CoV-S” refers to the CoV spike protein of the alpha (B.1.1.7) SARS-CoV-2 spike protein. The term “beta CoV-S” or “beta (B. 1.351) CoV-S” refers to the CoV spike protein of the beta (B. 1.351) SARS-CoV-2 spike protein. The term “gamma CoV-S” or “gamma (P. 1) CoV-S” refers to the CoV spike protein of the gamma (P. 1) SARS-CoV-2 spike protein. The term “delta CoV-S” or “delta (B. 1.617.2) CoV-S” refers to the CoV spike protein of the delta (B. 1.617.2) SARS-CoV-2 spike protein. The term “omicron CoV-S” or “omicron (BA. 1 or BA. 2) CoV-S” refers to the CoV spike protein of the omicron (BA. 1 or BA. 2) SARS-CoV-2 spike protein. The term also encompasses CoV spike protein or a fragment thereof coupled to, for example, a histidine tag, mouse or human Fc, or a signal sequence such as ROR1.
The term “RBD-CoV-S” , also called “RBD-S” or “RBD-S protein” refers to the RBD-domain spike protein of a coronavirus, and can refer to specific RBD-domains of S proteins such as RBD-SARS-CoV-2-S, RBD-MERS-CoV S, and RBD-SARS-CoV S. The RBD domain of the wild-type SARS-CoV-2-Spike protein facilitates binding of the S1 subunit to its cognate receptor. The amino acid sequence of the RBD domain of the wild-type SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 71. The amino acid sequence of the RBD domain of the alpha (B.1.1.7) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 73. The amino acid sequence of the RBD domain of the beta (B. 1.351) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 75. The amino acid sequence of the RBD domain of the gamma (P. 1) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 77. The amino acid sequence of the RBD domain of the delta (B. 1.617.2) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 79. The amino acid sequence of the RBD domain of the omicron (BA. 1) SARS-CoV-2 spike protein is exemplified by SEQ ID NO: 81. Unless further specified, the term “RBD-CoV-S” includes the RBD-domain of protein variants of CoV spike protein isolated from different CoV isolates as well as recombinant CoV spike protein or a fragment thereof. The term “alpha RBD-CoV-S” or “alpha (B. 1.1.7) RBD-CoV-S” refers to the RBD domain of the alpha (B. 1.1.7) SARS-CoV-2 spike protein. The term “beta RBD-CoV-S” or “beta (B. 1.351) RBD-CoV-S” refers to the RBD  domain of the beta (B. 1.351) SARS-CoV-2 spike protein. The term “gamma CoV-S” or “gamma (P.1) CoV-S” refers to the RBD domain of the gamma (P. 1) SARS-CoV-2 spike protein. The term “delta CoV-S” or “delta (B. 1.617.2) CoV-S” refers to the RBD domain of the delta (B.1.617.2) SARS-CoV-2 spike protein. The term “omicron CoV-S” or “omicron (BA. 1 or BA. 2) CoV-S” refers to the RBD domain of the omicron (BA. 1 or BA. 2) SARS-CoV-2 spike protein. The term also encompasses CoV spike protein or a fragment thereof coupled to, for example, a histidine tag, mouse or human Fc, or a signal sequence such as ROR1.
The term “coronavirus infection” or “CoV infection, ” as used herein, refers to infection with a coronavirus such as SARS-CoV-2, MERS-CoV, or SARS-CoV. The term includes coronavirus respiratory tract infections, often in the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastro-intestinal symptoms such as diarrhea, organ failure (kidney failure and renal dysfunction) , septic shock, and death in severe cases.
The term “alpha coronavirus infection” or “alpha SARS-CoV-2 infection, ” as used herein, refers to infection with an alpha coronavirus such as alpha (B. 1.1.7) SARS-CoV-2. The term “beta coronavirus infection” or “beta SARS-CoV-2 infection, ” as used herein, refers to infection with a beta coronavirus such as beta (B. 1.351) SARS-CoV-2. The term “gamma coronavirus infection” or “gamma SARS-CoV-2 infection, ” as used herein, refers to infection with a gamma coronavirus such as gamma (P. 1) SARS-CoV-2. The term “delta coronavirus infection” or “delta SARS-CoV-2 infection, ” as used herein, refers to infection with a delta coronavirus such as delta (B.1.617.2) SARS-CoV-2. The term “omicron coronavirus infection” or “omicron SARS-CoV-2 infection, ” as used herein, refers to infection with an omicron coronavirus such as omicron (BA. 1 or BA. 2) SARS-CoV-2.
The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody or antigen-binding protein, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F (ab’) 2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide) , or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies,  diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., as defined in WO08/020079 or WO09/138519) (e.g., monovalent nanobodies, bivalent nanobodies, etc. ) , small modular immunopharmaceuticals (SMIPs) , and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment, ” as used herein. In some embodiments, the antigen-binding fragment comprises three or more CDRs of an antibody of Table 1 (e.g., HCDR1, HCDR2 and HCDR3; or LCDR1, LCDR2 and LCDR3) .
Unless indicated otherwise, the terms “polypeptide” and “protein” are used interchangeably in this disclosure.
Viruses
The present disclosure includes methods for treating or preventing a viral infection in a subject. In some embodiments, the viral infection is caused by a respiratory infection virus. In some embodiments, the respiratory infection virus is influenza virus, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, or bocaviruses. In some embodiments, the term “virus” includes any virus whose infection in the body of a subject is treatable or preventable by administration of an respiratory viral antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof that bind to influenza virus antigens, respiratory syncytial virus antigens, parainfluenza virus antigens, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, or bocaviruses. In some embodiments, the term “virus” includes any virus whose infection in the body of a subject is treatable or preventable by administration of an anti-viral antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof of the present disclosure (e.g., of Table 1) , (e.g., wherein infectivity of the virus is at least partially dependent on CoV-S) . In some embodiments, a “virus” is any virus that expresses spike protein (e.g., CoV-S) . The term “virus” also includes a CoV-S-dependent respiratory virus which is a virus that infects the respiratory tissue of a subject (e.g., upper and/or lower respiratory tract, trachea, bronchi, lungs) and is treatable or preventable by administration of an anti-CoV-S antibody or antigen-binding fragment thereof. For example, In some embodiments, virus includes coronavirus, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) , SARS-CoV (severe acute respiratory syndrome coronavirus) , and MERS-CoV (Middle East respiratory syndrome (MERS) coronavirus) . Coronaviruses can include the genera of alphacoronaviruses, beta coronaviruses, gamma coronaviruses, delta coronaviruses, and/or omicron coronaviruses. In some embodiments, the antibodies or antigen-binding fragments provided herein can bind to and/or neutralize an alpha coronavirus, a beta coronavirus, a gamma coronavirus, a delta coronavirus, and/or an omicron coronavirus. In some embodiments, this binding and/or  neutralization can be specific for a particular genus of coronavirus or for a particular subgroup of a genus. “Viral infection” refers to the invasion and multiplication of a virus in the body of a subject.
Without being bound to theory or mechanism, coronavirus virions are spherical with diameters of approximately 125 nm. The most prominent feature of coronaviruses is the club-shape spike projections emanating from the surface of the virion. These spikes are a defining feature of the virion and give them the appearance of a solar corona, prompting the name, coronaviruses. Within the envelope of the virion is the nucleocapsid. Coronaviruses have helically symmetrical nucleocapsids, which is uncommon among positive-sense RNA viruses, but far more common for negative-sense RNA viruses. SARS-CoV-2, MERS-CoV, and SARS-CoV belong to the coronavirus family. The initial attachment of the virion to the host cell is initiated by interactions between the S protein and its receptor. The sites of receptor binding domains (RBD) within the S1 region of a coronavirus S protein vary depending on the virus, with some having the RBD at the C-terminus of S1. The S-protein/receptor interaction is the primary determinant for a coronavirus to infect a host species and also governs the tissue tropism of the virus. Many coronaviruses utilize peptidases as their cellular receptor. Following receptor binding, the virus must next gain access to the host cell cytosol. This is generally accomplished by acid-dependent proteolytic cleavage of S protein by a cathepsin, TMPRRS2 or another protease, followed by fusion of the viral and cellular membranes.
Pharmaceutical Compositions
In some embodiments, the present disclosure provides pharmaceutical compositions comprising: an active ingredient (e.g., a protein) , a cryoprotectant, a humectant, an emulsifier, a controlled release agent, and a buffer.
In some embodiments, such a pharmaceutical composition can retain the active ingredient or precursor thereof at the target site (e.g., nasal cavity) for a long time. In some embodiments, the mean retention time of the active ingredient or precursor thereof in the target site (e.g., nasal cavity) of a primate, when formulated in the pharmaceutical composition of the disclosure, is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, or at least 12 hours, after administration of the pharmaceutical composition to the primate. In some embodiments, the mean retention time is at least 1 hour. In some embodiments, the mean retention time is at least 2 hours. In some embodiments, the mean retention time is at least 2 hours. In some embodiments, the mean retention time is at least 3 hours. In some embodiments, the mean  retention time is at least 4 hours. In some embodiments, the mean retention time is at least 5 hours. In some embodiments, the mean retention time is at least 6 hours. In some embodiments, the mean retention time is at least 7 hours. In some embodiments, the mean retention time is at least 8 hours. In some embodiments, the mean retention time is at least 9 hours. In some embodiments, the mean retention time is at least 10 hours. In some embodiments, the mean retention time is at least 11 hours. In some embodiments, the mean retention time is at least 12 hours. In some embodiments, the primate is a human. In some embodiments, the primate is a rhesus macaque.
As used herein, the term “cryoprotectant” refers to a substance that can slow or prevent ice nucleation, ice-crystal growth, ice formation, or a combination thereof, in a composition. Accordingly, the cryoprotectant improves the post thaw viability of the active ingredient in the composition.
In some embodiments, the cryoprotectant is selected from the group consisting of dimethyl sulfoxide (DMSO) , glycerol, a polyethylene glycol (PEG) , a polysaccharide, a sugar, and an amino acid, or any combination thereof. In some embodiments, the cryoprotectant is selected from the group consisting of trehalose, maltose, sucrose, glucose, lactose, dextran, mannitol, and sorbitol, or any combination thereof. Additional disclosure of the cryoprotectant can be found, for example, in U.S. Pat. No. 9,707,204, which is incorporated by reference in its entirety. In some embodiments, the cryoprotectant comprises trehalose.
In some embodiments, the cryoprotectant is selected from the group consisting of trehalose, glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol, and sucrose, or a combination thereof. In some embodiments, the cryoprotectant is trehalose.
In some embodiments, the pharmaceutical composition comprises no more than 40% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 40% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 35% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.0001%(w/v) to about 30% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 25% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 20%(w/v) of the cryoprotectant.
In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.2% (w/v) of the cryoprotectant. In some embodiments,  the pharmaceutical composition comprises about 0.2% (w/v) to about 0.3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3%(w/v) to about 0.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2%(w/v) to about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 5% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 10%(w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 15% (w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 5%(w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 10% (w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 5% (w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3%(w/v) to about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2%(w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2%(w/v) to about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2%(w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 20% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 20% (w/v) of the cryoprotectant. In some  embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 20% (w/v) of the cryoprotectant.
In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.2%(w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.4% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.6% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.7% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.8% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 0.9%(w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1.1% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1.2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1.3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1.7% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2.2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 4% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 4.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 5.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 6% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 6.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 7% (w/v) of the cryoprotectant. In some  embodiments, the pharmaceutical composition comprises about 7.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 8% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 8.5%(w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 9% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 9.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 11% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 12% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 13% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 14%(w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 15% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 16% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 17% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 18% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 19% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 20% (w/v) of the cryoprotectant.
In some embodiments, the pharmaceutical composition comprises about 4% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3.8%(w/v) to about 4.2% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3.5% (w/v) to about 4.5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 5% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2.5%(w/v) to about 6% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 8% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3%(w/v) to about 6% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 7% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 8% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 3%(w/v) to about 10% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical  composition comprises about 2.5% (w/v) to about 6% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 6% (w/v) of the cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 6% (w/v) of the cryoprotectant.
In some embodiments, the cryoprotectant is trehalose, with a concentration of 0.0001-40%(w/v) . In some embodiments, the cryoprotectant is trehalose, with a concentration of 2%-8%(w/v) .
In some embodiments, the cryoprotectant is glycerol, with a concentration of 0.0001-40%(w/v) . In some embodiments, the cryoprotectant is glycerol, with a concentration of 1-20% (w/v) .
In some embodiments, the cryoprotectant is dimethyl sulfoxide, with a concentration of 0.0001-10% (w/v) . In some embodiments, the cryoprotectant is dimethyl sulfoxide, with a concentration of 1-10% (w/v) .
In some embodiments, the cryoprotectant is ethylene glycol, with a concentration of 0.0001-40% (w/v) . In some embodiments, the cryoprotectant is ethylene glycol, with a concentration of 4-30% (w/v) .
In some embodiments, the cryoprotectant is polyethylene glycol, with a concentration of 0.0001-20% (w/v) . In some embodiments, the cryoprotectant is polyethylene glycol, with a concentration of 5-15% (w/v) .
In some embodiments, the cryoprotectant is sucrose, with a concentration of 0.5 M. In some embodiments, the cryoprotectant is sucrose, with a concentration of 0.1-0.4 M.
As used herein, the term “humectant” refers to a substance that promotes the retention of moisture in a composition.
In some embodiments, the humectant is selected from the group consisting of glycerin, diglycerin, betaine, diols, propylene glycol, butylene glycol, pentylene glycol, propanediol, 1, 2-hexanediol, D-ribose, glucose, sorbitol, dextrose, urea, 2-Pyrrolidone-5-Carboxylic Acid and related salts, inorganic salts of lactic acid, ectoin, lactic acid, betaine, glycolic acid, and lactobionic acid, or any combination thereof. Additional disclosure of the humectant can be found, for example, in US 2020/0375870, which is incorporated by reference in its entirety.
In some embodiments, the humectant is selected from the group consisting of glycerin, mannitol, polyethylene glycol 400, polyethylene glycol 4000, D-sorbitol, chitosan, xylitol, and  sodium hyaluronate, or a combination thereof. In some embodiments, the humectant comprises glycerin. In some embodiments, the humectant is glycerin.
In some embodiments, when the cryoprotectant in the pharmaceutical composition comprises or is glycerol, the humectant does not comprise glycerin.
In some embodiments, when the humectant in the pharmaceutical composition comprises or is glycerin, the cryoprotectant does not comprise glycerol.
In some embodiments, the pharmaceutical composition comprises no more than 30% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.0001%(w/v) to about 30% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 25% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 20%(w/v) of the humectant.
In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 20% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 0.5%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 5% (w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 10% (w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 15% (w/v) to about 20%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5%(w/v) to about 2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 3% (w/v) of the humectant. In some embodiments, the  pharmaceutical composition comprises about 2% (w/v) to about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 10%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 5%(w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 10% (w/v) to about 20% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2%(w/v) to about 1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2%(w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 5% (w/v) to about 20% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1%(w/v) to about 1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 5%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) to about 20% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1%(w/v) to about 2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 10%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) to about 20% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 20%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5%(w/v) to about 20% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3%(w/v) to about 20% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 15% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 20% (w/v) of the humectant.
In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.2%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.4% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.6% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.7% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.8% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 0.9% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.3%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.4% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition  comprises about 1.6% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.7% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.8% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.9% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2.1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2.2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2.3%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2.4% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 2.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 3.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 4% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 4.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 5.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 6% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 6.5%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 7%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 7.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 8% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 8.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 9% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 9.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 10% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 11% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 12% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 13% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 14%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 15%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 16% (w/v) of the humectant. In some embodiments, the pharmaceutical composition  comprises about 17% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 18% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 19% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 20% (w/v) of the humectant.
In some embodiments, the pharmaceutical composition comprises about 1.7% (w/v) to about 2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.6% (w/v) to about 2.1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.5% (w/v) to about 2.2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.4% (w/v) to about 2.3%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.3% (w/v) to about 2.4% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.2% (w/v) to about 2.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.1% (w/v) to about 2.7% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.6% (w/v) to about 1.8% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.5% (w/v) to about 1.9% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.4% (w/v) to about 2%(w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.3% (w/v) to about 2.1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.2% (w/v) to about 2.2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.1% (w/v) to about 2.3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 2.4% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.9% (w/v) to about 2.1% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.8% (w/v) to about 2.2% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.7%(w/v) to about 2.3% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.6% (w/v) to about 2.4% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.5% (w/v) to about 2.5% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.4%(w/v) to about 2.6% (w/v) of the humectant. In some embodiments, the pharmaceutical composition comprises about 1.3% (w/v) to about 2.7% (w/v) of the humectant.
In some embodiments, the humectant is glycerin, with a concentration of 0.0001-30%(w/v) . In some embodiments, the humectant is glycerin, with a concentration of 2-6% (w/v) .
In some embodiments, the humectant is mannitol, with a concentration of 0.0001-7%(w/v) . In some embodiments, the humectant is mannitol, with a concentration of 3-5% (w/v) .
In some embodiments, the humectant is polyethylene glycol 400, with a concentration of 0.0001-20% (w/v) . In some embodiments, the humectant is polyethylene glycol 400, with a concentration of 10-18% (w/v) .
In some embodiments, the humectant is polyethylene glycol 4000, with a concentration of 5-15% (w/v) . In some embodiments, the humectant is polyethylene glycol 4000, with a concentration of 5-10% (w/v) .
In some embodiments, the humectant is D-sorbitol, with a concentration of 3-15% (w/v) . In some embodiments, the humectant is D-sorbitol, with a concentration of 3-6% (w/v) .
In some embodiments, the humectant is chitosan, with a concentration of 0.01-3% (w/v) . In some embodiments, the humectant is chitosan, with a concentration of 0.01-1% (w/v) .
In some embodiments, the humectant is xylitol, with a concentration of 0.0001-10% (w/v) . In some embodiments, the humectant is xylitol, with a concentration of 1-10% (w/v) .
In some embodiments, the humectant is sodium hyaluronate, with a concentration of 0.1-2.0% (w/v) . In some embodiments, the humectant is sodium hyaluronate, with a concentration of 0.1-1.0% (w/v) .
As used herein, the term “emulsifier” refers to a substance that promotes the formation and stabilization of an emulsion.
In some embodiments, the emulsifier is selected from the group consisting of a polysorbate, sodium dodecyl sulfate, a phospholipid, a glycolipid, a triglyceride, lecithin, sodium stearate, potassium stearate, ammonium stearate, sodium oleate, potassium oleate, ammonium oleate, sodium palmitate, potassium palmitate, and ammonium palmitate, or any combination thereof. In some embodiments, the emulsifier is selected from the group consisting of TWEEN 80, TWEEN 60, Vitamin E, Pluronic F68, Pluronic F127, Poloxamer 407, glycerol monostearate, Ascorbyl palmitate lecithin, egg yolk, a phospholipid, a phosphatidylcholine, and a polyethylene glycol-phosphatidyl ethanolamine conjugate, or any combination thereof. Additional disclosure of the emulsifier can be found, for example, in US 2018/0206504 and US 2011/0144578, which are incorporated by reference in their entireties.
In some embodiments, the emulsifier is selected from the group consisting of TWEEN 80, polysorbate 20, lecithin, sorbitan esters, mono-and/or diglycerides, and sodium stearoyl lactylate, or a combination thereof. In some embodiments, the emulsifier comprises TWEEN 80. In some embodiments, the emulsifier is TWEEN 80.
In some embodiments, the pharmaceutical composition comprises no more than 5% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0001%(w/v) to about 5% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 2%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0001% (w/v) to about 0.5% (w/v) of the emulsifier.
In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.002% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.003% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.005% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.01% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.02% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.03% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.03% (w/v) to about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.15%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.003% (w/v) of the emulsifier. In some embodiments, the pharmaceutical  composition comprises about 0.002% (w/v) to about 0.005% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.01%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.02% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.03% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.03%(w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.15% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.15%(w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.005%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.01% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.02% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.03%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.03% (w/v) to about 0.15% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.05%(w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.01% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.02% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.03%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.15% (w/v)  of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.03%(w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.02% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.03% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.05%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.15% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.03%(w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.03%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.15%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.03% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.15%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical  composition comprises about 0.002% (w/v) to about 0.15% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005%(w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.15%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.2% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.4% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.4% (w/v) of the emulsifier.
In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0015%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0025% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0035% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.004% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.0045% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.006% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.007%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.008% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.009% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.011% (w/v) of the emulsifier. In some embodiments, the  pharmaceutical composition comprises about 0.012% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.013% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.014% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.016%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.017% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.018% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.019% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.021% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.022% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.023% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.024% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.025%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.03% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.035% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.04% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.045% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.055% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.06% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.065% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.07%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.075% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.08% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.085% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.09% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.095% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.12% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v)  of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.2%(w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.25% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.35% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.4% (w/v) of the emulsifier.
In some embodiments, the pharmaceutical composition comprises about 0.009% (w/v) to about 0.011% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.008% (w/v) to about 0.012% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.013% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.006% (w/v) to about 0.014% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.015% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.004% (w/v) to about 0.016% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.017% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.018% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.02% (w/v) of the emulsifier.
In some embodiments, the pharmaceutical composition comprises about 0.019% (w/v) to about 0.021% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.018% (w/v) to about 0.022% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.017% (w/v) to about 0.023% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.016% (w/v) to about 0.024% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.025% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.014% (w/v) to about 0.026% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.013% (w/v) to about 0.027% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.028% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.011% (w/v) to about 0.029% (w/v) of the emulsifier. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.03% (w/v) of the emulsifier.
In some embodiments, the emulsifier is TWEEN 80, with a concentration of <5 % (w/v) . In some embodiments, the emulsifier is TWEEN 80, with a concentration of 0.005-2 % (w/v) .
In some embodiments, the emulsifier is Polysorbate 20, with a concentration of <5 %(w/v) . In some embodiments, the emulsifier is Polysorbate 20, with a concentration of 0.005-2 %(w/v) .
In some embodiments, the emulsifier is Lecithin, with a concentration of <5 % (w/v) . In some embodiments, the emulsifier is Lecithin, with a concentration of 0.005-2 % (w/v) .
In some embodiments, the emulsifier is Sorbitan esters, with a concentration of <5 %(w/v) . In some embodiments, the emulsifier is Sorbitan esters, with a concentration of 0.005-2 %(w/v) .
In some embodiments, the emulsifier is Mono-and/or di-glycerides, with a concentration of <5 % (w/v) . In some embodiments, the emulsifier is Mono-and/or di-glycerides, with a concentration of 0.005-2 % (w/v) .
In some embodiments, the emulsifier is Sodium stearoyl lactylate, with a concentration of <5 % (w/v) . In some embodiments, the emulsifier is Sodium stearoyl lactylate, with a concentration of 0.005-2 % (w/v) .
As used herein, the term “controlled release agent” refers to a substance that is capable of controlling the release of the active ingredient in a composition.
In some embodiments, the controlled release agent is selected from the group consisting of acetate succinate, a polyvinyl derivative, polyethylene oxide, polyacrylic acid, modified starch, cross-linked high amylose starch, hydroxypropyl starch, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, cellulose, microcrystalline cellulose, carboxymethylethyl cellulose, cellulose acetate, methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, cellulose phthalate, cellulose acetate, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate succinate, cellulose acetate butyrate, cellulose acetate trimellitate, poloxamer, povidone, alginic acid, sodium alginate, polyethylene glycol, polyethylene glycol alginate, gum (for example, xanthan gum) , polymethacrylate, a copolymer of methacrylic acid and ethyl acrylate, a copolymer of polymethyl vinyl ether and malonic acid anhydride, a copolymer of polymethyl vinyl ether and malonic acid or the ethyl-, isopropyl-, n-butylesters thereof, and zein, or any combination thereof. Additional disclosure of the controlled release agent can be found, for example, in U.S. Pat. No. 8,486,449, which is incorporated by reference in its entirety.
In some embodiments, the controlled release agent is selected from the group consisting of hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone K30, or a combination thereof. In some embodiments, the controlled release agent comprises hydroxypropyl methylcellulose. In some embodiments, the controlled release agent is hydroxypropyl methylcellulose.
In some embodiments, the pharmaceutical composition comprises no more than 10% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 10% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 9% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 8% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 7% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001%(w/v) to about 6% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 4%(w/v) of the controlled release agent.
In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.002% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.005% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.01% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.02% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.05% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.15%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.3% (w/v)  of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.005% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.01% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.02% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.05% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.15%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.01% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.02% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.05% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.15% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 0.5%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 1% (w/v) of the controlled release agent. In some  embodiments, the pharmaceutical composition comprises about 0.3% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.02% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.05% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.15% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02%(w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) to about 2%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.05% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.15% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.1%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.15% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.15%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05%(w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 1%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002%(w/v) to about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 1%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 2% (w/v) of the controlled release agent.
In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical  composition comprises about 0.003% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.004% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.006% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.008% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.009% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.014% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.016% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.018% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.022% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.025% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.03% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.035% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.04% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.045% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.06% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.07% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.08% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.09% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.12% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.14% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about  0.17% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.25% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.4% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.6% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.7% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.8% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.9% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 1% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 1.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 1.5% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 1.7% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 2% (w/v) of the controlled release agent.
In some embodiments, the pharmaceutical composition comprises about 0.019% (w/v) to about 0.021% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.018% (w/v) to about 0.022% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.017% (w/v) to about 0.023% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.025% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.013% (w/v) to about 0.027% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.03% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.05% (w/v) of the controlled release agent.
In some embodiments, the pharmaceutical composition comprises about 0.09% (w/v) to about 0.11% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.08% (w/v) to about 0.12% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.07% (w/v) to about  0.13% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.06% (w/v) to about 0.15% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.17% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.04% (w/v) to about 0.2% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.03% (w/v) to about 0.25%(w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.3% (w/v) of the controlled release agent. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.4% (w/v) of the controlled release agent.
In some embodiments, the controlled release agent is hydroxypropyl methylcellulose, with a concentration of 0.1-5.0% (w/v) . In some embodiments, the controlled release agent is hydroxypropyl methylcellulose, with a concentration of 0.1-1% (w/v) .
In some embodiments, the controlled release agent is polyvinylpyrrolidone K30, with a concentration of 2-10% (w/v) . In some embodiments, the controlled release agent is polyvinylpyrrolidone K30, with a concentration of 3-6% (w/v) .
As used herein, the term “buffering agent” refers to an acid or base component (usually a weak acid or weak base) that is capable of maintaining the pH of a composition at or near a pre-determined value. In some embodiments, a buffering agent is present in a mixture of a weak acid and its conjugate base or a in a mixture of a weak base and its conjugated acid. A “buffer, ” when used in connection with the composition, refers to the buffering agent that is dissolved/mixed into the composition.
In some embodiments, the buffering agent is selected from the group consisting of sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, magnesium oxide, magnesium aluminate, magnesium carbonate, magnesium silicate, magnesium citrate, aluminum hydroxide, aluminum hydroxide/magnesium carbonate, potassium carbonate, potassium citrate, aluminum hydroxide/sodium bicarbonate coprecipitate, aluminum magnesium hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogenphosphate, dipotassium hydrogenphosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, calcium acetate, calcium glycerophosphate, calcium hydroxide, calcium lactate, calcium carbonate, calcium gluconate, calcium bicarbonate, calcium citrate, potassium phosphate, and sodium phosphate, or any combination thereof. In some  embodiments, the buffering agent is selected from the group consisting of citric acid, sodium citrate, sodium acetate, acetic acid, sodium phosphate, phosphoric acid, sodium ascorbate, tartaric acid, maleic acid, glycine, sodium lactate, lactic acid, ascorbic acid, imidazole, sodium bicarbonate and carbonic acid, sodium succinate and succinic acid, histidine, and sodium benzoate and benzoic acid, and any combination thereof. In some embodiments, the buffer is a carbonate buffer, a citrate buffer, a phosphate buffer, an acetate buffer, a hydrochloric acid buffer, a lactic acid buffer, and a tartric acid buffer.
In some embodiments, the buffer is selected from the group consisting of a phosphate buffer, a tris buffer, and a glycine buffer. In some embodiments, the buffer agent comprises phosphate buffered saline. In some embodiments, the buffer agent is phosphate buffered saline.
In some embodiments, the buffer has a pH of about 4-8. In some embodiments, the buffer has a pH of about 4-4.5. In some embodiments, the buffer has a pH of about 4.5-5. In some embodiments, the buffer has a pH of about 5-5.5. In some embodiments, the buffer has a pH of about 5.5-6. In some embodiments, the buffer has a pH of about 6-6.5. In some embodiments, the buffer has a pH of about 6.5-7. In some embodiments, the buffer has a pH of about 7-7.5. In some embodiments, the buffer has a pH of about 7.5-8. In some embodiments, the buffer has a pH of about 4-5. In some embodiments, the buffer has a pH of about 4.5-5.5. In some embodiments, the buffer has a pH of about 5-6. In some embodiments, the buffer has a pH of about 5.5-6.5. In some embodiments, the buffer has a pH of about 6-7. In some embodiments, the buffer has a pH of about 6.5-7.5. In some embodiments, the buffer has a pH of about 7-8. In some embodiments, the buffer has a pH of about 4-5.5. In some embodiments, the buffer has a pH of about 4.5-6. In some embodiments, the buffer has a pH of about 5-6.5. In some embodiments, the buffer has a pH of about 5.5-7. In some embodiments, the buffer has a pH of about 6-7.5. In some embodiments, the buffer has a pH of about 6.5-8. In some embodiments, the buffer has a pH of about 4-6. In some embodiments, the buffer has a pH of about 4.5-6.5. In some embodiments, the buffer has a pH of about 5-7. In some embodiments, the buffer has a pH of about 5.5-7.5. In some embodiments, the buffer has a pH of about 6-8. In some embodiments, the buffer has a pH of about 4-6.5. In some embodiments, the buffer has a pH of about 4.5-7. In some embodiments, the buffer has a pH of about 5-7.5. In some embodiments, the buffer has a pH of about 5.5-8. In some embodiments, the buffer has a pH of about 4-7. In some embodiments, the buffer has a pH of about 4.5-7.5. In some embodiments, the buffer has a pH of about 5-8. In some embodiments, the buffer has a pH of about 4-7.5. In some embodiments, the buffer has a pH of about 4.5-8.
In some embodiments, the buffer has a pH of about 4. In some embodiments, the buffer has a pH of about 4.1. In some embodiments, the buffer has a pH of about 4.2. In some embodiments, the buffer has a pH of about 4.3. In some embodiments, the buffer has a pH of about 4.4. In some embodiments, the buffer has a pH of about 4.5. In some embodiments, the buffer has a pH of about 4.6. In some embodiments, the buffer has a pH of about 4.7. In some embodiments, the buffer has a pH of about 4.8. In some embodiments, the buffer has a pH of about 4.9. In some embodiments, the buffer has a pH of about 5. In some embodiments, the buffer has a pH of about 5.1. In some embodiments, the buffer has a pH of about 5.2. In some embodiments, the buffer has a pH of about 5.3. In some embodiments, the buffer has a pH of about 5.4. In some embodiments, the buffer has a pH of about 5.5. In some embodiments, the buffer has a pH of about 5.6. In some embodiments, the buffer has a pH of about 5.7. In some embodiments, the buffer has a pH of about 5.8. In some embodiments, the buffer has a pH of about 5.9. In some embodiments, the buffer has a pH of about 6. In some embodiments, the buffer has a pH of about 6.1. In some embodiments, the buffer has a pH of about 6.2. In some embodiments, the buffer has a pH of about 6.3. In some embodiments, the buffer has a pH of about 6.4. In some embodiments, the buffer has a pH of about 6.5. In some embodiments, the buffer has a pH of about 6.6. In some embodiments, the buffer has a pH of about 6.7. In some embodiments, the buffer has a pH of about 6.8. In some embodiments, the buffer has a pH of about 6.9. In some embodiments, the buffer has a pH of about 7. In some embodiments, the buffer has a pH of about 7.1. In some embodiments, the buffer has a pH of about 7.2. In some embodiments, the buffer has a pH of about 7.3. In some embodiments, the buffer has a pH of about 7.4. In some embodiments, the buffer has a pH of about 7.5. In some embodiments, the buffer has a pH of about 7.6. In some embodiments, the buffer has a pH of about 7.7. In some embodiments, the buffer has a pH of about 7.8. In some embodiments, the buffer has a pH of about 7.9. In some embodiments, the buffer has a pH of about 8.
In some embodiments, the buffer has a pH of about 5.9-6.1. In some embodiments, the buffer has a pH of about 5.8-6.2. In some embodiments, the buffer has a pH of about 5.7-6.3. In some embodiments, the buffer has a pH of about 5.6-6.4. In some embodiments, the buffer has a pH of about 5.5-6.5. In some embodiments, the buffer has a pH of about 5.4-6.6. In some embodiments, the buffer has a pH of about 5.3-6.7. In some embodiments, the buffer has a pH of about 5.2-6.8. In some embodiments, the buffer has a pH of about 5.1-6.9. In some embodiments, the buffer has a pH of about 5-7.
In some embodiments, the pharmaceutical composition comprises about 1 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 5 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 10 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 15 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 20 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM to about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 50 mM to about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 100 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 200 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 300 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 400 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 10 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 15 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 20 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 50 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 100 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 200 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 300 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 15 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 20 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM  to about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 50 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 100 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 200 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 20 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 50 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 100 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 50 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 100 mM of the buffering agent. In some embodiments, the  pharmaceutical composition comprises about 5 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 200 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 15 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 300 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 500 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 400 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 500 mM of the buffering agent.
In some embodiments, the pharmaceutical composition comprises about 15 mM to about 25 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM to about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 7 mM to about 40 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 2 mM to about 100 mM of the buffering agent.
In some embodiments, the pharmaceutical composition comprises about 5 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 6 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 7 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 8 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 9 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 10 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 12 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 14 mM of the buffering agent. In  some embodiments, the pharmaceutical composition comprises about 16 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 18 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 20 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 22 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 25 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 30 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 35 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 40 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 45 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 50 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 60 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 70 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 80 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 90 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 100 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 120 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 140 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 160 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 180 mM of the buffering agent. In some embodiments, the pharmaceutical composition comprises about 200 mM of the buffering agent.
In some embodiments, the buffer is a phosphate buffer, with a concentration of 10-100 mM.In some embodiments, the buffer is a phosphate buffer, with a concentration of 10-40 mM.
In some embodiments, the buffer is a tris buffer, with a concentration of 10-100 mM. In some embodiments, the buffer is a tris buffer, with a concentration of 10-40 mM.
In some embodiments, the buffer is a glycine buffer, with a concentration of 0.1-0.5 M. In some embodiments, the buffer is a glycine buffer, with a concentration of 0.1-0.2 M.
In some embodiments, the pharmaceutical composition comprises an antibiotic. In some embodiments, the antibiotic is selected from the group consisting of benzalkonium chloride, benzyl alcohol, and chlorobutanol, or a combination thereof. In some embodiments, the antibiotic comprises benzalkonium chloride. In some embodiments, the antibiotic is benzalkonium chloride.
In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 3.0% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 2.5% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 2.0% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 1.5% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 1.0% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.5% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.4% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.3% (w/v) of the antibiotic.
In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.002% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.005% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.007% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.01% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.012% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.015% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.07% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.15% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.005% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.007% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.01% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.012% (w/v) of the antibiotic. In some embodiments,  the pharmaceutical composition comprises about 0.01% (w/v) to about 0.015% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.07% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.1% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.007% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.01% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.012% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.015% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.07% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.01% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.012% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.015% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.05% (w/v) to about  0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.012% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.015% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.02% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.015% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.015% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.012% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.05% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the  pharmaceutical composition comprises about 0.01% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.07% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.1% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.2% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.15% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.2% (w/v) of the antibiotic.
In some embodiments, the pharmaceutical composition comprises about 0.01% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.009%(w/v) to about 0.011% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.008% (w/v) to about 0.012% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.007% (w/v) to about 0.015%(w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.005% (w/v) to about 0.02% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.003% (w/v) to about 0.03% (w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.002% (w/v) to about 0.05%(w/v) of the antibiotic. In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.1% (w/v) of the antibiotic.
In some embodiments, the antibiotic is benzalkonium chloride, with a concentration of 0.002-0.02% (w/v) . In some embodiments, the antibiotic is benzalkonium chloride, with a concentration of 0.005-0.02% (w/v) .
In some embodiments, the antibiotic is benzyl alcohol, with a concentration of 0.001-3.0%(v/v) . In some embodiments, the antibiotic is benzyl alcohol, with a concentration of 1.0-3.0%(v/v) .
In some embodiments, the antibiotic is chlorobutanol, with a concentration of 0.5-2.0%(w/v) . In some embodiments, the antibiotic is chlorobutanol, with a concentration of 1.0-2.0%(w/v) .
In some embodiments, the active ingredient comprises a protein. In some embodiments, the protein comprises an antibody. In some embodiments, the protein comprises the antigen binding fragment of an antibody. In some embodiments, the protein is capable of treating a respiratory disease. In some embodiments, the protein is an antibody or comprises the antigen binding fragment of the antibody. In some embodiments, the antibody neutralizes SARS-CoV-2. In some embodiments, the protein is an anti-CoV-S antibodies or antigen-binding fragment thereof described herein. In some embodiments, the antibody is an antibody described in Table 1. In some embodiments, the protein is or comprises Antibody B.
In some embodiments, the active ingredient has a concentration of about 0.1-100 mg/ml in the pharmaceutical composition. In some embodiments, the active ingredient has a concentration of about 0.1 to about 0.2 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 0.5 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 1 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 2 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 3 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 4 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 5 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 6 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 7 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 8 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 10 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 20 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 50 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 0.5 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 1 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 2 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 3 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 4 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 5 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 6 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 7 to about 10 mg/ml. In some embodiments, the active ingredient has a  concentration of about 8 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 10 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 20 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 1 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 2 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 3 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 4 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 5 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 6 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 7 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 8 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 10 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 2 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 3 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 4 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 5 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 6 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 7 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 8 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 3 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 4 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 5 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 20 mg/ml. In some embodiments, the active ingredient has a  concentration of about 6 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 7 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 4 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 5 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 6 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 5 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 5 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 7 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 10 mg/ml. In some embodiments, the active ingredient has a  concentration of about 0.5 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 20 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 100 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.1 to about 50 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.2 to about 100 mg/ml.
In some embodiments, the active ingredient has a concentration of about 5 mg/ml in the pharmaceutical composition. In some embodiments, the active ingredient has a concentration of about 4.5 to about 5.5 mg/ml. In some embodiments, the active ingredient has a concentration of about 4 to about 6 mg/ml. In some embodiments, the active ingredient has a concentration of about 3 to about 8 mg/ml. In some embodiments, the active ingredient has a concentration of about 2 to about 10 mg/ml. In some embodiments, the active ingredient has a concentration of about 1 to about 25 mg/ml. In some embodiments, the active ingredient has a concentration of about 0.5 to about 50 mg/ml.
Non-limiting examples of various components that may be used in the pharmaceutical composition, and their non-limiting concentration ranges, is listed in Table 3 below.
Table 3: Non-limiting Examples of Formulation Components and Concentration Ranges

Without being bound to theory or mechanism, while the exemplary pharmaceutical composition developed according to the Examples of this application initially aims at intranasal administration (e.g., to the mucosa in the nose) , the pharmaceutical composition of the disclosure can be for other purposes –for example, mucosal administration. In some embodiments, the pharmaceutical composition is for application to mucosa of a subject. Mucosa is the soft tissue that lines the body’s canals and organs in the digestive, respiratory and reproductive systems. In some embodiments, the mucosa is located in the nose, mouth, throat, ear, genital, or anus. In some embodiments, the mucosa is located in the esophagus, lungs, stomach, intestine, bladder, or uterus. In some embodiments, the mucosa is located in the mouth, esophagus, or stomach. In some  embodiments, the mucosa is located in the nose, mouth, pharynx, trachea, or lung. In some embodiments, the mucosa is nasal mucosa.
In some embodiments, the pharmaceutical composition is suitable for intranasal administration to a subject (e.g., a mammal) . In some embodiments, the mammal is a human.
In some embodiments, the cryoprotectant is trehalose between about 1% (w/v) and about 8%(w/v) , and the humectant is glycerin between about 0.01% (w/v) and about 0.1% (w/v) , and the emulsifier is TWEEN 80 between about 1% (w/v) and about 3% (w/v) , the controlled release agent is hydroxypropyl methylcellulose between about 0.01% (w/v) and about 0.5% (w/v) , and the buffer is phosphate buffered saline between about 10 mM and about 30 mM, and the protein is an antibody described herein, such as in Table 1, such as Antibody B.
In some embodiments, the disclosure provides a pharmaceutical composition which provides an efficacious dose of an antibody between about 0.0001 mg/kg to about 2 mg/kg per dose. In some embodiments, the disclosure provides a pharmaceutical composition which provides a low yet efficacious dose of an antibody which is about 0.4 mg per dose to a human.
Anti-CoV-S Antibodies and Antigen-Binding Fragments
The present disclosure provides antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that specifically bind to CoV spike protein or an antigenic fragment thereof. Such antigen-binding proteins can be used as active ingredients in a pharmaceutical composition for the prevention or treatment of coronavirus infection. Examples include: wild-type-CoV-S antigen-binding proteins, such as wild-type-CoV-S antibodies and wild-type-CoV-S antigen-binding fragments thereof, that specifically bind to wild-type-CoV spike protein or an antigenic fragment thereof; alpha-CoV-S antigen-binding proteins (alpha (B. 1.1.7) -CoV-S antigen-binding proteins) , such as alpha-CoV-S antibodies (alpha (B. 1.1.7) -CoV-S antibodies) and alpha-CoV-S antigen-binding fragments (alpha (B. 1.1.7) -CoV-S antigen-binding fragments) thereof, that specifically bind to alpha-CoV spike protein or an antigenic fragment thereof; beta-CoV-S antigen-binding proteins (beta (B. 1.351) -CoV-S antigen-binding proteins) , such as beta-CoV-S antibodies (beta (B. 1.351) -CoV-S antibodies) and beta-CoV-S antigen-binding fragments (beta (B. 1.351) -CoV-S antigen-binding fragments) thereof, that specifically bind to beta-CoV spike protein or an antigenic fragment thereof; gamma-CoV-S antigen-binding proteins (gamma (P.1) -CoV-S antigen-binding proteins) , such as gamma-CoV-S antibodies (gamma (P. 1) -CoV-S antibodies) and gamma-CoV-S antigen-binding fragments (gamma (P. 1) -CoV-S antigen-binding fragments) thereof, that specifically bind to gamma-CoV spike protein or an antigenic fragment  thereof; delta-CoV-S antigen-binding proteins (delta (B. 1.617.2) -CoV-S antigen-binding proteins) , such as delta-CoV-S antibodies (delta (B. 1.617.2) -CoV-S antibodies) and delta-CoV-S antigen-binding fragments (delta (B. 1.617.2) -CoV-S antigen-binding fragments) thereof, that specifically bind to delta-CoV spike protein or an antigenic fragment thereof; omicron-CoV-S antigen-binding proteins (omicron (BA. 1 or BA. 2) -CoV-S antigen-binding proteins) , such as omicron-CoV-S antibodies (omicron (BA. 1 or BA. 2) -CoV-S antibodies) and omicron-CoV-S antigen-binding fragments (omicron (BA. 1 or BA. 2) -CoV-S antigen-binding fragments) thereof, that specifically bind to omicron-CoV spike protein or an antigenic fragment thereof. The present disclosure provides antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that specifically bind to the RBD-domain of the CoV spike protein.
The term “antibody” , as used herein, refers to immunoglobulin molecules comprising four polypeptide chains, two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds (i.e., “full antibody molecules” ) , as well as multimers thereof (e.g. IgM) . Exemplary antibodies include, for example, those listed in Table 1. Each heavy chain comprises a heavy chain variable region ( “HVR” or “VH” ) and a heavy chain constant region (comprised of domains CH1, CH2 and CH3) . Each light chain is comprised of a light chain variable region ( “LVR or “VL” ) and a light chain constant region (CL) . The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) . Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Heavy chain CDRs can also be referred to as HCDRs or CDR-Hs, and numbered as described above (e.g., HCDR1, HCDR2, and HCDR3 or CDR-H1, CDR-H2, and CDR-H3) . Likewise, light chain CDRs can be referred to as LCDRs or CDR-Ls, and numbered LCDR1, LCDR2, and LCDR3, or CDR-L1, CDR-L2, and CDR-L3. In some embodiments, the assignment of amino acids to each domain is in accordance with the definitions of the international ImMunoGeneTics information  (see www. imgt. org) .
In some embodiments, the FRs of the antibody (or antigen binding fragment thereof) are identical to the human germline sequences, or are naturally or artificially modified. Exemplary human germline sequences include, but are not limited to, VH3-66 and Vkl-33. Thus, the present disclosure provides anti-CoV-S antibodies or antigen-binding fragments thereof (e.g., anti-SARS-CoV-2-S antibodies or antigen-binding fragments thereof) comprising HCDR and LCDR sequences of Table 1 within a VH3-66 or Vkl-33 variable heavy chain or light chain region.
The present disclosure further provides anti-CoV-S antibodies or antigen-binding fragments thereof (e.g., anti-SARS-CoV-2-S antibodies or antigen-binding fragments thereof) comprising HCDR and LCDR sequences of Table 1. The present disclosure further provides anti-CoV-S antibodies or antigen-binding fragments thereof (e.g., anti-SARS-CoV-2-S antibodies or antigen-binding fragments thereof) comprising HVR and LVR sequences of Table 1 within a combination of a light chain described herein, and a heavy chain described herein.
The present disclosure includes monoclonal anti-CoV-S antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, as well as monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins. The term “monoclonal antibody” , as used herein, refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. A “plurality” of such monoclonal antibodies and fragments in a composition refers to a concentration of identical (i.e., as discussed above, in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts) antibodies and fragments which is above that which would normally occur in nature, e.g., in the blood of a host organism such as a mouse or a human.
In some embodiments, an anti-CoV-S antigen-binding protein, e.g., antibody or antigen-binding fragment comprises a heavy chain constant domain, e.g., of the type IgA (e.g., IgA1 or IgA2) , IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 and IgG4) or IgM. In some embodiments, antibody or antigen-binding fragment comprises a heavy chain constant domain of the type IgG.
In some embodiments, the IgG is a IgG1, IgG2, IgG3, or IgG4 type. In some embodiments, the IgG is IgG1. In some embodiments, the IgG is IgG2. In some embodiments, the IgG is IgG3. In some embodiments, the IgG is IgG4.
In some embodiments, an antigen-binding protein, e.g., antibody or antigen-binding fragment comprises a light chain constant domain, e.g., of the type kappa or lambda.
In some embodiments, the antibody is a human antibody. The term “human” antigen-binding protein, such as an antibody, as used herein, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences whether in a human cell or grafted into a non-human cell, e.g., a mouse cell. See e.g., US8502018, US6596541 or US5789215. The human mAbs of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) , for example in the CDRs and, in particular, CDR3. However, the term “human antibody” , as used herein, is not intended to include  mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject. See below.
The present disclosure includes anti-CoV-S chimeric antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, and methods of use thereof. As used herein, a “chimeric antibody” is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species. (US4816567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855) .
The term “recombinant” antigen-binding proteins, such as antibodies or antigen-binding fragments thereof, refers to such molecules created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression. The term includes antibodies expressed in a nonhuman mammal (including transgenic non-human mammals, e.g., transgenic mice) , or a cell (e.g., CHO cells) expression system, or a non-human cell expression system, or isolated from a recombinant combinatorial human antibody library. In some embodiments, a recombinant antibody shares a sequence with an antibody isolated from an organism (e.g., a mouse or a human) , but has been expressed via recombinant DNA technology. Such antibodies may have post-translational modifications (e.g., glycosylation) that differ from the antibody as isolated from the organism.
In some embodiments, the active ingredient comprises a recombinant and/or chimeric antigen-binding protein.
Recombinant anti-CoV-S antigen-binding proteins, e.g., antibodies and antigen-binding fragments, disclosed herein may also be produced in an E. coli/T7 expression system. In some embodiments, nucleic acids encoding the anti-CoV-S antibody immunoglobulin molecules of the disclosure (e.g., as found in Table 1) may be inserted into a pET-based plasmid and expressed in the E. coli/T7 system. For example, the present disclosure includes methods for expressing an antibody or antigen-binding fragment thereof or immunoglobulin chain thereof in a host cell (e.g., bacterial host cell such as E. coli such as BL21 or BL21DE3) comprising expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding an immunoglobulin chain that is operably linked to a T7 promoter. For example, In some embodiments, a bacterial host cell, such as an E. coli, includes a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside) . See US4952496 and  US5693489 or Studier &Moffatt, Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol. 1986 May 5; 189 (1) : 113-30.
There are several methods by which to produce recombinant antibodies which are known in the art. One example of a method for recombinant production of antibodies is disclosed in US4816567.
Transformation can be by any known method for introducing polynucleotides (e.g., DNA or RNA, including mRNA) into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) in liposomes, lipid nanoparticle technology, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors such as lentivirus or adeno-associated virus. Methods of transforming cells are well known in the art. See, for example, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455. In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure can be introduced to a subject in nucleic acid form (e.g, DNA or RNA, including mRNA) , such that the subject’s own cells produce the antibody. The present disclosure further provides modifications to nucleotide sequences encoding the anti-CoV-S antibodies described herein that result in increased antibody expression, increased antibody stability, increased nucleic acid (e.g., mRNA) stability, or improved affinity or specificity of the antibodies for the CoV spike protein.
Thus, the present disclosure includes recombinant methods for making an anti-CoV-S antigen-binding protein, such as an antibody or antigen-binding fragment thereof of the present disclosure, or an immunoglobulin chain thereof, comprising (i) introducing one or more polynucleotides described herein encoding light and/or heavy immunoglobulin chains, or CDRs, of the antigen-binding protein, wherein the polynucleotide is in a vector; and/or integrated into a host cell chromosome and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., CHO or Pichia or Pichiapastoris) under condition favorable to expression of the polynucleotide and, (iii) optionally, isolating the antigen-binding protein, (e.g., antibody or fragment) or chain from the host cell and/or medium in which the host cell is grown. For example, a polynucleotide can be integrated into a host cell chromosome through targeted insertion with a vector such as adeno-associated virus (AAV) , e.g., after cleavage of the chromosome using a gene editing system (e.g., CRISPR (for example, CRISPR-Cas9) , TALEN, megaTAL, zinc finger, or Argonaute) . Targeted insertions can take place, for example, at host cell loci such as an albumin  or immunoglobulin genomic locus. Alternatively, insertion can be at a random locus, e.g., using a vector such as lentivirus. When making an antigen-binding protein (e.g., antibody or antigen-binding fragment) comprising more than one immunoglobulin chain, e.g., an antibody that comprises two heavy immunoglobulin chains and two light immunoglobulin chains, co-expression of the chains in a single host cell leads to association of the chains, e.g., in the cell or on the cell surface or outside the cell if such chains are secreted, so as to form the antigen-binding protein (e.g., antibody or antigen-binding fragment) . The methods include those wherein only a heavy immunoglobulin chain or only a light immunoglobulin chain (e.g., any of those discussed herein including mature fragments and/or variable domains thereof) is expressed. Such chains are useful, for example, as intermediates in the expression of an antibody or antigen-binding fragment that includes such a chain. For example, the present disclosure also includes anti-CoV-S antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, comprising a heavy chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by a polynucleotide comprising a nucleotide sequence described herein and a light chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by a nucleotide sequence described herein which are the product of such production methods, and, optionally, the purification methods set forth herein. For example, in some embodiments, the product of the method is an anti-CoV-S antigen-binding protein which is an antibody or fragment comprising an HVR comprising an amino acid sequence set forth in Table 1 and an LVR comprising an amino acid sequence set forth in Table 1, wherein the HVR and LVR sequences are selected from a single antibody listed in Table 1. In some embodiments, the product of the method is an anti-CoV-S antigen-binding protein which is an antibody or fragment comprising HCDR1, HCDR2, and HCDR3 comprising amino acid sequences set forth in Table 1 and LCDR1, LCDR2, and LCDR3 comprising amino acid sequences set forth in Table 1, wherein the six CDR sequences are selected from a single antibody listed in Table 1. In some embodiments, the product of the method is an anti-CoV-S antigen-binding protein which is an antibody or fragment comprising a heavy chain comprising an HC amino acid sequence set forth in Table 1 and a light chain comprising an LC amino acid sequence set forth in Table 1.
Eukaryotic and prokaryotic host cells, including mammalian cells, may be used as hosts for expression of an anti-CoV-S antigen-binding protein. Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC) . These host cells include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS) , human hepatocellular carcinoma cells (e.g., Hep G2) , A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells  include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Other cell lines that may be used are insect cell lines (e.g., Spodopterafrugiperda or Trichoplusia ni) , amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri) , Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. The present disclosure includes an isolated host cell (e.g., a CHO cell) comprising an antigen-binding protein, such as those of Table 1; or a polynucleotide encoding such a polypeptide thereof.
The term “specifically binds” refers to those antigen-binding proteins (e.g., mAbs) having a binding affinity to an antigen, such as a CoV-S protein (e.g., SARS-CoV-2-S) or the RBD-domain of a CoV-S protein (e.g., RBD-SARS-CoV-2-S) , expressed as KD, of at least about 10- 8M, as measured by real-time, label free bio-layer interferometry assay, for example, at 25℃ or 37℃, e.g., an HTX biosensor, or by surface plasmon resonance, e.g., BIACORETM, or by solution-affinity ELISA. The present disclosure includes antigen-binding proteins that specifically bind to a CoV-S protein, or to the RBD-domain of a CoV-S protein.
An antigen-binding fragment of an antibody, in some embodiments, comprises at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH -VH, VH -VL or VL -VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
In some embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (V) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (Viii) VL-CH1; (ix) VL- CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond (s) ) .
In some embodiments, the present disclosure provides an isolated protein (e.g., an antibody or antigen binding fragment thereof) . “Isolated” antigen-binding proteins, antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides and vectors, are at least partially free of other biological molecules from the cells or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments. In some embodiments, “Isolated” antigen-binding proteins, antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides and vectors, are essentially free of other biological molecules from the cells or cell culture from which they are produced. In some embodiments, “Isolated” antigen-binding proteins, antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides and vectors, are free of other biological molecules from the cells or cell culture from which they are produced.
The term “epitope” refers to an antigenic determinant (e.g., a CoV-S polypeptide) that interacts with a specific antigen-binding site of an antigen-binding protein, e.g., a variable region of an antibody molecule, known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be  defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may be linear or conformational, that is, composed of non-linear amino acids. In some embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, In some embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
Methods for determining the epitope of an antigen-binding protein, e.g., antibody or fragment or polypeptide, include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63) , peptide cleavage analysis, crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496) . Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding protein (e.g., antibody or fragment or polypeptide) (e.g., coversin) interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antigen-binding protein, e.g., antibody or fragment or polypeptide, to the deuterium-labeled protein. Next, the CoV-S protein/antigen-binding protein complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antigen-binding protein interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface. After dissociation of the antigen-binding protein (e.g., antibody or fragment or polypeptide) , the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antigen-binding protein interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.
The term “compete” as used herein, refers to an antigen-binding protein (e.g., antibody or antigen-binding fragment thereof) that binds to an antigen (e.g., CoV-S) and inhibits or blocks the binding of another antigen-binding protein (e.g., antibody or antigen-binding fragment thereof) to the antigen. The term also includes competition between two antigen-binding proteins e.g., antibodies, in both orientations, i.e., a first antibody that binds and blocks binding of second  antibody and vice versa. In some embodiments, the first antigen-binding protein (e.g., antibody) and second antigen-binding protein (e.g., antibody) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) may bind to different, but, for example, overlapping epitopes, wherein binding of one inhibits or blocks the binding of the second antibody, e.g., via steric hindrance. Competition between antigen-binding proteins (e.g., antibodies) may be measured by methods known in the art, for example, by a real-time, label-free bio-layer interferometry assay. Epitope mapping (e.g., via alanine scanning or hydrogen-deuterium exchange (HDX) ) can be used to determine whether two or more antibodies are non-competing (e.g., on a spike protein receptor binding domain (RBD) monomer) , competing for the same epitope, or competing but with diverse micro-epitopes (e.g., identified through HDX) . In some embodiments, competition between a first and second anti-CoV-S antigen-binding protein (e.g., antibody) is determined by measuring the ability of an immobilized first anti-CoV-S antigen-binding protein (e.g., antibody) (not initially complexed with CoV-S protein) to bind to soluble CoV-S protein complexed with a second anti-CoV-S antigen-binding protein (e.g., antibody) . A reduction in the ability of the first anti-CoV-S antigen-binding protein (e.g., antibody) to bind to the complexed CoV-S protein, relative to uncomplexed CoV-S protein, indicates that the first and second anti-CoV-S antigen-binding proteins (e.g., antibodies) compete. The degree of competition can be expressed as a percentage of the reduction in binding. Such competition can be measured using a real time, label-free bio-layer interferometry assay, e.g., on an Octet RED384 biosensor (Pall ForteBio Corp. ) , ELISA (enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance) .
Binding competition between anti-CoV-S antigen-binding proteins (e.g., monoclonal antibodies (mAbs) ) can be determined using a real time, label-free bio-layer interferometry assay on an Octet RED384 biosensor (Pall ForteBio Corp. ) . For example, to determine competition between two anti-CoV-S monoclonal antibodies, the anti-CoV-S mAb can be first captured onto anti-hFc antibody coated Octet biosensor tips (Pall ForteBio Corp., #18-5060) by submerging the tips into a solution of anti-CoV-S mAb (subsequently referred to as “mAb 1” ) . As a positive-control for blocking, the antibody captured biosensor tips can then be saturated with a known blocking isotype control mAb (subsequently referred to as “blocking mAb” ) by dipping into a solution of blocking mAb. To determine if mAb2 competes with mAb 1, the biosensor tips can then be subsequently dipped into a co-complexed solution of CoV-S polypeptide and a second anti-CoV-S mAb (subsequently referred to as “mAb2” ) , that had been pre-incubated for a period of time and binding of mAbl to the CoV-S polypeptide can be determined. The biosensor tips can be washed in buffer in between every step of the experiment. The real-time binding response can  be monitored during the course of the experiment and the binding response at the end of every step can be recorded.
For example, in some embodiments, the competition assay is conducted at 25 ℃ and pH about 7, e.g., 7.4, e.g., in the presence of buffer, salt, surfactant and a non-specific protein (e.g., bovine serum albumin) .
Typically, an antibody or antigen-binding fragment of the disclosure which is modified in some way retains the ability to specifically bind to CoV-S, e.g., retains at least 10%of its CoV-S binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment of the disclosure retains at least 20%, 50%, 70%, 80%, 90%, 95%or 100%or more of the CoV-S binding affinity as the parental antibody. It is also intended that an antibody or antigen-binding fragment of the disclosure can include conservative or non-conservative amino acid substitutions (referred to as “conservative alternates” or “function conserved alternates” of the antibody) that do not substantially alter its biologic activity.
An “alternate” of a polypeptide” , such as an immunoglobulin chain refers to a polypeptide comprising an amino acid sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to a referenced amino acid sequence that is set forth herein (e.g., SEQ ID NO: 2, 10, 18, 20, 22, 30, 38, 40, 42, 50, 58, or 60) ; when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expect threshold: 10; word size: 3; max matches in a query range: 0; BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional compositional score matrix adjustment) .
An “alternate” of a polynucleotide refers to a polynucleotide comprising a nucleotide sequence that is at least about 70-99.9% (e.g., at least about 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9%) identical to a referenced nucleotide sequence that is set forth herein (e.g., SEQ ID NO: 1, 9, 17, 19, 21, 29, 37, 39, 41, 49, 57, or 59) ; when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expect threshold: 10; word size: 28; max matches in a query range: 0; match/mismatch scores: 1, -2; gap costs: linear) .
Anti-CoV-S antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof of the present disclosure, In some embodiments, include a heavy chain immunoglobulin  variable region having at least 70% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the HVR amino acid sequences set forth in Table 1; and/or a light chain immunoglobulin variable region having at least 70% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the LVR amino acid sequences set forth in Table 1.
In addition, an alternate anti-CoV-S antigen-binding protein may include a polypeptide comprising an amino acid sequence that is set forth herein except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or more) mutations such as, for example, missense mutations (e.g., conservative substitutions) , non-sense mutations, deletions, or insertions. For example, the present disclosure includes antigen-binding proteins which include an immunoglobulin light chain alternate comprising an LVR amino acid sequence set forth in Table 1 but having one or more of such mutations and/or an immunoglobulin heavy chain alternate comprising an HVR amino acid sequence set forth in Table 1 but having one or more of such mutations. In some embodiments, an alternate anti-CoV-S antigen-binding protein includes an immunoglobulin light chain alternate comprising LCDR1, LCDR2 and LCDR3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g., conservative substitutions) and/or an immunoglobulin heavy chain alternate comprising HCDR1, HCDR2, and HCDR3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g., conservative substitutions) . Substitutions can be in a CDR, framework, or constant region.
The disclosure further provides alternate anti-CoV-S antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof, comprising one or more alternate CDRs (e.g., any one or more of LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and/or HCDR3) that are set forth herein with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%sequence identity to, e.g., the heavy chain and light chain CDRs of Table 1.
Embodiments of the present disclosure also include alternate antigen-binding proteins, e.g., anti-CoV-S antibodies and antigen-binding fragments thereof, that comprise immunoglobulin VHs and VLs; or HCs and LCs, which comprise an amino acid sequence having 70%or more (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) overall amino acid sequence identity or similarity to the amino acid sequences of the corresponding VHs, VLs, HCs or LCs specifically set forth herein, but wherein the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of such immunoglobulins are not alternates and  comprise CDR amino acid sequence set forth in Table 1. Thus, in such embodiments, the CDRs within alternate antigen-binding proteins are not, themselves, alternates.
Conservatively/Function-conservative modified alternate anti-CoV-S antibodies and antigen-binding fragments thereof are also part of the present disclosure. A “conservatively modified alternate” or a “conservative substitution” refers to an alternate wherein there is one or more substitutions of amino acids in a polypeptide with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc. ) . Such changes can frequently be made without significantly disrupting the biological activity of the antibody or fragment. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4’ Ed. ) ) . In addition, substitutions of structurally or functionally similar amino acids are less likely to significantly disrupt biological activity.
Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45.
Function-conservative alternates of the anti-CoV-S antibodies and antigen-binding fragments thereof are also part of the present disclosure. Any of the alternates of the anti-CoV-S antibodies and antigen-binding fragments thereof (as discussed herein) may be “function-conservative alternates” . Such function-conservative alternates may, in some cases, also be characterized as conservatively modified alternates. “Function-conservative alternates, ” as used herein, refers to alternates of the anti-CoV-S antibodies or antigen-binding fragments thereof in which one or more amino acid residues have been changed without significantly altering one or more functional properties of the antibody or fragment. In some embodiments, a function-conservative alternate anti-CoV-S antibody or antigen-binding fragment thereof of the present  disclosure comprises an alternate amino acid sequence and exhibits one or more of the following functional properties:
● Inhibits growth of coronavirus (e.g., SARS-CoV-2, SARS-CoV, and/or MERS-CoV) in ACE2-and/or TMPRSS2-expressing cells (e.g., Calu-3 cells) ;
● Does not significantly bind to MDCK/Tet-on cells which do not express ACE2 and/or TMPRSS2;
● Inhibits the binding or interaction of coronavirus or coronavirus pseudovirus with ACE2-and/or TMPRSS2-expressing cells.
● Limits spread of coronavirus infection (e.g., by SARS-CoV-2, SARS-CoV, and/or MERS-CoV) of cells, e.g., Calu-3, in vitro;
● Protects a mouse engineered to express the human TMPRSS2 and/or ACE2 protein from death caused by coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) , for example, wherein the mice are infected with an otherwise lethal dose of the virus, optionally when combined with a second therapeutic agent;
● Protects a mouse engineered to express the human TMPRSS2 and/or ACE2 protein from weight loss caused by coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) , for example, wherein the mice are infected with a dose of the virus that would otherwise cause weight loss, optionally when combined with a second therapeutic agent;
● Protects a hamster from death caused by coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) , for example, wherein the animal is infected with an otherwise lethal dose of the virus, optionally when combined with a second therapeutic agent;
● Protects a hamster from weight loss caused by coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) , for example, wherein the animal is infected with a dose of the virus that would otherwise cause weight loss, optionally when combined with a second therapeutic agent; and/or
● Protects a human subject from coronavirus infection.
A “neutralizing” or “antagonist” anti-CoV-S antigen-binding protein, e.g., antibody or antigen-binding fragment, refers to a molecule that inhibits an activity of CoV-S to any detectable  degree, e.g., inhibits the ability of CoV-S to bind to a receptor such as ACE2, to be cleaved by a protease such as TMPRSS2, or to mediate viral entry into a host cell or viral reproduction in a host cell. In some embodiments, the neutralizing anti-CoV-S antigen-binding protein inhibits the ability of CoV-S to bind to a receptor such as ACE2 by between about 15%to about 25%, or between about 25%to about 35%, or between about 35%to about 45%, or between about 45%to about 55%, or between about 55%to about 65%, or between about 65%to about 75%, or between about 65%to about 80%, or between about 75%to about 85%, or between about 85%to about 95%, or between about 95%to about 99%, or between about 90%to about 99%, or between about 95%to 100%.
Table 1 refers to antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that comprise the heavy chain or VH (or an alternate thereof) and light chain or VL (or an alternate thereof) as set forth below; or that comprise a VH that comprises the CDRs thereof (HCDR1 (or an alternate thereof) , HCDR2 (or an alternate thereof) and HCDR3 (or an alternate thereof) ) and a VL that comprises the CDRs thereof (LCDR1 (or an alternate thereof) , LCDR2 (or an alternate thereof) and LCDR3 (or an alternate thereof) ) , e.g., wherein the immunoglobulin chains, variable regions and/or CDRs comprise the specific amino acid sequences described below.
The antibodies described herein also include embodiments wherein the VH is fused to a wild-type IgG4 (e.g., wherein residue 108 is S) or to IgG4 alternates (e.g., wherein residue 108 is P) .
Antibodies and antigen-binding fragments of the present disclosure comprise immunoglobulin chains including the amino acid sequences set forth herein as well as cellular and in vitro post-translational modifications to the antibody. For example, the present disclosure includes antibodies and antigen-binding fragments thereof that specifically bind to CoV-S comprising heavy and/or light chain amino acid sequences set forth herein (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3) as well as antibodies and fragments wherein one or more amino acid residues is glycosylated, one or more Asn residues is deamidated, one or more residues (e.g., Met, Trp and/or His) is oxidized, the N-terminal Gln is pyroglutamate (pyroE) and/or the C-terminal Lysine is missing.
The amino acid and nucleotide sequences of exemplary anti-SARS-CoV-2-Spike protein (SARS-CoV-2-S) antibodies are shown in the Table 1: Exemplary Sequences, below.
Table 1: Exemplary Sequences







Additional antibodies or antigen binding fragments can be found, for example, in: Chinese Pat. No. 111909261B, Chinese Pat. No. 111909263B, Chinese Pat. No. 111925444B, Chinese Pat. No. 111909260B, Chinese Pat. No. 111925440B, Chinese Pat. No. 111925441B, Chinese Pat. Publ. No. 114989293A, Chinese Pat. No. CN111925442B, Chinese Pat. No. CN111925443B, WO2022037616, WO2022036788, WO2022037033, the content of each of which is incorporated by reference in its entirety. Further antibodies or antigen binding fragments include those that comprises the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of any one of the antibodies disclosed in these patents and applications.
Addition descriptions of the antibodies or antigen binding fragments can be found in the research articles: “Potent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants, ” Li et al., Nat Commun, 2021 Nov 2; 12 (1) : 6304; and “A cocktail containing two synergetic antibodies broadly neutralizes SARS-CoV-2 and its variants including Omicron BA. 1 and BA. 2, ” by Zhang et al., bioRxiv, doi: 10.1101/2022.04.26.489529, the content of each of which is incorporated by reference in its entirety.
In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable domain (VH) comprising:
- an HCDR1 comprising the amino acid sequence of GFTFSGSA (SEQ ID NO: 13) ;
- an HCDR2 comprising the amino acid sequence of IVVGSGNT (SEQ ID NO: 14) ; and
- an HCDR3 comprising the amino acid sequence of AAPYCSSTSCRDGFDI (SEQ ID NO: 15) .
In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain variable domain (VL) comprising:
- a LCDR1 comprising the amino acid sequence of QSVRSSY (SEQ ID NO: 10) ;
- a LCDR2 comprising the amino acid sequence of GAS (SEQ ID NO: 11) ; and
- a LCDR3 comprising the amino acid sequence of QQYGRSPWT (SEQ ID NO: 12) .
In some embodiments, the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 18, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto. In some embodiments, the VH comprises, consists essentially of, or consists of a sequence having at least 95%identity to SEQ ID NO: 18. In some embodiments, the VL comprises, consists essentially of, or consists  of the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto. In some embodiments, the VL comprises, consists essentially of, or consists of a sequence having at least 95%identity to SEQ ID NO: 16.
In some embodiments, the antibody or antigen binding fragment is capable of binding to a SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike proteins comprises the amino acid sequence set forth in SEQ ID NO: 76, SEQ ID NO: 78, or SEQ ID NO: 80, or a sequence having at least 95%, at least 98%, or at least 99%sequence identity thereto.
In some embodiments, the antibody is an IgG, an IgM, or an IgA. In some embodiments, the antigen binding fragment is an Fab fragment, an F (ab’) 2 fragment, an Fd fragment, an Fv fragment, or a single-chain Fv (scFv) molecules.
In some embodiments, the antibody or antigen binding fragment is for intranasal, subcutaneous, intravenous, or intramuscular administration. In some embodiments, the antibody or antigen binding fragment is for intranasal administration.
The disclosure further provides method of producing the antibody or antigen binding fragment of the disclosure, comprising culturing a cell comprising a vector encoding the antibody or antigen binding fragment thereof and harvesting the antibody or antigen binding fragment from the culture medium of the cell.
The disclosure provides a combination of antibodies or antigen binding fragments of the disclosure. In some embodiments, the combination displays a synergistic effect against one or more coronavirus strains. In some embodiments, the combination displays a synergistic effect against COVID-19 Omicron BA. 2, BA. 2.12.1, or BA. 5 strain. In some embodiments, the combination comprises at least two different antibodies or antigen binding fragments, each comprising the HCDRs and LCDRs of one of the antibodies listed in Table 1. In some embodiments, the combination comprises one of the following embodiments in Table 2 below. In some embodiments, the combination comprises at least two antibodies and/or antigen binding fragments, each comprising the HCDRs or LCDRs of the antibodies according to one of combinations #1 to #10 of Table 2. In some embodiments, the combination comprises at least three antibodies and/or antigen binding fragments, each comprising the HCDRs and LCDRs of the antibodies according to one of combinations #11 to #20 of Table 2.
Table 2: Exemplary Combinations of Antibodies and/or Antigen Binding Fragments
In some embodiments, the combination comprises (i) an antibody or antigen binding fragment comprising the HCDRs and LCDRs of antibody B in Table 1; and (ii) an antibody or antigen binding fragment comprising the HCDRs and LCDRs of antibody D/E in Table 1. See, for examples, one of combinations #6, #12, #17, and #19 in Table 2. In some embodiments, the ratio of (i) an antibody or antigen binding fragment comprising the HCDRs and LCDRs of antibody B in Table 1 to (ii) an antibody or antigen binding fragment comprising the HCDRs and LCDRs of antibody D/E is between about 1: 1 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 2. In some embodiments, the ratio is between about 1: 2 to about 1: 3. In some embodiments, the ratio is between about 1: 3 to about 1: 4. In some embodiments, the ratio is between about 1: 4 to about 1: 5. In some embodiments, the ratio is between about 1: 5 to about 1: 6. In some embodiments, the ratio is between about 1: 6 to about 1: 7. In some embodiments, the ratio is between about 1: 7 to about 1: 8. In some embodiments,  the ratio is between about 1: 8 to about 1: 9. In some embodiments, the ratio is between about 1: 9 to about 1: 10. In some embodiments, the ratio is between about 1: 10 to about 1: 15. In some embodiments, the ratio is between about 1: 15 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 3. In some embodiments, the ratio is between about 1: 2 to about 1: 4. In some embodiments, the ratio is between about 1: 3 to about 1: 5. In some embodiments, the ratio is between about 1: 4 to about 1: 6. In some embodiments, the ratio is between about 1: 5 to about 1: 7. In some embodiments, the ratio is between about 1: 6 to about 1: 8. In some embodiments, the ratio is between about 1: 7 to about 1: 9. In some embodiments, the ratio is between about 1: 8 to about 1: 10. In some embodiments, the ratio is between about 1: 9 to about 1: 15. In some embodiments, the ratio is between about 1: 10 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 4. In some embodiments, the ratio is between about 1: 2 to about 1: 5. In some embodiments, the ratio is between about 1: 3 to about 1: 6. In some embodiments, the ratio is between about 1: 4 to about 1: 7. In some embodiments, the ratio is between about 1: 5 to about 1: 8. In some embodiments, the ratio is between about 1: 6 to about 1: 9. In some embodiments, the ratio is between about 1: 7 to about 1: 10. In some embodiments, the ratio is between about 1: 8 to about 1: 15. In some embodiments, the ratio is between about 1: 9 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 5. In some embodiments, the ratio is between about 1: 2 to about 1: 6. In some embodiments, the ratio is between about 1: 3 to about 1: 7. In some embodiments, the ratio is between about 1: 4 to about 1: 8. In some embodiments, the ratio is between about 1: 5 to about 1: 9. In some embodiments, the ratio is between about 1: 6 to about 1: 10. In some embodiments, the ratio is between about 1: 7 to about 1: 15. In some embodiments, the ratio is between about 1: 8 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 6. In some embodiments, the ratio is between about 1: 2 to about 1: 7. In some embodiments, the ratio is between about 1: 3 to about 1: 8. In some embodiments, the ratio is between about 1: 4 to about 1: 9. In some embodiments, the ratio is between about 1: 5 to about 1: 10. In some embodiments, the ratio is between about 1: 6 to about 1: 15. In some embodiments, the ratio is between about 1: 7 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 7. In some embodiments, the ratio is between about 1: 2 to about 1: 8. In some embodiments, the ratio is between about 1: 3 to about 1: 9. In some embodiments, the ratio is between about 1: 4 to about 1: 10. In some embodiments, the ratio is between about 1: 5 to about 1: 15. In some embodiments, the ratio is between about 1: 6 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 8. In some embodiments, the ratio is between about 1: 2 to about 1: 9. In some embodiments, the ratio is between about 1: 3 to about 1: 10. In some embodiments, the ratio is between about  1: 4 to about 1: 15. In some embodiments, the ratio is between about 1: 5 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 9. In some embodiments, the ratio is between about 1: 2 to about 1: 10. In some embodiments, the ratio is between about 1: 3 to about 1: 15. In some embodiments, the ratio is between about 1: 4 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 10. In some embodiments, the ratio is between about 1: 2 to about 1: 15. In some embodiments, the ratio is between about 1: 3 to about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 15. In some embodiments, the ratio is between about 1: 2 to about 1: 20. In some embodiments, the ratio is about 1: 1. In some embodiments, the ratio is about 1: 1.1. In some embodiments, the ratio is about 1: 1.3. In some embodiments, the ratio is about 1: 1.5. In some embodiments, the ratio is about 1: 1.7. In some embodiments, the ratio is about 1: 2. In some embodiments, the ratio is about 1: 2.2. In some embodiments, the ratio is about 1: 2.5. In some embodiments, the ratio is about 1: 2.7. In some embodiments, the ratio is about 1: 3. In some embodiments, the ratio is about 1: 3.5. In some embodiments, the ratio is about 1: 4. In some embodiments, the ratio is about 1: 4.5. In some embodiments, the ratio is about 1: 5. In some embodiments, the ratio is about 1: 5.5. In some embodiments, the ratio is about 1: 6. In some embodiments, the ratio is about 1: 6.5. In some embodiments, the ratio is about 1: 7. In some embodiments, the ratio is about 1: 7.5. In some embodiments, the ratio is about 1: 8. In some embodiments, the ratio is about 1: 8.5. In some embodiments, the ratio is about 1: 9. In some embodiments, the ratio is about 1: 9.5. In some embodiments, the ratio is about 1: 10. In some embodiments, the ratio is about 1: 12. In some embodiments, the ratio is about 1: 14. In some embodiments, the ratio is about 1: 16. In some embodiments, the ratio is about 1: 18. In some embodiments, the ratio is about 1: 20. In some embodiments, the ratio is between about 1: 1 to about 1: 8. In some embodiments, the ratio is between about 1: 2 to about 1: 6. In some embodiments, the ratio is between about 1: 3 to about 1: 5. In some embodiments, the ratio is between about 1: 3.5 to about 1: 4.5. In some embodiments, the ratio is about 1: 4.
Preparation of the Pharmaceutical Compositions
In some embodiments, to prepare pharmaceutical compositions described herein, a protein described herein is admixed with the cryoprotectant, humectant, emulsifier, controlled release agent, and/or buffer described herein. In some embodiments, to prepare pharmaceutical compositions of the anti-CoV-S antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof (e.g., of Table 1) , antigen-binding protein is admixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington’s Pharmaceutical Sciences and U.S.  Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984) ; Hardman, et al. (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds. ) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds. ) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds. ) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y. In some embodiments, the pharmaceutical composition is sterile. Such compositions are part of the present disclosure.
The scope of the present disclosure includes desiccated, e.g., freeze-dried, compositions comprising an anti-CoV-S antigen-binding proteins, e.g., antibody or antigen-binding fragment thereof (e.g., of Table 1) , or a pharmaceutical composition thereof that includes a pharmaceutically acceptable carrier but substantially lacks water. The scope of the disclosure also includes proteins provided as part of an aqueous solution or a suspension.
The mode of administration can vary. In some embodiments, routes of administration include intranasal, inhalation, insufflation, and oral. In some embodiments, routes of administration include parenteral, intramuscular, and subcutaneous.
In some embodiments, the disclosure provides intranasal administration of an antibody described herein. In some embodiments, the disclosure provides intranasal administration of an antigen-binding protein, e.g., antibody or antigen-binding fragment thereof (e.g., of Table 1) , described herein. In some embodiments, the disclosure provides inhaled administration of an antigen-binding protein, e.g., antibody or antigen-binding fragment thereof (e.g., of Table 1) , described herein. In some embodiments, the disclosure provides insufflated administration of an antigen-binding protein, e.g., antibody or antigen-binding fragment thereof (e.g., of Table 1) , described herein.
In an embodiment of the present disclosure, an antibody of the disclosure, e.g., antibody described herein (e.g., of Table 1) , is administered in association with one or more further therapeutic agents.
In some embodiments, the anti-CoV-S antigen-binding protein, e.g., antibody or antigen-binding fragment thereof of the present disclosure, along with another agent, can be formulated into a single composition, e.g., for simultaneous delivery, or formulated separately into two or more compositions (e.g., a kit) . Each component can be administered to a subject at a different time than when the other component is administered; for example, each administration may be  given non-simultaneously (e.g., separately or sequentially) at intervals over a given period of time. Moreover, the separate components may be administered to a subject by the same or by a different route e.g., wherein an anti-CoV-S antibody or antigen-binding fragment thereof.
In some embodiments, the present disclosure provides a vessel (e.g., a plastic or glass vial, e.g., with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising any of the anti-CoV-S antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof (e.g., of Table 1) , polypeptides (e.g., an HC, LC, VH or VL of Table 1) or polynucleotides or vectors set forth herein or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier.
In some embodiments, the present disclosure also provides an injection device comprising one or more antigen-binding proteins (e.g., antibody or antigen-binding fragment) that bind specifically to CoV-S, e.g., those of Table 1, or a pharmaceutical composition thereof. The injection device may be packaged into a kit. An injection device is a device that introduces a substance into the body of a subject via a parenteral route, e.g., intramuscular, subcutaneous or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., comprising the antibody or fragment or a pharmaceutical composition thereof) , a needle for piecing skin and/or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore. In some embodiments, an injection device that comprises an antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof, from a combination of the present disclosure, or a pharmaceutical composition thereof is an intravenous (IV) injection device. Such a device can include the antigen-binding protein or a pharmaceutical composition thereof in a cannula or trocar/needle which may be attached to a tube which may be attached to a bag or reservoir for holding fluid (e.g., saline) introduced into the body of the subject through the cannula or trocar/needle. The antibody or fragment or a pharmaceutical composition thereof may, in some embodiments, be introduced into the device once the trocar and cannula are inserted into the vein of a subject and the trocar is removed from the inserted cannula. The IV device may, for example, be inserted into a peripheral vein (e.g., in the hand or arm) ; the superior vena cava or inferior vena cava, or within the right atrium of the heart (e.g., a central IV) ; or into a subclavian, internal jugular, or a femoral vein and, for example, advanced toward the heart until it reaches the superior vena cava or right atrium (e.g., a central venous line) . In some embodiments, an injection device is an autoinjector; a jet injector or an external infusion pump. A jet injector uses a high-pressure  narrow jet of liquid which penetrate the epidermis to introduce the antibody or fragment or a pharmaceutical composition thereof to a subject’s body. External infusion pumps are medical devices that deliver the antibody or fragment or a pharmaceutical composition thereof into a subject’s body in controlled amounts. External infusion pumps may be powered electrically or mechanically. Different pumps operate in different ways, for example, a syringe pump holds fluid in the reservoir of a syringe, and a moveable piston controls fluid delivery, an elastomeric pump holds fluid in a stretchable balloon reservoir, and pressure from the elastic walls of the balloon drives fluid delivery. In a peristaltic pump, a set of rollers pinches down on a length of flexible tubing, pushing fluid forward. In a multi-channel pump, fluids can be delivered from multiple reservoirs at multiple rates.
Administration
The present disclosure provides methods for administering an anti-CoV-S antigen-binding protein of the present disclosure, e.g., those of Table 1, comprising introducing the antigen-binding protein into the body of a subject (e.g., a human) . For example, the method comprises the human subject taking the anti-CoV-S antigen-binding protein of the present disclosure via an intranasal, inhaled, or insufflated route. In some embodiments, an antibody described herein is intranasally administered to the human subject. In some embodiments, Antibody A or Antibody B or Antibody C or Antibody D or Antibody E or Antibody F, or a combination of any two, or three, or four, antibodies thereof, is intranasally administered to the human subject. In some embodiments, Antibody B is intranasally administered to the human subject.
The present disclosure provides methods for administering an anti-CoV-S antigen-binding protein, e.g., antibody or antigen-binding fragment thereof (e.g., of Table 1) , comprising introducing the protein into the body of a subject. For example, the method comprises piercing the body of the subject with a needle of a syringe and injecting the antigen-binding protein into the body of the subject, e.g., into the vein, artery, tumor, muscular tissue or subcutis of the subject.
The present disclosure provides methods for administering the pharmaceutical composition, or one or more antibodies or antigen binding fragments to a nasal cavity or a mucosa of a subject. In some embodiments, the pharmaceutical composition or the one or more antibodies or antigen binding fragment (s) is/are administrated to a mucosa location. Mucosa is the soft tissue that lines the body’s canals and organs in the digestive, respiratory and reproductive systems. In some embodiments, the mucosa is located in the nose, mouth, throat, ear, genital, or anus. In some embodiments, the mucosa is located in the esophagus, lungs, stomach, intestine, bladder, or uterus. In some embodiments, the mucosa is located in the mouth, esophagus, or stomach. In some  embodiments, the mucosa is located in the nose, mouth, pharynx, trachea, or lung. In some embodiments, the mucosa is nasal mucosa. In some embodiments, the pharmaceutical composition or the one or more antibodies or antigen binding fragment (s) is/are administrated intranasally.
Therapeutic Methods
In one aspect, the disclosure provides methods of preventing a disease in a subject in need thereof, comprising administering the pharmaceutical composition, the antibody or antigen binding fragment, or a combination thereof to the subject.
In one aspect, the disclosure provides methods of treating a disease in a subject in need thereof, comprising administering the pharmaceutical composition, the antibody or antigen binding fragment, or a combination thereof to the subject.
In some embodiments, the pharmaceutical composition is administered to a mucosa of the subject. In some embodiments, the mucosa is located in the nose, mouth, throat, ear, genital, or anus. In some embodiments, the mucosa is located in the esophagus, lungs, stomach, intestine, bladder, or uterus. In some embodiments, the mucosa is located in the mouth, esophagus, or stomach. In some embodiments, the mucosa is located in the nose, mouth, pharynx, trachea, or lung. In some embodiments, the mucosa is nasal mucosa.
In some embodiments, the administration is intranasal administration.
In some embodiments, the administration starts prior to the onset of any symptom of the disease.
In some embodiments, the pharmaceutical composition is a vaccine. In some embodiments, the vaccine comprises a protein antigen as an active ingredient.
In some embodiments, the pharmaceutical composition comprises an active ingredient (e.g., an antibody) that neutralizes an infectious agent (e.g., a virus) .
In some embodiments, the administration starts after the onset of at least one symptom of the disease. In some embodiments, the symptom is a respiratory symptom. In some embodiments, the respiratory symptom is allergy, nasal congestion, nasal infection, or a combination thereof.
In some embodiments, the disease is a respiratory infection caused by a virus, a bacterial, a fungus, or a combination thereof. In some embodiments, the disease is a viral infection. In some embodiments, the disease is a chronic viral infection. In some embodiments, the disease is an  acute viral infection. In some embodiments, the disease is a coronavirus infection. In some embodiments, the disease is SARS-CoV-2 infection.
In some embodiments, the present disclosure provides methods for treating or preventing viral infection (e.g., coronavirus infection) by administering a therapeutically effective amount of the active ingredient in the pharmaceutical composition (e.g., an antigen-binding protein, e.g., antibody or antigen-binding fragment, (e.g., of Table 1) ) to a subject (e.g., a human) in need of such treatment or prevention.
Coronavirus infection may be treated or prevented, in a subject, by administering an anti-CoV-S antigen-binding protein of the present disclosure to a subject.
An effective or therapeutically effective dose of the antigen-binding protein, e.g., antibody or antigen-binding fragment (e.g., of Table 1) , for treating or preventing a viral infection refers to the amount of the antibody or fragment sufficient to alleviate or prevent one or more signs and/or symptoms of the infection in the treated subject, whether by inducing the regression or elimination of such signs and/or symptoms or by inhibiting the progression of such signs and/or symptoms. The dose amount may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. In some embodiments, an effective or therapeutically effective dose of antibody or antigen-binding fragment thereof of the present disclosure, for treating or preventing viral infection, e.g., in an adult human subject, is about 0.01 mg to about 200 mg per dose.
In some embodiments, about 0.01 mg to about 200 mg of the antigen-binding protein is delivered to a nasal cavity of a human per administration. In some embodiments, the dose per administration is about 0.01 mg to about 0.05 mg. In some embodiments, the dose per administration is about 0.05 mg to about 0.1 mg. In some embodiments, the dose per administration is about 0.1 mg to about 0.2 mg. In some embodiments, the dose per administration is about 0.2 mg to about 0.3 mg. In some embodiments, the dose per administration is about 0.3 mg to about 0.5 mg. In some embodiments, the dose per administration is about 0.5 mg to about 0.7 mg. In some embodiments, the dose per administration is about 0.7 mg to about 1 mg. In some embodiments, the dose per administration is about 1 mg to about 2 mg. In some embodiments, the dose per administration is about 2 mg to about 5 mg. In some embodiments, the dose per administration is about 5 mg to about 10 mg. In some embodiments, the dose per administration is about 10 mg to about 30 mg. In some embodiments, the dose per administration is about 30 mg to about 100 mg. In some embodiments, the dose per administration is about 100 mg to about 200 mg. In some  embodiments, the dose per administration is about 0.01 mg to about 0.1 mg. In some embodiments, the dose per administration is about 0.05 mg to about 0.2 mg. In some embodiments, the dose per administration is about 0.1 mg to about 0.3 mg. In some embodiments, the dose per administration is about 0.2 mg to about 0.5 mg. In some embodiments, the dose per administration is about 0.3 mg to about 0.7 mg. In some embodiments, the dose per administration is about 0.5 mg to about 1 mg. In some embodiments, the dose per administration is about 0.7 mg to about 2 mg. In some embodiments, the dose per administration is about 1 mg to about 5 mg. In some embodiments, the dose per administration is about 2 mg to about 10 mg. In some embodiments, the dose per administration is about 5 mg to about 30 mg. In some embodiments, the dose per administration is about 10 mg to about 100 mg. In some embodiments, the dose per administration is about 30 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 0.2 mg. In some embodiments, the dose per administration is about 0.05 mg to about 0.3 mg. In some embodiments, the dose per administration is about 0.1 mg to about 0.5 mg. In some embodiments, the dose per administration is about 0.2 mg to about 0.7 mg. In some embodiments, the dose per administration is about 0.3 mg to about 1 mg. In some embodiments, the dose per administration is about 0.5 mg to about 2 mg. In some embodiments, the dose per administration is about 0.7 mg to about 5 mg. In some embodiments, the dose per administration is about 1 mg to about 10 mg. In some embodiments, the dose per administration is about 2 mg to about 30 mg. In some embodiments, the dose per administration is about 5 mg to about 100 mg. In some embodiments, the dose per administration is about 10 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 0.3 mg. In some embodiments, the dose per administration is about 0.05 mg to about 0.5 mg. In some embodiments, the dose per administration is about 0.1 mg to about 0.7 mg. In some embodiments, the dose per administration is about 0.2 mg to about 1 mg. In some embodiments, the dose per administration is about 0.3 mg to about 2 mg. In some embodiments, the dose per administration is about 0.5 mg to about 5 mg. In some embodiments, the dose per administration is about 0.7 mg to about 10 mg. In some embodiments, the dose per administration is about 1 mg to about 30 mg. In some embodiments, the dose per administration is about 2 mg to about 100 mg. In some embodiments, the dose per administration is about 5 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 0.5 mg. In some embodiments, the dose per  administration is about 0.05 mg to about 0.7 mg. In some embodiments, the dose per administration is about 0.1 mg to about 1 mg. In some embodiments, the dose per administration is about 0.2 mg to about 2 mg. In some embodiments, the dose per administration is about 0.3 mg to about 5 mg. In some embodiments, the dose per administration is about 0.5 mg to about 10 mg. In some embodiments, the dose per administration is about 0.7 mg to about 30 mg. In some embodiments, the dose per administration is about 1 mg to about 100 mg. In some embodiments, the dose per administration is about 2 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 0.7 mg. In some embodiments, the dose per administration is about 0.05 mg to about 1 mg. In some embodiments, the dose per administration is about 0.1 mg to about 2 mg. In some embodiments, the dose per administration is about 0.2 mg to about 5 mg. In some embodiments, the dose per administration is about 0.3 mg to about 10 mg. In some embodiments, the dose per administration is about 0.5 mg to about 30 mg. In some embodiments, the dose per administration is about 0.7 mg to about 100 mg. In some embodiments, the dose per administration is about 1 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 1 mg. In some embodiments, the dose per administration is about 0.05 mg to about 2 mg. In some embodiments, the dose per administration is about 0.1 mg to about 5 mg. In some embodiments, the dose per administration is about 0.2 mg to about 10 mg. In some embodiments, the dose per administration is about 0.3 mg to about 30 mg. In some embodiments, the dose per administration is about 0.5 mg to about 100 mg. In some embodiments, the dose per administration is about 0.7 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 2 mg. In some embodiments, the dose per administration is about 0.05 mg to about 5 mg. In some embodiments, the dose per administration is about 0.1 mg to about 10 mg. In some embodiments, the dose per administration is about 0.2 mg to about 30 mg. In some embodiments, the dose per administration is about 0.3 mg to about 100 mg. In some embodiments, the dose per administration is about 0.5 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 5 mg. In some embodiments, the dose per administration is about 0.05 mg to about 10 mg. In some embodiments, the dose per administration is about 0.1 mg to about 30 mg. In some embodiments, the dose per administration is about 0.2 mg to about 100 mg. In some embodiments, the dose per administration is about 0.3 mg to about 200 mg. In some embodiments, the dose per  administration is about 0.01 mg to about 10 mg. In some embodiments, the dose per administration is about 0.05 mg to about 30 mg. In some embodiments, the dose per administration is about 0.1 mg to about 100 mg. In some embodiments, the dose per administration is about 0.2 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 30 mg. In some embodiments, the dose per administration is about 0.05 mg to about 100 mg. In some embodiments, the dose per administration is about 0.1 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg to about 100 mg. In some embodiments, the dose per administration is about 0.05 mg to about 200 mg. In some embodiments, the dose per administration is about 0.01 mg. In some embodiments, the dose per administration is about 0.02 mg. In some embodiments, the dose per administration is about 0.03 mg. In some embodiments, the dose per administration is about 0.04 mg. In some embodiments, the dose per administration is about 0.05 mg. In some embodiments, the dose per administration is about 0.06 mg. In some embodiments, the dose per administration is about 0.07 mg. In some embodiments, the dose per administration is about 0.08 mg. In some embodiments, the dose per administration is about 0.09 mg. In some embodiments, the dose per administration is about 0.1 mg. In some embodiments, the dose per administration is about 0.12 mg. In some embodiments, the dose per administration is about 0.14 mg. In some embodiments, the dose per administration is about 0.17 mg. In some embodiments, the dose per administration is about 0.2 mg. In some embodiments, the dose per administration is about 0.23 mg. In some embodiments, the dose per administration is about 0.27 mg. In some embodiments, the dose per administration is about 0.3 mg. In some embodiments, the dose per administration is about 0.35 mg. In some embodiments, the dose per administration is about 0.4 mg. In some embodiments, the dose per administration is about 0.45 mg. In some embodiments, the dose per administration is about 0.5 mg. In some embodiments, the dose per administration is about 0.6 mg. In some embodiments, the dose per administration is about 0.7 mg. In some embodiments, the dose per administration is about 0.8 mg. In some embodiments, the dose per administration is about 0.9 mg. In some embodiments, the dose per administration is about 1 mg. In some embodiments, the dose per administration is about 0.01 mg to about 5 mg. In some embodiments, the dose per administration is about 0.05 mg to about 2 mg. In some embodiments, the dose per administration is about 0.1 mg to about 1 mg. In some embodiments, the dose per administration is about 0.15 mg to about 0.75 mg. In some embodiments, the dose per administration is about 0.2 mg to about 0.6 mg. In some embodiments, the dose per administration is about 0.25 mg to about 0.5 mg. In some  embodiments, the dose per administration is about 0.3 mg to about 0.4 mg. In some embodiments, the dose per administration is about 0.35 mg.
In some embodiments, about 0.01-2 mL of the pharmaceutical composition is administered to a nasal cavity of a human; that is, the volume per administration is about 0.01 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.02 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.04 mL.In some embodiments, the volume per administration is about 0.04 mL to about 0.06 mL. In some embodiments, the volume per administration is about 0.06 mL to about 0.08 mL. In some embodiments, the volume per administration is about 0.08 mL to about 0.1 mL. In some embodiments, the volume per administration is about 0.1 mL to about 0.2 mL. In some embodiments, the volume per administration is about 0.2 mL to about 0.4 mL. In some embodiments, the volume per administration is about 0.4 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.6 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.8 mL to about 1 mL. In some embodiments, the volume per administration is about 1 mL to about 1.5 mL. In some embodiments, the volume per administration is about 1.5 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.04 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.06 mL. In some embodiments, the volume per administration is about 0.04 mL to about 0.08 mL. In some embodiments, the volume per administration is about 0.06 mL to about 0.1 mL. In some embodiments, the volume per administration is about 0.08 mL to about 0.2 mL. In some embodiments, the volume per administration is about 0.1 mL to about 0.4 mL. In some embodiments, the volume per administration is about 0.2 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.4 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.6 mL to about 1 mL. In some embodiments, the volume per administration is about 0.8 mL to about 1.5 mL. In some embodiments, the volume per administration is about 1 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.06 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.08 mL. In some embodiments, the volume per administration is about 0.04 mL to about 0.1 mL. In some embodiments, the volume per administration is about 0.06 mL to about 0.2 mL. In some embodiments, the volume per administration is about 0.08 mL to about 0.4 mL. In some embodiments, the volume per administration is about 0.1 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.2 mL to about 0.8 mL. In some embodiments, the volume per  administration is about 0.4 mL to about 1 mL. In some embodiments, the volume per administration is about 0.6 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.8 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.08 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.1 mL. In some embodiments, the volume per administration is about 0.04 mL to about 0.2 mL. In some embodiments, the volume per administration is about 0.06 mL to about 0.4 mL. In some embodiments, the volume per administration is about 0.08 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.1 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.2 mL to about 1 mL. In some embodiments, the volume per administration is about 0.4 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.6 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.1 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.2 mL. In some embodiments, the volume per administration is about 0.04 mL to about 0.4 mL. In some embodiments, the volume per administration is about 0.06 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.08 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.1 mL to about 1 mL. In some embodiments, the volume per administration is about 0.2 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.4 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.2 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.4 mL. In some embodiments, the volume per administration is about 0.04 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.06 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.08 mL to about 1 mL. In some embodiments, the volume per administration is about 0.1 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.2 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.4 mL. In some embodiments, the volume per administration is about 0.02 mL to about 0.6 mL. In some embodiments, the volume per administration is about 0.04 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.06 mL to about 1 mL. In some embodiments, the volume per administration is about 0.08 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.1 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.6 mL. In some embodiments, the volume per  administration is about 0.02 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.04 mL to about 1 mL. In some embodiments, the volume per administration is about 0.06 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.08 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.02 mL to about 1 mL. In some embodiments, the volume per administration is about 0.04 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.06 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 1 mL. In some embodiments, the volume per administration is about 0.02 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.04 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.02 mL to about 2 mL. In some embodiments, the volume per administration is about 0.01 mL. In some embodiments, the volume per administration is about 0.05 mL. In some embodiments, the volume per administration is about 0.1 mL. In some embodiments, the volume per administration is about 0.15 mL. In some embodiments, the volume per administration is about 0.2 mL. In some embodiments, the volume per administration is about 0.25 mL. In some embodiments, the volume per administration is about 0.3 mL. In some embodiments, the volume per administration is about 0.4 mL. In some embodiments, the volume per administration is about 0.5 mL. In some embodiments, the volume per administration is about 0.6 mL. In some embodiments, the volume per administration is about 0.7 mL. In some embodiments, the volume per administration is about 0.8 mL. In some embodiments, the volume per administration is about 0.9 mL. In some embodiments, the volume per administration is about 1 mL. In some embodiments, the volume per administration is about 1.1 mL. In some embodiments, the volume per administration is about 1.2 mL. In some embodiments, the volume per administration is about 1.3 mL. In some embodiments, the volume per administration is about 1.4 mL. In some embodiments, the volume per administration is about 1.5 mL. In some embodiments, the volume per administration is about 1.6 mL. In some embodiments, the volume per administration is about 1.7 mL. In some embodiments, the volume per administration is about 1.8 mL. In some embodiments, the volume per administration is about 1.9 mL. In some embodiments, the volume per administration is about 2 mL. In some embodiments, the volume per administration is about 0.1 mL to about 2 mL. In some embodiments, the volume per administration is about 0.2 mL to about 1.5 mL. In some embodiments, the volume per administration is about 0.3 mL to about 1.2 mL. In some embodiments, the volume per  administration is about 0.4 mL to about 1 mL. In some embodiments, the volume per administration is about 0.5 mL to about 0.9 mL. In some embodiments, the volume per administration is about 0.6 mL to about 0.8 mL. In some embodiments, the volume per administration is about 0.7 mL.
In some embodiments, about 0.01-2 mL of the pharmaceutical composition is administered by nasal spray to the nasal cavity.
Depending on the likelihood and/or severity of the infection, the frequency and the duration of the treatment can be adjusted. In some embodiments, the pharmaceutical composition or antigen-binding protein of the present disclosure can be administered at an initial dose, followed by one or more secondary doses, (e.g., once a day, twice a day, 3 times a day, 4 times a day, once every at least one day) . In some embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-2 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-3 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 3-4 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 4-5 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 5-7 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 7-10 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 10-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 14-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 20-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-3 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-4 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 3-5 days. In some embodiments, the pharmaceutical composition or antigen-binding  protein is administered for about 4-7 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 5-10 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 7-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 10-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 14-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-4 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-5 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 3-7 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 4-10 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 5-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 7-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 10-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-5 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-7 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 3-10 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 4-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 5-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 7-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-7 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-10 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 3-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 4-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 5-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-10 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 3-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 4-30 days. In some  embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-14 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 3-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1-20 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 2-30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
In some embodiments, the pharmaceutical composition or antigen-binding protein is administered for about 7-14 days.
In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 1-10 times a day, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 1-6 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 2-4 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 2-3 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 3-4 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about once a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about twice a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 3 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 4 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject about 5 times a day. In some embodiments, the  pharmaceutical composition or antigen-binding protein is administered to the subject about 6 times a day. In some embodiments, the pharmaceutical composition or antigen-binding protein is administered to the subject no more than 6, 5, 4, 3, 2, or 1 time (s) a day.
As used herein, the term “subject” refers to a mammal (e.g., rat, mouse, cat, dog, cow, pig, sheep, horse, goat, rabbit) , preferably a human, for example, in need of prevention and/or treatment of a disease or disorder such as viral infection or cancer. The subject may have a viral infection, or be predisposed to developing an infection. Subjects predisposed to developing an infection, or subjects who may be at elevated risk for contracting an infection (e.g., of coronavirus or influenza virus) , include subjects with compromised immune systems because of autoimmune disease, subjects receiving immunosuppressive therapy (for example, following organ transplant) , subjects afflicted with human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS) , subjects with forms of anemia that deplete or destroy white blood cells, subjects receiving radiation or chemotherapy, or subjects afflicted with an inflammatory disorder. Additionally, subjects of very young (e.g., 5 years of age or younger) or old age (e.g., 65 years of age or older) are at increased risk. Moreover, a subject may be at risk of contracting a viral infection due to proximity to an outbreak of the disease, e.g., subject resides in a densely populated city or in close proximity to subjects having confirmed or suspected infections of a virus, or choice of employment, e.g. hospital worker, pharmaceutical researcher, traveler to infected area, or frequent flier.
“Treat” or “treating” means to administer a composition comprising an active ingredient, e.g., antibody or antigen-binding fragment of the present disclosure (e.g., of Table 1) , to a subject having one or more signs or symptoms of a disease or infection, e.g., viral infection, for which the active ingredient is effective when administered to the subject at an effective or therapeutically effective amount or dose (as discussed herein) .
The present disclosure also encompasses prophylactically administering a composition comprising an active ingredient, e.g., antibody or antigen-binding fragment thereof of the present disclosure (e.g., of Table 1) , to a subject who is at risk of a disease (e.g., viral infection) so as to prevent such a disease. Passive antibody-based immunoprophylaxis has proven an effective strategy for preventing subject from viral infection. See e.g., Berry et al., Passive broad-spectrum influenza immunoprophylaxis. Influenza Res Treat. 2014; 2014: 267594. Epub 2014 Sep 22; and Jianqiang et al., Passive immune neutralization strategies for prevention and control of influenza A infections, Immunotherapy. 2012 February; 4 (2) : 175-186; Prabhu et al., Antivir Ther.  2009; 14 (7) : 911-21, Prophylactic and therapeutic efficacy of a chimeric monoclonal antibody specific for H5 hemagglutinin against lethal H5N1 influenza.
“Prevent” or “preventing” means to administer a composition comprising an active ingredient, e.g., antibody or antigen-binding fragment of the present disclosure (e.g., of Table 1) , to a subject to inhibit the manifestation of a disease (e.g., viral infection) in the body of a subject, for which the active ingredient is effective when administered to the subject at an effective or therapeutically effective amount or dose (as discussed herein) .
In some embodiments, a sign or symptom of a viral infection in a subject is survival or proliferation of virus in the body of the subject, e.g., as determined by viral titer assay (e.g., coronavirus propagation in embryonated chicken eggs or coronavirus spike protein assay) . Other signs and symptoms of viral infection are discussed herein.
As noted above, in some embodiments the subject may be a non-human animal, and the active ingredient or the antigen-binding proteins (e.g., antibodies and antigen-binding fragments) discussed herein may be used in a veterinary context to treat and/or prevent disease in the non-human animals (e.g., cats, dogs, pigs, cows, horses, goats, rabbits, sheep, and the like) .
The present disclosure provides a method for treating or preventing viral infection (e.g., coronavirus infection) or for inducing the regression or elimination or inhibiting the progression of at least one sign or symptom of viral infection such as: --fever or feeling feverish/chills; --cough; --sore throat; --runny or stuffy nose; --sneezing; --muscle or body aches; --headaches; --fatigue (tiredness) ; --vomiting; --diarrhea; --respiratory tract infection; --chest discomfort; --shortness of breath; --bronchitis; and/or --pneumonia, which sign or symptom is secondary to viral infection, in a subject in need thereof (e.g., a human) , by administering a therapeutically effective amount of the active ingredient (e.g., anti-CoV-S antigen-binding protein (e.g., of Table 1) ) to the subject, for example, by administering of the active ingredient into the body of the subject.
In some embodiments, the disclosure provides a method of treating a respiratory infection, said method comprising administering an active ingredient (e.g., an antibody) described herein intranasally to a mammal with the respiratory infection, thereby treating the respiratory infection. In some embodiments, the disclosure provides a method of treating a SARS-CoV-2 infection, said method comprising administering active ingredient (e.g., an antibody) described herein intranasally to a mammal infected with SARS-CoV-2, thereby treating the SARS-CoV-2 infection. In some embodiments, the disclosure provides a method of treating a SARS-CoV-2 infection, said method comprising administering an active ingredient (e.g., an antibody) to a  mammal infected with SARS-CoV-2, thereby treating the SARS-CoV-2 infection. In some embodiments, the disclosure provides a method of delivering high local concentration of an active ingredient (e.g., an antibody) in the nasal cavity while having a low exposure in the blood circulation (systemic exposure) . In some embodiments, the active ingredient comprises an antibody or antigen binding fragment thereof.
Diagnostic Uses of the Antibodies
The anti-CoV-S antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof of the present disclosure (e.g., of Table 1) , may be used to detect and/or measure CoV-S in a sample. Exemplary assays for CoV-S may include, e.g., contacting a sample with an anti-CoV-S antigen-binding protein of the disclosure, wherein the anti-CoV-S antigen-binding protein is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate CoV-S from samples. The presence of an anti-CoV-S antigen-binding protein complexed with CoV-S indicates the presence of CoV-S in the sample. Alternatively, an unlabeled anti-CoV-S antibody can be used in combination with a secondary antibody which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure CoV-S in a sample include neutralization assays, enzyme-linked immunosorbent assay (ELISA) , radioimmunoassay (MA) , and fluorescence-activated cell sorting (FACS) . Thus, the present disclosure includes a method for detecting the presence of spike protein polypeptide in a sample comprising contacting the sample with an anti-CoV-S antigen-binding protein and detecting the presence of a CoV-S/anti-CoV-S antigen-binding protein wherein the presence of the complex indicates the presence of CoV-S.
An anti-CoV-S antigen-binding protein of the disclosure (e.g., of Table 1) may be used in a Western blot or immune-protein blot procedure for detecting the presence of CoV-S or a fragment thereof in a sample. Such a procedure forms part of the present disclosure and includes the steps of e.g.:
(1) providing a membrane or other solid substrate comprising a sample to be tested for the presence of CoV-S, e.g., optionally including the step of transferring proteins from a sample to be tested for the presence of CoV-S (e.g., from a PAGE or SDS-PAGE electrophoretic separation of the proteins in the sample) onto a membrane or other solid substrate using a method known in the art (e.g., semi-dry blotting or tank blotting) ; and  contacting the membrane or other solid substrate to be tested for the presence of CoV-S or a fragment thereof with an anti-CoV-S antigen-binding protein of the disclosure. Such a membrane may take the form, for example, of a nitrocellulose or vinyl-based (e.g., polyvinylidene fluoride (PVDF) ) membrane to which the proteins to be tested for the presence of CoV-S in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel have been transferred (e.g., following electrophoretic separation in the gel) . Before contacting the membrane with the anti-CoV-S antigen-binding protein, the membrane is optionally blocked, e.g., with non-fat dry milk or the like so as to bind non-specific protein binding sites on the membrane.
(2) washing the membrane one or more times to remove unbound anti-CoV-S antigen-binding protein and other unbound substances; and
(3) detecting the bound anti-CoV-S antigen-binding protein.
Detection of the bound antigen-binding protein indicates that the CoV-S protein is present on the membrane or substrate and in the sample. Detection of the bound antigen-binding protein may be by binding the antigen-binding protein with a secondary antibody (an anti-immunoglobulin antibody) which is detectably labeled and, then, detecting the presence of the secondary antibody label.
The anti-CoV-S antigen-binding proteins (e.g., antibodies and antigen-binding fragments (e.g., of Table 1) ) disclosed herein may also be used for immunohistochemistry. Such a method forms part of the present disclosure and comprises, e.g.,
(1) contacting tissue to be tested for the presence of CoV-S protein with an anti-CoV-S antigen-binding protein of the disclosure; and
(2) detecting the antigen-binding protein on or in the tissue.
If the antigen-binding protein itself is detectably labeled, it can be detected directly. Alternatively, the antigen-binding protein may be bound by a detectably labeled secondary antibody wherein the label is then detected.
Kits
Further provided are kits comprising one or more components that include, but are not limited to, a pharmaceutical composition described herein suitable for intranasal administration, and a nasal administration device for accurate delivery of said intranasal pharmaceutical composition. In some embodiments, the nasal administration device is a nasal pump device. In  some embodiments, the nasal administration device delivers about 0.01 mg/dose to about 20 mg/dose of an intranasal pharmaceutical composition described herein, by a single or multiple actuation (s) e.g., 0.1 mL per spray to each nostril, spray twice to each nostril (for a total of 0.4 mL) of antibody of 1 mg/mL, gives 0.4 mg per dose.
In some embodiments, the kit comprises a pharmaceutical composition described herein suitable for intranasal administration, and a nasal administration device. In some embodiments, the nasal administration device is a nasal pump device. In some embodiments, the disclosure provides a kit comprising a pharmaceutical composition described herein suitable for intranasal administration, and a nasal administration device, and instructions for the use of the kit.
The intranasal compositions of this disclosure are most effective when proper product delivery design is utilized. The preferred product design includes a composition of the disclosure contained within a delivery system, such as a bottle and a pump, for nasal delivery of the formulation in a mist of spray droplets to coat the mucosa of the nasal cavity upon administration. Preferred pumps for use in such products of the disclosure are metered multi-dose pumps; however, single unit dose containers are also acceptable to deliver an intranasal pharmaceutical composition described herein to the nasal cavity. The selection of the pump is based on the desired dose/spray volume and spray pattern appropriate for local delivery to the nasal mucosa. In some embodiments of the disclosure, the compositions can be effectively contained in a package comprising a bottle fitted with a cap, and the cap can be screwed on or snapped on or sealed with a pump head, and are delivered by a metered-dose spray pump designed for intranasal application in volumes of about 0.02 to 0.2 ml per press. Suitable compositions of the present disclosure include about 0.02 mg/dose to about 200 mg/dose of an intranasal pharmaceutical formulation described herein relative to the total weight of the composition.
Further provided are kits comprising one or more components that include, but are not limited to, an anti-CoV-S antigen-binding protein, e.g., an antibody or antigen-binding fragment as discussed herein (e.g., of Table 1) , in association with one or more additional components including, optionally, a further therapeutic agent.
In some embodiments, the kit of the disclosure includes a pharmaceutical composition for parenteral administration to a subject and a device (e.g., an injection device) for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above containing the anti-CoV-S antigen-binding protein, e.g., antibody or antigen-binding fragment thereof of the present disclosure (e.g., of Table 1) .
The kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the disclosure may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.
Spike Protein sequences
The sequence of the spike protein of wild-type SARS-CoV-2 is according to SEQ ID NO: 70:
The sequence of the RBD-domain of the spike protein of wild-type SARS-CoV-2 is according to SEQ ID NO: 71:
The sequence of the spike protein of alpha (B. 1.1.7) SARS-CoV-2 is according to SEQ ID NO: 72:
The sequence of the RBD-domain of the spike protein of alpha (B. 1.1.7) SARS-CoV-2 is according to SEQ ID NO: 73:
The sequence of the spike protein of beta (B. 1.351) SARS-CoV-2 is according to SEQ ID NO: 74:
The sequence of the RBD-domain of the spike protein of beta (B. 1.351) SARS-CoV-2 is according to SEQ ID NO: 75:

The sequence of the spike protein of gamma (P. 1) SARS-CoV-2 is according to SEQ ID NO: 76:
The sequence of the RBD-domain of the spike protein of gamma (P. 1) SARS-CoV-2 is according to SEQ ID NO: 77:
The sequence of the spike protein of delta (B. 1.617.2) SARS-CoV-2 is according to SEQ ID NO: 78:
The sequence of the RBD-domain of the spike protein of delta (B. 1.617.2) SARS-CoV-2 is according to SEQ ID NO: 79:
The sequence of the spike protein of omicron (BA. 1) SARS-CoV-2 is according to SEQ ID NO: 80:
The sequence of the RBD-domain of the spike protein of omicron (BA. 1) SARS-CoV-2 is according to SEQ ID NO: 81:
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, room temperature is about 25℃, and pressure is at or near atmospheric.
EXAMPLE 1
Antibody B nasal spray composition
The active ingredient of Antibody B is IgG1 antibody, and the dosage form is nasal spray, with a specification of 5mg/mL and 70μL per press. Excipients are: disodium hydrogen phosphate, sodium dihydrogen phosphate, Tween 80, trehalose, glycerol and hydroxypropyl methylcellulose. The excipients used are all approved excipients with known safety. See Table 4A.
Table 4A. Intranasal Formulation Excipients
Dose study
The formulation above was utilized to make different concentrations (1 mg/mL, 2 mg/mL, 5 mg/mL and 10 mg/mL) of Antibody B as shown in Table 4B:
Table 4B: Formulations of Antibody B






Antibody B in an aqueous solution at a concentration of about 5 mg/mL was nasally sprayed to Cynomolgus monkeys twice a day for 14 days, 0.38 mL per dose. Blood samples and nasal swab samples were collected. The concentration of Antibody B in the samples was determined (Figure 1G) . The concentration of Antibody B in the nasal cavity was on average at least about 50 times higher than in the circulating blood within 12 hour of antibody administration. Administration of Antibody B via nasal spray provided a high local concentration of Antibody B in the nasal cavity that was about 1000 times higher than the Antibody B concentration needed for 50%neutralization (IC50) of SARS-CoV-2 viruses, while at the same time kept a low systemic exposure for improved safety.
Stability test
In order to verify whether Antibody B has the storage stability that can be used as a drug, this experiment uses ultra-high performance liquid chromatography-size exclusion chromatography (UPLC-SEC) to determine the antibody content. The results show that the Antibody B solution of about 1 mg/ml is stable after 31 days of storage at 37 ℃, and no antibody decomposition or polymerization occurs.
EXAMPLE 2
Animal protection experiments-
Cells and virus
Vero (catalog no. CCL-81, ATCC) , Vero E6 (catalog no. CRL-1586TM) , 293T cells (catalog no. GDC187, CCTCC) , L-929 (catalog no. GDC034, CCTCC) , HEp-2 (catalog no. GDC004, CCTCC) , and Huh7 cells (catalog no. GDC134, CCTCC) were cultured in DMEM (Gibco) with 10%FBS (Gibco) , 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco) at 37℃ and 5%CO2. OVCAR-3 cells (catalog no. TCHu228, CCTCC) were cultured in RPMI Medium 1640 (Gibco) with 20%FBS at 37℃ and 5%CO2. HEK293F cells (Thermo) were cultured using Freestyle 293 expression medium (Invitrogen) in an incubator at 37℃ and 8%CO2 with shaking at 180 rpm/min. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, strain: GWHBEBW01000000) was originally isolated from a swab sample of a 33 year old man travelling from Indonesia to Wuhan in June 2021. All processes in this study involving authentic SARS-CoV-2 were performed in a biosafety level 3 (BSL-3) facility.
Ethics statements
Twenty-four female Syrian golden hamsters (five-to-six weeks old) were randomly assigned to different groups in this study. Viral infections were performed according to the standard operating procedures of the biosafety level 3 (BSL-3) facility. All procedures of the animal experiments were in line with recommendations for the care and use of laboratory animals.
Administration to hamsters
Groups of 6 female Syrian golden hamsters were challenged intranasally with 104 PFU of the Delta and Omicron variants per animal in 100 μl of PBS after anesthetization with isoflurane. For the prophylactic group (Group 1) , hamsters were intranasally administered 100 μl of Antibody B (5 mg/kg for Delta challenge and 15 mg/kg for Omicron challenge) 30 min before challenge and given two more doses at 24 h and 48 h post challenge. The activity of Antibody B was also assessed in an in vivo therapeutic experiment. Alternatively, hamsters were intranasally administered 100 μl of 5 mg/kg (Delta challenge) or 15 mg/kg (Omicron challenge) Antibody B (Group 2) or PBS (Group 3, this group was established only in the Delta challenge but not the omicron challenge) at 3 h post challenge, followed by two doses of 100 μl of 5mg/kg (Delta challenge) or 15 mg/kg (Omicron challenge) Antibody B at 24 h and 48 h post challenge. The negative control group (Group 4; this group was named Group 3 in the Omicron challenge) was treated the same as Groups 2 and 3 but with the equivalent volume of PBS instead of antibody. At 3 dpi, three animals were euthanized, and nasal washes, throat swabs and tissues (lung, nasal turbinate and trachea) were harvested for analysis. The remaining three hamsters were monitored for weight loss until 8 dpi in protection experiment against Delta or 6 dpi in protection experiment against Omicron.
Virus RNA copies and titers
Viral RNA in the nasal washes, throat swabs, and tissue homogenates was quantified by one-step real-time RT–PCR as described previously (Feng, L. et al. An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques. Nature communications 11, 1-11 (2020) ) . Briefly, viral RNA was purified using a QIAamp Viral RNA Mini Kit (Qiagen) and quantified with a II One-Step qRT–PCR Green Kit (Vazyme Biotech Co., Ltd. ) with the primers ORF1ab-F (5’-CCCTGTGGGTTTTACACTTAA-3’; SEQ ID NO: 82) and ORF1ab-R (5’-ACGATTGTGCATCAGCTGA-3’; SEQ ID NO: 83) . The amplification procedure was set up as follows: 50℃ for 3 min, 95℃ for 30 s, and 40 cycles consisting of 95℃ for 10 s and 60℃ for 30 s. The expression level of cytokines in lung tissues of infected animals was determined similarly, with the primers shown in the supplementary material.
The virus titer was determined with a plaque assay as previously described, with slight modification. (Zhang, Q. et al. A serological survey of SARS-CoV-2 in cat in Wuhan. Emerg Microbes Infect 9, 2013-2019, doi: 10.1080/22221751.2020.1817796 (2020) ) . Briefly, virus samples were serially 10-fold diluted with DMEM with 2.5%FBS and inoculated onto Vero E6 cells seeded overnight at 1.5 × 105/well in 24-well plates; after incubation at 37℃ for 1 h, the inoculum was replaced with DMEM containing 2.5%FBS and 0.9%carboxymethyl-cellulose. The plates were fixed with 8%paraformaldehyde and stained with 0.5%crystal violet 3 days later. Virus titer was calculated with the dilution gradient with 10～100 plaques.
Histological Analysis
Lung tissue was fixed with 10%neutral formaldehyde for H&E staining. Histologic lesion severity was scored per lung lobe according to a standardized scoring system evaluating the presence of interstitial pneumonia, type II pneumocyte hyperplasia, edema and fibrin deposition, and perivascular lymphoid cuffing.
Statistical analysis
All statistical analyses were performed in GraphPad Prism 9. An unpaired t test followed by Welch’s correction for unequal standard deviations (SD) was used for comparisons of two groups. The asterisks shown in the figures refer to the level of significance (zeros after the decimal point of the p value) : *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.
Results
Antibody B shows protective efficacy in vivo in hamster model through intranasal administration
Antibody B potently and broadly neutralizes different SARS-CoV-2 VOCs
The neutralization activity of Antibody B was first measured with pseudotyped SARS-CoV-2 viruses, including SARS-CoV-2 WT, Delta and Omicron variants (Figure 1B) . Antibody B showed broadly neutralizing activities against all tested pseudoviruses, with IC50 values ranging from 4.75 to 165 ng/ml. In a live viral plaque reduction assay, Antibody B neutralized authentic SARS-CoV-2 WT, Delta, and Omicron with IC50 values of 2.63 ng/ml, 3.38 ng/ml, and 108.6 ng/ml, respectively (Figure 1C) . Remarkably, the IC50 of Antibody B against omicron was lower than that of other tested antibodies (Cao Y, et al. Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature, (2021) ) .
Antibody B delivered via intranasal administration shows protective efficacy against Delta variant in vivo in a hamster model
In our previous study, Antibody B exhibited protective efficacy in hACE2-transgenic mice (K18 mice) challenged with SARS-CoV-2 WT and the Beta variant when administered intraperitoneally. While K18 mice served as a very useful animal model for evaluating vaccines and therapeutics in the early stage of the pandemic, they are much more expensive, have more limited supplies, and have recently been replaced/or partially replaced by golden (Syrian) hamsters. SARS-CoV-2 can cause lethality in K18 mice and severe damage to various organs, including the lung, liver, kidney, and even brain (McCray PB, Jr., et al. Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus. Journal of virology 81, 813-821 (2007) ) . Therefore, this model might not be suitable for the evaluation of drugs administered nasally. The golden (Syrian) hamster can be naturally infected by SARS-CoV-2, which results in the presence of viral antigens in the nasal mucosa, bronchial epithelial cells and areas of lung consolidation after inoculation with SARS-CoV-2 (Sia SF, et al. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters. Nature 583, 834-838 (2020) ) . It has been widely accepted as an animal model by the field for the development of vaccines and therapies against SARS-CoV-2. Hence, we conducted an in vivo study with hamsters. In the prophylactic group (Group 1) , hamsters were intranasally administered 5 mg/kg Antibody B 30 min before challenge with SARS-CoV-2 Delta and given two more doses at 24 h and 48 h post challenge (Figure 1A) . In the therapeutic efficacy assessment, the hamsters were intranasally administered 5 mg/kg Antibody B (Group 2) or buffer only (Group 3) at 3 h post infection. Then, these two groups were given two more doses of Antibody B at 24 h and 48 h post infection. Another group (Group 4) was set as Group 1 but administered buffer only instead of antibody (Figure 1A) .
We monitored animal weight and found that the body weight in groups with antibody intervention (prophylaxis or treatment) either did not decrease or recovered faster than that in untreated groups (Figure 1B) . Viral RNA levels in the nasal wash (Figure 1C) , different tissue homogenates (Figure 1D) , and throat swab (Figure 1E) at 3 dpi were quantified by one-step real-time RT-PCR, while viral titers in different tissue homogenates (Figure 1F) were determined with a plaque assay. In general, the viral RNA levels in the nasal wash, all tested tissue homogenates, and throat swab (Figures 1C-1E) were significantly lower in the prophylactic group (Group 1) than in the other groups. Consistently, the viral titer in the prophylactic group at 3 dpi was under the limit of detection (L.O.D. ) (Figure 1F) .
Antibody B reduces lung injury caused by Delta variant infection
Histological analysis was performed on the lung samples from the three intervention groups and the control (buffer) group at 3 dpi (Figure 2A) . After hematoxylin-eosin (H&E) staining, the lungs from the buffer group (Group 4) displayed lung pathology with increased infiltration of inflammatory cells around blood vessels and branches, extensive alveolar wall broadening and thickening, and a small amount of exudation. In contrast, no increase in the number of perivascular inflammatory cells was seen in Group 1 (prophylactic group) , and lung pathology was characterized by a slight increase in the number of perivascular inflammatory cells for Group 2 (3-dose Antibody B group) . In Group 3 (2-dose Antibody B group) , a modest increase in the number of perivascular inflammatory cells and a slightly widened and thickened alveolar wall were observed. All sections were also pathologically scored for general evaluation of histological changes and inflammation progression (Figure 2B) . The prophylactic group (Group 1) showed a significantly reduced pathology score. One of the therapeutic groups (Group 2) also showed better outcomes, although the statistical significance was not very obvious. In general, these data demonstrate that Antibody B could reduce lung pathology following SARS-CoV-2 infection via nasal delivery.
Antibody B delivered via intranasal administration also shows protective efficacy against Omicron variant in vivo in a hamster model
Since the emergence of the SARS-CoV-2 Omicron variant seriously challenges the current nmAbs approved for clinical use and those under development, we also performed an animal study to evaluate the protective efficacy of Antibody B against Omicron (Figures 3A-3E) . The experiments were performed similarly to the procedure described for animals challenged by the Delta variant but with a 3-fold higher antibody dose and without the 2-dose Antibody B group (Figure 3A) . In general, Antibody B could also protect against Omicron variant challenge in hamsters. The viral RNA levels in the nasal wash, all tested tissue homogenates, and throat swabs (Figures 3B-3D) were significantly lower in the prophylactic group than that in the other groups. In consistence, viral titers in prophylactic group at 3 dpi were significantly lower than that in other groups or under the L.O.D. (Figure 3E) . We did not observe animal body weight decrease in all groups, which was consistent with recent report (Diamond M, et al. The SARS-CoV-2 B. 1.1.529 Omicron virus causes attenuated infection and disease in mice and hamsters. Research square, (2021) ) . Therefore, Antibody B is a promising candidate against Omicron variant infection in  vivo, although most nmAbs lose neutralizing activities against Omicron variant, as mentioned above.
Discussion
Our data indicate that Antibody B can efficiently neutralize authentic Delta and Omicron variants and prevent viral infection in vivo at a low dose when delivered through nasal administration. Hence, Antibody B is a good clinical candidate for use against infections caused by newly emerging SARS-CoV-2 variants (e.g., Delta and Omicron) in humans. In contrast with currently approved nmAbs, Antibody B could relieve patients’ burden due to the low dose used in animal studies and be more acceptable to different patients due to its convenient and safe nasal administration. More importantly, Antibody B is a broad nmAb that can inhibit SARS-CoV-2 and all currently recognized VOCs. Due to the conserved sites in the RBD recognized by Antibody B, it is very possible that Antibody B is capable of neutralizing additional SARS-CoV-2 variants that will appear in the future.
EXAMPLE 3
a) Antibody B Generation
Antibody B is a fully humanized monoclonal antibody that is expressed in mammalian cells transfected with plasmids carrying its heavy chain and light chain genes. Antibody B was expressed by Chinese hamster ovarian cells CHOK1Q. The final cell bank virus safety test proved that there was no risk of contamination by exogenous factors.
CHOK1Q cells were resuscitated with CD02 medium and were cryopreserved after expanded culture to establish the primary cell bank.
The heavy chain and light chain amino acid sequences of Antibody B are shown below. The corresponding DNA sequence are shown afterward each.
B monoclonal antibody heavy chain amino acid sequence
B monoclonal antibody heavy chain DNA sequence
B monoclonal antibody light chain amino acid sequence
B monoclonal antibody light chain DNA sequence

Plasmid transfection of host cells
In order to construct engineering cells, the plasmids carrying the target gene were transfected into the host cell by electric transfer. Cells were cultured in BalanCD CHO GrowthA medium containing 4 mM Glutamine for 3 days, diluted and passaged once, and sampled and counted after 3 days of continuous nurturing. The viable cell density is 7.01 × 10 5 cells/mL, and the cell viability is 97.82%. Prepare for electric transfer. Take 13 mL of host cell suspension and centrifuge at 200 g at room temperature for 5 min. Discard the supernatant, resuspend and mix with 500 μL EX-CELL CD CHO Fusion for later use. Add 250 μL EX-CELL CD CHO Fusion, 10 μg of linearized B1 HC plasmid digested with PvuI and 15 μg of linearized B2 LC plasmid digested with PvuI into a new centrifuge tube and mix well. Mix the host cell and plasmid suspension well and add them to the electric shock cup. The voltage is 300 V, the capacitance is 650 μF, and the resistance is infinite.
EXAMPLE 4
Method
Cells and virus
Vero E6 cells (catalog no. CRL-1586TM) were cultured in DMEM (Gibco) with 10%FBS (Gibco) , 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco) at 37℃ and with 5%CO2. The SARS-CoV-2 WIV04 strain was originally isolated from a COVID-19 patient in 2019 (GISAID, accession no. EPI_ISL_402124) . The SARS-CoV-2 Omicron virus was isolated from a throat swab of a patient from Hong Kong (CCPM-B-V-049-2112-18) . All processes in this study involving authentic SARS-CoV-2 were performed in a BSL-3 facility.
ELISA
ELISA plates (corning 96well clear flat bottom, Cat no. 9018) were coated with 50μl/well of SARS-CoV-2 RBD recombinant protein at 4 μg/ml in 0.05 M PBS, pH 9.6, 4℃ overnight. Serially diluted Antibody B solution was added to each well and incubated at 37℃ for 1h. Peroxidase-conjugated Mouse Anti-Human IgG Fc Antibody [HRP] , mAb (GenScript, Clone ID 50B4A9) was used as the detection antibody. The absorbance was measured at 450 nm using a microplate reader.
Inhibition of pseudotyped SARS-CoV-2 infection
The experiments were performed as described in Li T, et al. Nature Communications 12, 6304 (2021) . In brief, serially diluted mAbs in a volume of 50 μl were incubated with the same volume of Lenti-X293T cell supernatants containing pseudovirus (WT SARS-CoV-2, Delta, and Omicron) for 1 h at 37℃. These Antibody B mixtures were added to ACE2-expressing Lenti-X293 T cells (293 T/ACE2) . After 72 h, the luciferase activities of infected 293T/ACE2 cells were detected with the Bright-Luciferase Reporter Assay System (Promega, E2650) . The IC50 values of the evaluated mAbs were tested determined with a Varioskan LUX Microplate Spectrophotometer (ThermoFisher) and calculated by four-parameter logistic regression using GraphPad Prism 8.0.
Neutralizing activities against authentic SARS-CoV-2
An authentic SARS-CoV-2 neutralization assay was performed to evaluate the inhibition of virus attachment by antibodies with a plaque assay in a BSL-3 laboratory (Zhao, S. et al. Identification of potent human neutralizing antibodies against SARS-CoV-2 implications for development of therapeutics and prophylactics. Nature communications 12, 4887, doi: 10.1038/s41467-021-25153-x (2021) ) . In brief, Vero cells were seeded at 1.5×105 per well in 24-well culture plates overnight. The antibody samples were serially diluted threefold in DMEM with 2.5%FBS. An equal volume including 600 PFU/ml SARS-CoV-2 was added, and the antibody-virus mixture was incubated at 37℃ for 1 h. Then, the mixture was added to a 24-well culture plate containing Vero cells. Cells infected with 300 PFU/ml SARS-CoV-2 only and cells without virus were used as positive and uninfected controls, respectively. After another incubation at 37℃ for 1 h, the antibody-virus mixture was removed, and the medium was replenished with DMEM with 2.5%FBS plus 0.9%carboxymethyl cellulose. The plates were fixed with 8%paraformaldehyde and stained with 0.5%crystal violet 3 days later.
Results
Antibody B binds to conserved region in the SARS-CoV-2 S protein RBD
The SARS-CoV-2 S protein is the major antigenic protein and target for the development of neutralizing antibodies and vaccines (Walls et al. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281-292 e286 (2020) ) . It utilizes human angiotensin-converting enzyme II (hACE2) as its cellular receptor to enter the host through its receptor-binding domain (RBD) (Zhou P, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273 (2020) ) . The region in the RBD that directly participates in the interaction between the RBD and ACE2 is termed the receptor-binding motif (RBM) (Lan J, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215-220 (2020) ; Shang J, et al. Structural basis of receptor recognition by SARS-CoV-2. Nature 581, 221-224 (2020) ; Wang Q, et al. Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. Cell 181, 894-904 e899 (2020) ) . Although most mutations occur in the RBM, there is still an opportunity for the identification of broad nmAbs recognizing some conserved regions in the RBD (Cameroni E, et al. Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift. Nature, (2021) ) . By a single-B cell-sorting method, we previously identified a human monoclonal antibody that could bind to RBDs from SARS-CoV-2 WT and the Alpha, Beta and Gamma variants (Li T, et al. Potent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants. Nature communications 12, 6304 (2021) ) . Here, we found that Antibody B retained its binding activity with EC50 values of 22.5 ng/ml for the Delta RBD and 24.3 ng/ml for SARS-CoV-2 WT RBD, respectively, and still strongly bound to the Omicron RBD, with an EC50 of 90.0 ng/ml (Figure 4A) . Based on our previous study, Antibody B recognizes two major regions in the RBD (Figures 4D-4E) (Li T, et al. Potent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants. Nature communications 12, 6304 (2021) ) .
Discussion
The nmAbs are powerful weapons to fight against infectious diseases. To date, a panel of neutralizing antibodies against SARS-CoV-2 from transgenic mice, convalescent patients and phage display have been identified and characterized. Among these antibodies, some of them have already been approved for treatment of COVID-19. However, these mAbs had either lost or significantly decreased their protective efficacy against the two most dominant VOCs, delta and omicron (Cao Y, et al. Omicron escapes the majority of existing SARS-CoV-2 neutralizing  antibodies. Nature, (2021) ; Liu L, et al. Striking Antibody Evasion Manifested by the Omicron Variant of SARS-CoV-2. Nature, (2021) ) . Among these approved mAbs, S309 (VIR-7831) (Tortorici MA, et al. Science 370, 950-957 (2020) ) maintained its neutralizing activity against Delta and Omicron in a pseudovirus assay (Cao Y, et al. Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature, (2021) ) Currently, S309 (sotrovimab) is approved under an Emergency Use Authorization (EUA) for the treatment of mild-to-moderate COVID-19. However, the benefit of treatment with sotrovimab has not been observed in patients hospitalized due to COVID-19. Therefore, broader nmAbs against SARS-CoV-2 that yield better clinical outcomes are still highly desired.
EXAMPLE 5: Preclinical animal challenge and pharmacokinetics tests of antibody B
Antibody B (B066) preparation was a clear, colorless to light yellow solution, which was contained in 6R vials and could be administered by nasal spraying for many times. The solution in each vial was required to be administered at least 56 times (70μL±14μL each time) . In order to meet the specified times of administration, the labeled amount was set to 6.0mL. For the composition of the preparation, see Table 5. There was no excessive feed in the preparation process.
Table 5: A Composition Comprising Antibody B
Auxiliary materials hydroxypropyl methylcellulose (HPMC) , glycerin and benzalkonium chloride were added to the original solution to obtain the preparation formulation that met the  purpose of multiple times of nasal spraying administration. Through formulation development, the preparation formulation was determined as follows: 5 mg/ml of B066 antibody, 20mM of phosphate buffer (PB) , 4% (w/v) of trehalose (calculated by anhydrous trehalose) , 1.7% (w/v) of glycerin, 0.1% (w/v) of HPMC, 0.01% (w/v) of benzalkonium chloride, and 0.02% (w/v) of PS80, with pH of 6.0.
Golden hamster in-vivo challenge tests -Delta strain: Antibody B coronavirus neutralizing antibodies were administered to golden hamsters (5 mg/mL, 100 μL/hamster) by nasal dripping. Delta strains (1×104 PFU, 100 μL in total, 50 μL/nostril) were used for animal challenge.
Administration was performed on Group 1 (G-1) at 30 min before challenge, and 1d and 2 d after challenge. Administration was performed on Group 2 (G-2) at 3 h, 1 d and 2 d after challenge. Administration was performed on Group 3 (G-3) at 1 d and 2 d after challenge. Group 4 (G-4) was the control group. The body weight data and the turbinate, trachea and lung tissue samples were collected. The virus RNA level in the tissue samples was detected by qRT-PCR and the virus titer was determined by plaque assay.
The results were as follows:
Body weight: at 3 d after golden hamsters were infected with coronavirus Delta strains, the average weight gain of G-1, G-2 and G-3 golden hamsters was 9.46±3.83%, 4.31±2.35%and 2.01±3.69%respectively, and the average weight loss of golden hamsters in the control group (G-4) was 1.93±7.15%. At 6 d after infection, the weight gain of golden hamsters in G-1 and G-2 was 13.88±6.20%and 6.15±9.36%respectively, and the weight loss of golden hamsters in the control group (G-4) was 12.11±10.92%. For details, see Figure 5.
Virus RNA level: at 3 d after challenge, the virus RNA load Log (copies/g) in turbinate, trachea and left and right lung tissues of each group was as shown in Figure 6.
Virus titer: at 3 d after challenge, the Log (virus titer PFU/g) in turbinate, trachea and left and right lung tissues of each group was as shown in Figure 7.
Golden hamster in-vivo challenge tests -Omicron strain: Antibody B coronavirus neutralizing antibodies were administered to golden hamsters (100 μL/hamster) by nasal dripping. Omicron strains (1×104 PFU, 100 μL in total, 50 μL/nostril) were used for animal challenge. 10 mg/mL, 5 mg/mL and 2 mg/mL of Antibody B antibodies were respectively administered to G-1, G-2 and G-3 at 3 h before challenge and 1 d and 2 d after challenge. 10 mg/kg were administered to G-4 at 3 h before challenge by intraperitoneal injection. G-5 was the control group. The body  weight data and the trachea and lung tissue samples were collected. The virus RNA level in the tissue samples was detected by qRT-PCR and the virus titer was determined by plaque assay.
The results were as follows:
Body weight: at 3 d after golden hamsters were infected with coronavirus Omicron strains, the weight gain of the 10 mg/kg, 5 mg/kg and 2 mg/kg nasal dripping administration groups was 5.29±2.24%, 4.70±0.68%and 5.49±2.94%respectively, the weight gain of the 10 mg/kg intraperitoneal injection group was 5.99±1.19%, and the weight gain of golden hamster of the control group was 4.62±2.32%. For details, see Figure 8.
Virus RNA level: at 3 d after challenge, the virus RNA load Log (copies/g) in trachea and left and right lung tissues of each group is as shown in Figure 9.
Virus titer: at 3 d after challenge, the Log (virus titer) in trachea and left and right lung tissues of each group is as shown in Figure 10.
Pharmacokinetics (PK) :
Analysis method: The study on Antibody B in blood samples and tissue samples of mice and nasal swab samples of cynomolgus macaques was based on enzyme-linked immunosorbent assay (ELISA) . The pharmacokinetic study in New Zealand rabbits adopted the SPECT/CT imaging method of radiopharmaceuticals.
Absorption: The clinical route of Antibody B was nasal spraying. The mechanism of action was mainly through its retention in the nasal mucosa to neutralize the coronavirus that entered the human body through the nasal cavity.
Nasal dripping administration to mice: Antibody B coronavirus neutralizing antibodies were administered to mice by nasal dripping. In this experiment, 33 C57BL/6 male mice were divided into 5 groups, 6 mice/group, and the other 3 blank mice were used for the follow-up dilution of Antibody B standard curve samples without any treatment. The concentration of Antibody B was 3 mg/mL, 1 mg/mL, 0.3 mg/mL, 0.1 mg/mL and 0 mg/mL respectively. One dose was given by nasal dripping. Each dose included two times of nasal dripping for left and right nostrils (7.5 μL/drop each time) , 30 μL in total per dose. The content of Antibody B in the nasal mucosa, blood, trachea, lung and liver tissues of mice was detected by enzyme-linked immunosorbent assay (ELISA) at 6 h and 24 h after administration. The results show that the content of Antibody B in nasal mucosa of 3 mg/mL, 1 mg/mL, 0.3 mg/mL and 0.1 mg/mL dose groups was respectively 15.1 μg/mL, 8.26 μg/mL, 1.99 μg/mL, and 0.443 μg/mL at 6 h after administration, indicating that the content of Antibody B in nasal mucosa was positively  correlated with the dose. The content of Antibody B in trachea is respectively 2.7%, 4.5%, 4.5%and 15.5%of that in nasal mucosa. The content of Antibody B in blood is respectively 0.28%, 0.20%, 0.36%and 0.85%of that in nasal mucosa. Only a trace amount of Antibody B is detected in 3 mg/mL and 1 mg/mL dose groups in lung and liver. It shows that Antibody B administered through nasal dripping is mainly distributed in the nasal mucosa. The content of Antibody B in nasal mucosa at 24 h after administration is near the LOB (Limit of blank) 0.073 μg/mL, and there is no significant change in Antibody B in blood, indicating that the nasal mucosa of mice subjected to administration by nasal dripping maintains a high concentration of the Antibody B antibody for at least 6 h. For details, see Figures 11A-11C.
SPECT/CT imaging of New Zealand rabbits: The SPECT/CT imaging of experimental rabbits (supine position) was carried out by adopting chemically synthesized radiopharmaceuticals. The test was divided into a 131I-antibody nasal dripping test group and a Na131I nasal dripping control group. Nasal dripping was carried out after intraperitoneal injection of chloral hydrate for anesthesia, respectively. The effects at 1 h, 3 h, 6 h, 12 h and 24 h after nasal dripping were collected. The results were as follows: 131I-Antibody B nasal cavity had maintained a strong radioactivity within 24 h of nasal dripping, and the signal at the pharynx and larynx was also higher than that of the control group (Na131I) . However, at 6 h after Na131I nasal dripping, the radioactivity in the nasal cavity decreased significantly, and basically disappeared at 24 h. There was no significant concentration of radioactivity in the pharynx and larynx, and the difference was significant (for details, see Figures 12A-12D) .
Nasal spraying administration to cynomolgus macaques: Antibody B coronavirus neutralizing antibodies (5 mg/mL) were administered in a single dose to cynomolgus macaques by nasal spraying (3 females and 3 males) . The content of Antibody B in the nasal swab collection solution of cynomolgus macaques after administration was detected by ELISA. The results show that after single nasal spraying of 1.4 mg/dose (5 mg/mL) of Antibody B to cynomolgus macaques, the drug concentration of the nasal swab reaches a high level (12554 to 52424 ng/mL) at 0.25 h or 1 h, the drug concentration gradually decreases at 3 h after administration, and the drug concentration decreases to a relatively low level (194 to 9693 ng/mL) at 6 h after administration, where t1/2 is 1.35 h, AUC0-6h is 86513 ng/mL·h. For details, see Figure 13. (Note: 3h-L represents the second collection of the left nostril after administration, 6h-L represents the third collection of the left nostril after administration, and 6h-R represents the second collection of the right nostril after administration. The drug concentration of 0 h nasal swab is the average of left and right nasal swabs. The drug concentration of 6 h nasal swab is the data of the right nostril. )
Discussion and conclusion:
In mice and cynomolgus macaques, the Antibody B neutralizing antibodies were administered through nasal cavities to study its pharmacokinetics and provide a reference for clinical dose, administration time and frequency. According to the understanding of different strains of coronaviruses, the transmissibility, infectivity and pathogenicity of wild-type, Delta and Omicron strains are different. Therefore, Antibody B is designed as a product with a target concentration in the nasal cavity that can achieve 100%inhibition rate against coronavirus wild type strain and Delta strain, and more than 90%inhibition rate against the Omicron strain (BA. 2.12.1) .
When Antibody B is administered to mice by nasal dripping (0.1 mg/mL to 3mg/mL, single time, 15 uL for each of the left and right nostrils) , at 6 h after administration, the content of Antibody B in the nasal mucosa is the highest and positively correlated with the dose. At the dose of 3 mg/mL, the concentration in the nasal cavity can reach 15 ug/mL. This concentration can achieve 100%inhibition rate against the wild-type coronavirus strain and Delta strain, and more than 90%inhibition rate against the Omicron strain (BA. 2.12.1) . The content of Antibody B in the nasal mucosa decreases to a level near the background value of the blank sample at 24 h after administration. This shows that the Antibody B antibody in the nasal mucosa of mice subjected to administration by nasal dripping within 6 h can maintain a predetermined target concentration (which can meet the setting of three doses in the daytime) .
When Antibody B is administered by nasal spraying to cynomolgus macaques (5 mg/mL, 280 uL in a single dose) , the concentration reaches the highest concentration in the nasal cavity (35 ug/mL) at 15 min to 1 h after administration, then gradually decreases with time, and reaches 10 ug/mL at 3 h after administration. It is verified by the pseudovirus neutralization experiment that this concentration can achieve 100%inhibition rate against the coronavirus wild-type strain and Delta strain, and more than 95%inhibition rate against the Omicron strain (BA. 2.12.1) . At 6 h after administration, the concentration in the nasal cavity decreases to 3 ug/mL. This concentration can achieve 100%inhibition rate against the coronavirus wild-type strain and Delta strain, but the inhibition rate against the Omicron strain (BA. 2.12.1) decreased to 80%. Since the goal of Antibody B is to achieve 100%inhibition against the coronavirus wild-type strain and Delta strain and 90%inhibition rate against the Omicron strain (BA. 2.12.1) , it can be preliminarily inferred from the above data that Antibody B can maintain the target concentration within 4 h after administration.
The SPECT/CT imaging study was carried out by using 131I-labeled Antibody B (131I-Antibody B) after nasal spraying to healthy adult rabbits. The results are similar to the results of the above studies by nasal dripping to mice and nasal spraying to monkeys. Antibody B is mainly distributed in the nasal cavity within 1-6h after nasal dripping administration, and a small amount is distributed in the pharynx. The concentration decreases rapidly from 6h to 24h, and then decreases to a level near the background level after 24h.
The distribution of the Antibody B neutralizing antibody in various tissues was studied by nasal dripping administration to mice. At 6 h after nasal administration (0.1 mg/mL to 3mg/mL, single dose, 15 uL for each of left and right nostrils) , the content of Antibody B in the trachea was 2.7%to 15.4%of the content in the nasal mucosa; the content of Antibody B in blood was 0.28%to 0.84%of the content in the nasal mucosa; in the lung and liver, only a small amount of Antibody B was detected in the high-dose group (near the lower limit of detection) . It shows that Antibody B administered through nasal dripping is mainly distributed in the nasal mucosa. At 24 h after administration, the content of Antibody B in the nasal mucosa and trachea decreases to a level near the background value of the blank sample, and the concentration of Antibody B in the blood, lung and liver remains at a low level without changing significantly. At 1 h after nasal cavity administration to cynomolgus macaques (5 mg/mL, 280 uL in a single dose) , the concentration of Antibody B in the blood is less than 1%of the nasal cavity concentration. From 1 h to 24 h after administration, the concentration of Antibody B in the blood remains at this low concentration and changes little. These data show that after nasal cavity administration, the Antibody B neutralizing antibody is mainly distributed in the nasal mucosa, a small amount is distributed in the pharynx, and a low amount is distributed in other tissues such as liver, lung, and blood. Low-level system exposure provides a theoretical data support for the safety of Antibody B.
The pharmacokinetics of drug distribution in serum was studied by intravenous injection administration of Antibody B. After intravenous injection of 1 mg/kg Antibody B in mice, the half-life of serum was 16 d, and the 0-14 d AUC was 2579.2 μg·h/mL, AUC (0-∞) was 5655.7 μg·h/mL; the 0-14 d clearance CL was 0.177 mL/h/kg; the distribution volume Vd was 98 mL/kg.
EXAMPLE 6: Clinical Experiment of Antibody B
Clinical pharmacological study: This Investigator Initiated Trial (IIT) study was a randomized, double-blind, placebo-controlled clinical study on the safety and tolerance of the Antibody B neutralizing antibody nasal spray in 72 healthy subjects. The main purpose of this study was to evaluate the safety and tolerance of the Antibody B neutralizing antibody nasal spray  in healthy subjects, and the secondary purpose is to evaluate the drug concentration of the Antibody B neutralizing antibody nasal spray in serum and nasal and pharyngeal swabs of healthy subjects. This trial was a randomized, double-blind, placebo-controlled clinical study. 72 healthy subjects (male and female) were included. After screening qualified subjects into the group, they were randomly assigned to Cohort 1, Cohort 2, and Cohort 3, and blood and nasopharyngeal swabs were collected at the corresponding time points from 1 d before administration to 14 d after the last time of administration for drug concentration detection; safety check was performed on Cohort 1 on Day 2 and at 14 d after the last time of administration, safety check was performed on Cohort 2 on Day 4 and at 14 d after the last time of administration, and safety check was performed on Cohort 3 on Day 10 and at 14 d after the last time of administration. Administration dose and frequency were as follows: for the test group, Antibody B neutralizing antibody nasal spray; specification: 5 mg/mL, 5 mL/vial, 100 uL per spray; nasal administration, two times of spraying to each of left and right nostrils as one dose, 400 uL/dose; placebo group: only containing excipients; specification: 0 mg/mL, 5 mL/vial, 100 uL per spray; nasal administration, two times of spraying to each of left and right nostrils as one dose, 400 uL/dose. Only 1 dose was administered to Cohort 1; 2 doses were administered to Cohort 2 per day for 3 consecutive days; 2 doses were administered to Cohort 3 per day for 7 consecutive days.
Main clinical pharmacological characteristics: The results of this IIT clinical study show the following pharmacokinetic characteristics:
1. The concentration of the Antibody B neutralizing antibody in the nasal cavity can be maintained at 5-11 ug/mL at 6 h after administration of the first dose. According to the in-vitro antiviral neutralizing activity study, the concentration in this range can achieve an inhibition rate of more than 99%against the coronavirus wild-type strain and Delta strain, and an inhibition rate of 80%against the Omicron strain BA. 2.
2. The second dose is administered at 6 h after administration of the first dose, and the concentration of the second dose can be maintained at 5-11 ug/mL at 6 h after administration of the second dose.
3. The concentration of the Antibody B neutralizing antibody in the nasal cavity decreases to the background level at 24 h after administration.
4. The antibody concentration in the blood of 72 subjects does not significantly increase after administration. Considering the nasal administration of the drug in this study, combined with the results observed in the preclinical pharmacokinetics in cynomolgus macaques, it is  speculated that it basically does not enter the blood or only a small amount enters the blood in the human body.
5. The antibody concentration in the pharynx of 72 subjects is not detected or the detected value is near the lower limit of quantitation. It is speculated that this product can be well attached to the surface of the nasal mucosa after nasal administration, and only a small amount may enter the pharynx and larynx.
Clinical pharmacological experiment design: 72 healthy volunteers were selected for this IIT study, who were negative for coronavirus nucleic acid detection, aged 18 to 65 (critical value included) , with an appropriate gender ratio and body weight index in a range of 19.0-30.0 (critical value included) . The selection of clinical dose was mainly based on the results of preclinical pharmacokinetic and pharmacodynamic studies of mice and cynomolgus macaques. The Antibody B neutralizing antibody has a high binding ability to the S1 recombinant protein of the coronavirus wild-type strain S and mutant strains Alpha strain S, Beta strain S, Indian strain S, Delta strain S, Gamma strain S1, Lambda strain S1, and D614G strain. Antibody B has strong pseudovirus neutralizing activity against these coronavirus strains, with IC50 of less than 10 ng/mL, and IC50 of only 1.18 ng/mL for the Delta strain. Antibody B also has super strong live virus neutralizing activity against the coronavirus wild-type strain and Beta strain live viruses, with IC50 of 6.03 ng/mL and 1.66 ng/mL respectively. The live virus in-vivo challenge test shows that Antibody B has a highly effective preventive protection ability to golden hamsters infected with coronavirus Delta and Omicron strains. After nasal dripping of Antibody B with different concentrations to mice, the content of the antibody in the nasal mucosa of mice increased with the increase of the dose. The nasal concentration of Antibody B in the 0.3 mg/mL 50 uL dose group at 6 h after administration was more than 1 ug/mL. At 3 h after single nasal spraying of 380 μL of 10 mg/mL Antibody B to cynomolgus macaques, the concentration of the Antibody B neutralizing antibody in the nasal cavity was about 39 μg/mL. It is predicted that after nasal spraying of 140 μL of 5 mg/mL Antibody B to the human body, the concentration of Antibody B in the nasal cavity will remain much higher than the concentration required by the antibody to neutralize the coronavirus within 6 h. Toxicity tests in ICR mice show that after two doses of 150 mg/kg Antibody B neutralizing antibody are injected intraperitoneally or 5 mg/mL Antibody B is administered by nasal dripping for 14 consecutive days with two doses per day and 25 μL per dose, there is no influence on general physical signs, blood routine, blood biochemistry and histopathology. After a single-dose intravenous injection of 10 mg/kg Antibody B, the blood biochemistry, blood routine and coagulation function of the cynomolgus macaques are normal. After nasal spraying of 5 mg/mL Antibody B for 14 consecutive days with 3 doses per day and  380 μL per dose, the clinical status of cynomolgus macaques is normal, and the blood biochemistry, blood routine and coagulation function are normal. In the Phase-I clinical trial design, the content of each dose of Antibody B was 0.7 mg (140 μL of 5 mg/mL Antibody B) , the maximum amount was three doses per day for 14 consecutive days, accumulating 29.4 mg of Antibody B in total, approximately amounting to 0.69 mg/kg. After nasal dripping to mice or cynomolgus macaques, the amount of the Antibody B antibody entering the blood circulation was less than 1/100. Even if all of them enter the blood circulation according to the dose, the safety margin of Antibody B is respectively 300 and 20 times higher than that in mice and cynomolgus macaques. Therefore, there is sufficient safety margin for nasal spraying of 140 μL of 5mg/mL Antibody B for 14 consecutive days.
Summary of various clinical studies: This IIT study was divided into three cohorts, conducted at The Second Affiliated Hospital of Chongqing Medical University. See Table 6.
In Cohort 1, the mean concentration of the neutralizing antibody in the blood during the baseline period is 1503.0±2508.3 ng/mL, the mean concentration of the neutralizing antibody on the second day of the test is 1427.7±2433.2n g/mL, and the mean concentration of the neutralizing antibody during the follow-up period is 1482.0±2015.7 ng/mL. Although there is a large difference in the concentration of the antibody among subjects, the concentration of the antibody in the blood during the whole test period changes little and tends to be stable. The data of the concentration of the antibody in nasal swabs after administration show a significant difference between the test group and the control group, and the concentration of the antibody in nasal swabs in the test group gradually decreases with time. The data of the concentration of the neutralizing antibody in pharyngeal swabs in the test group and the control group are basically lower than the lower limit of quantitation.
In Cohort 2, the mean concentration of the neutralizing antibody in the blood during the baseline period is 1435.3±1954.5 ng/mL, the mean concentration of the neutralizing antibody on the fourth day of the test is 1355.9±1874.1 ng/mL, and the mean concentration of the neutralizing antibody during the follow-up period is 1220.1±1713.3 ng/mL. Although there is a large difference in the concentration of the antibody among subjects, the concentration of the antibody in the blood during the whole test period changes little and tends to be stable. The mean concentration of the neutralizing antibody in nasal swabs during the baseline period is 4.28±1.79 ng/mL, and the mean concentration of the neutralizing antibody in the left nose on the third day of the test period (the day of the last time of administration) is 9323.1±12636 ng/mL, where the mean of the placebo group is 5.59±0.97 ng/mL, and the mean of the test group is 7940.7±15348 ng/mL; on the fourth day of the test period, the mean concentration of the neutralizing antibody in the left and right noses of the test group is respectively 2780.5±5989.3 ng/mL and 6618.0±14222 ng/mL, the mean concentration of the neutralizing antibody in the left and right noses of the placebo group is respectively 4.86 ng/mL and 4.71±2.71 ng/mL. The data of the concentration of the neutralizing antibody in pharyngeal swabs are basically lower than the lower limit of quantitation.
In Cohort 3, the mean concentration of the neutralization antibody in the blood during the baseline period is 2037.7±4676.6 ng/mL, the mean concentration of the neutralization antibody on the eighth day is 2025.0±4305.8 ng/mL, the mean concentration of the neutralization antibody on the tenth day is 1984.7±4223.9 ng/mL, the mean concentration of the neutralization antibody in the follow-up period is 1743.5±3436.0 ng/mL, and although there are significant differences in inter-individual antibody concentration among subjects, the concentration of the antibody in  blood is changed little and tends to be stable during the whole test. The mean concentration of the nose swab neutralization antibody in the baseline period is 9.79±10.76 ng/mL; the mean concentration of the left nose neutralization antibody in the test period on the seventh day (on the day of the last administration) is 4713.5±6744.3 ng/mL, wherein the mean of the placebo group is 10.56±7.14 ng/mL, and the mean of the test group is 5418.9±6969.2 ng/mL; the mean concentration of the right nose neutralization antibody in the test period on the eighth day is 1272.5±2677.0 ng/mL, wherein the mean of the test group is 1304.9±2704.1 ng/mL, and the mean of the placebo group is 8.25 ng/mL; the mean concentration of the left nose neutralization antibody in the test period on the ninth day is 1496.7±8093.3 ng/mL, wherein the mean of the test group is 1634.5±8462.3 ng/mL, the mean of the placebo group is 27.54±38.31 ng/mL, and there is a significant difference between the test group and the placebo group; and in the test period on the tenth day, the mean concentration of the left nose neutralization antibody is 72.36±152.29 ng/mL, and the mean concentration of the right nose neutralization antibody is 55.90±124.72 ng/mL, wherein the mean concentrations of the left and right nose neutralization antibodies of the test group respectively are 82.88±161.36 ng/mL and 56.97±126.78 ng/mL, the mean concentrations of the left and right nose neutralization antibodies of the placebo group respectively are 3.95±1.88 ng/mL and 24.89 ng/mL, and there is a certain difference between the test group and the placebo group. The concentration data of the pharyngeal swab neutralization antibody is substantially lower than the lower limit of quantitation.
Analysis and comparison of various clinical study results: during the entire study of this IIT experiment, the concentration of the antibody in the blood of subjects is changed little, but there are larger inter-individual differences among the subjects. At the same time, the antibody concentration is almost not detected in three patients who are not inoculated with the coronavirus vaccines (M332, M339, and M342) , and it is speculated that the existing analysis method possibly cannot effectively identify the MY-596 neutralization antibody and the antibody produced after the coronavirus vaccine is injected, and the larger inter-individual antibody differences may be related to the individuals own factors, the types of injected vaccines, and injection time. There is no significant increase in concentration of the antibodies in the blood of 72 subjects, and in consideration of intranasal administration of this study drug, it is speculated that it substantially does not enter the blood or only enters the blood a little. For the 72 subjects, the concentration of the pharyngeal antibody is not detected or the detected value is near the lower limit of quantitation, and it is preculated that this product can be well attached to the surface of the nasal mucous after intranasal administration and only a small amount may enter the throats. For Cohorts 1, 2, and 3, 6 hours after administration of a first dose, the nasal Antibody B concentration can reach 5-11  ug/mL. According to the in vitro antiviral neutralization activity study, the concentration in this range can reach an inhibition rate of over 99%on the coronavirus wild strain and Delta strain, and can also reach an inhibition rate of 80%on Omicron BA. 2. Results of Cohort 2 and Cohort 3 show that in case of two doses of administration every day, the nasal concentration can be kept at concentration with a higher virus neutralization activity within 12 hours during administration. There is no significant drug aggregation in nasal cavities in case of continuous administration for days. For Cohort 1 D2 and Cohort 2 D4, nose swab results show that: on the same day, the antibody concentration of the left nose is significantly lower than the antibody concentration of the right nose, and it is speculated that the difference may be caused by a case that a nose swab sample was collected from the left nose the day before the test. 14 days after the last administration of three cohorts, only a small amount of neutralization antibodies are detected from all the nose swabs, and for Cohort 3, after the last administration, it can be obviously seen that the antibody concentration in the nose is gradually decreased (72 h after the last administration, the antibody amount is basically close to the baseline level) . It can be seen that this study drug is mainly attached to the nasal cavity site and the antibody concentration is gradually reduced over time.
EXAMPLE 7: Preclinical Experiment of Antibody B and Antibody D mixture (A8G6)
1. Introduction
This embodiment illustrates a preclinical experimental result of an antibody B and antibody D mixture. In this embodiment, the mixture is named as A8G6; contained Antibody B and Antibody D, both obtained by sorting viral-specific memory B cells in a peripheral blood sample of a coronavirus patient in the convalescent period in a flow manner, and the sorted individual cells are subjected to antibody gene analysis; then an antibody gene sequence is cloned by a linear gene cassette; and finally, an antibody for nasal spraying is prepared by carrying out stable transfection expression on the antibody gene. The Antibody B and the Antibody D are noncompetitively bound with different and complementary epitopes of a Receptor Binding Domain (RBD) of a SARS-Cov-2 spike protein, so that viruses are synergistically prevented from invading the cells through specific binding with an angiotensin-transferase 2 (ACE2) on human cells, and meanwhile, an immune complex formed by the viruses and a neutralization antibody is easier to phagocytose and eliminate by macrophage, thereby achieving the effect of inhibiting the viruses from infecting the cells.
2. Pharmacology
2.1 Binding Activity
The following several binding activity studies are respectively performed on the coronavirus neutralization antibodies contained in the A8G6:
(1) The binding conditions of the Antibody D antibody with Spike proteins of a wild-type strain and multiple mutant strains are detected by Enzyme Linked Immunosorbent Assay (ELISA) : the experimental result can be found in Figure 14, the Antibody D has higher binding activities (EC50 < 500 ng/mL) with S1 recombinant proteins of multiple coronavirus mutant strains D614G and Gamma (P. 1) and S recombinant proteins of the wild-type strain, the Alpha strain (B. 1.1.7) , the Beta strain (B. 1.351) , the Kappa strain (B. 1.617) , the Delta strain (B. 1.617.2) , the Omicron BA.1 strain (BA. 1.1.529.1) , Omicron BA. 2 (BA. 1.1.529.2) , BA. 4/5 (BA. 1.1.529.4 or BA.1.1.529.5) , and the EC50 thereof respectively are 25.39 ng/mL, 19.45 ng/mL, 43.94 ng/mL, 17.82 ng/mL, 15.84 ng/mL, 254.5 ng/mL, 122.4 ng/mL, 102.5 ng/mL, 89.07 ng/mL, and 193.7 ng/mL.
(2) The binding conditions of the Antibody B antibody with Spike proteins of a wild-type strain and multiple mutant strains are detected by ELISA: the experimental result can be found in Figure 15, the binding activities of the Antibody B with Spike recombinant proteins of the coronavirus wild-type strain and mutant strains Alpha (B. 1.1.7) , Beta (B. 1.351) , Gamma (P. 1) , Kappa (B. 1.617) , Delta (B. 1.617.2) , Lambda (C. 37) , and D614G have no significant difference, and the IC50 respectively are 5.431 ng/mL, 5.574 ng/mL, 6.516 ng/mL, 6.06 ng/mL, 6.411 ng/mL, 6.246 ng/mL, 6.197 ng/mL, and 6.509 ng/mL.
(3) Affinities of the Antibody D for the coronavirus wild-type strain, the Delta strain, the Omicron BA. 1 strain, the Omicron BA. 2 strain, and the Omicron BA. 4/BA. 5 strain are detected by Bio-Layer interferometry (BLI) : the BLI detection result can be found in Figure 16, and the result shows that: the dissociation constants (KD) of the Antibody D antibody with the spike proteins of coronavirus wild-type strain, the Delta strain, the Omicron BA. 1 strain, the Omicron BA.2 strain, and the Omicron BA. 4/BA. 5 strain are respectively less than 1x10-3 nM, equal to 2.58 nM, less than 1x10-3 nM, less than 1x10-3 nM, equal to 2.22 nM. The result above indicates that: the Antibody D has strong affinities for the spike proteins of the coronavirus wild-type strain, the Delta strain, the Omicron BA. 1 strain, the Omicron BA. 2 strain, and the Omicron BA. 4/BA. 5 strain (KD < 10-8 M) .
(4) Affinities of the Antibody B for the spike proteins of the coronavirus Delta strain and the Omicron (BA. 1 and BA. 2) strains are detected by BLI: the BLI detection result can be found in Figure 17, and the result shows that the dissociation constants (KD) of the Antibody B antibody  with the spike proteins of the coronavirus Delta strain and Omicron (BA. 1 and BA. 2) strains respectively are 0.162 nM, 5.86 nM, and 2.37 nM. The dissociation constants are all less than 10-8 M. The result above indicates that the Antibody B has strong affinities for the spike proteins of the coronavirus Delta strain and the Omicron (BA. 1 and BA. 2) strains.
2.2 In-vitro Neutralization Activity
The following several in-vitro neutralization activity studies are respectively performed on the coronavirus neutralization antibodies contained in the A8G6:
(1) By Using the BLI method, it is confirmed whether the A8G6 can effectively compete with ACE2 so as to block binding of the ACE2 with a coronavirus Omicron RBD/Srecombinant protein. The BLI detection result can be found in Figure 18. The result shows that the Antibody D partially competes with the ACE2 so as to blocking the binding of the ACE2 with the coronavirus spike (S) recombinant protein; the Antibody D does not block the binding of the ACE2 with the coronavirus Receptor Binding Domain (RBD) recombinant protein; the Antibody B completely competes with the ACE2 so as to blocking the binding of the ACE2 with the coronavirus RBD or S recombinant protein; and the A8G6 completely competes with the ACE2 so as to block the binding of the ACE2 with the coronavirus RBD or S recombinant protein.
(2) By cryo-electron microsocopy, epitopes where the Antibody B (58G6) /Antibody D (55A8) antibody is bound with the coronavirus mutant strain Omicron S protein, and details of intermolecular interactions are analyzed. From a complex of a Antibody B/Antibody D antigen-binding fragment (hereinafter referred to as Antibody B/Antibody D Fab) and the coronavirus mutant strain Omicron spike protein (hereinafter referred to as S) , it can be observed that three Antibody D Fabs and one Antibody B Fab are bound to receptor binding domains (hereinafter referred to as RBDs) of S protein trimers, and these RBDs are located in two conformations of 1 “up” /2 “down” or 2 “up” /1 “down” ; the Antibody D and the Antibody B can be simultaneously bound to one “up” RBD; and the Antibody D Fab identifies spatial epitopes of RBD proteins S345-352 and S440-450 by using antibody complementary determining regions (hereinafter referred to as CDRs) CDRH3, CDRL1 and CDRL3, and the Antibody B Fab identifies spatial epitopes of RBD proteins S475-480 AND s483-495 by using CDRH2, CDRH3 and CDRL1. The Antibody B and the Antibody D can be simultaneously bound to the same Omicron S protein. This test provides a molecular structure action mechanism and illuminates that the Antibody B/Antibody D antibody is bound with the S protein so as to block binding of the virus with the receptor ACE2 and produces the synergistic neutralization activity by different binding sites.
(3) The study on the neutralization activities of the Antibody D for pseudoviruses of coronavirus wild-type and mutant strains Beta, Gamma, Kappa, Delta, Lambda and Mu is performed: the detection result shows that: the Antibody D has extremely strong neutralization capacity against the pseudoviruses of the wild-type (WT) and mutant strains Beta (B. 1.351) , Gamma (P. 1) , Kappa (B. 1.617.1) , Delta (B. 1.617.2) , Lambda (C. 37) and Mu (B. 1.621) , and IC50 thereof respectively is 37.95 ng/mL, 15.17 ng/mL, 20.42 ng/mL, 117.9 ng/mL, 194.7 ng/mL, 594.8 ng/mL, and 24.47 ng/mL. The experimental result can be found in Figure 19A.
(4) The study on the neutralization activities of the Antibody B for pseudoviruses of coronavirus wild-type and mutant strains Alpha, Beta, Gamma, Kappa, Delta, Lambda and Mu is performed: the detection result shows that: the Antibody B has extremely strong neutralization capacity against the pseudoviruses of the wild-type and mutant strains Alpha (B. 1.1.7) , Neta (B.1.351) , Gamma (P. 1) , Kappa (B. 1.617) , Delta (B. 1.617.2) , Lambda (C. 37) , and Mu (B. 1.612) , and IC50 thereof respectively is 3.48 ng/mL, 7.89 ng/mL, 9.91 ng/mL, 0.49 ng/mL, 9.78 ng/mL, 8.77 ng/mL, 0.61 ng/mL, and 10.13 ng/mL. The experimental result can be found in Figure 19B.
(5) The study on the neutralization activities of the A8G6 for pseudoviruses of the coronavirus mutant strains Delta and Omicron (BA. 2, BA. 2.12.1, and BA. 5) is performed so as to determine the neutralization activities of the A8G6 coronavirus neutralization antibody against the coronavirus mutant strains Delta and Omicron (BA. 2, BA. 2.12.1, and BA. 5) . The detection result shows that: the mean IC50 (mean ± standard deviation) of the A8G6 coronavirus neutralization antibody for pseudoviruses of coronavirus mutant strains Delta and Omicron (B.1.1.529, BA. 2, BA. 2.12.1, and BA. 5) respectively is 6.4±1.0 ng/mL, 13.3±0.4 ng/mL, 13.2±4.9 ng/mL, 36.3±6.1 ng/mL, and 407.6±92.1 ng/mL, and the A8G6 antibody can effectively neutralize the coronavirus mutant strains described above. The specific experimental result is listed in Figure 20.
(6) A Antibody B and Antibody D combined effect verification and pseudovirus neutralization experiment is performed: in order to determine the combined effect of the major active ingredients Antibody B and Antibody D in the coronavirus neutralization antibody nasal spray A8G6, a Antibody B and Antibody D combined BA. 2 coronavirus pseudovirus neutralization experiment is developed. Pseudovirus neutralization experimental data obtained after the Antibody B and the Antibody D are combined for use is analyzed by an analysis template MacSynergy II according to a Bliss independence model so as to determine a judgment standard of the combined effect in this experimental system, and the possible synergistic effect and antagonistic effect when the Antibody B and the Antibody D are combined for use are analyzed.  The Antibody B and Antibody D combined pseudovirus neutralization experiment result shows that the synergy volume is 263.0 ±31.1 ng/mL2%and is higher than a threshold (synergy volume >100 ng/mL2%) of the strong synergistic effect defined in MacSyerngy II, which indicates that combination of two antibodies has the strong synergistic effect; and the Antibody B and Antibody D combined pseudovirus neutralization experiment result shows that the the antagonism volume is -0.1±0.1 ng/mL2%and is within an interval range of an additive volume (-25 ng/mL2%to 25 ng/mL2%) defined by MacSyerngy II, which indicates that combination of the two antibodies has no antagonistic effect. Meanwhile, the CCK-8 cell activity detection result shows that in case of any concentration combination, the cell activity is about 100%, which indicates that when the antibodies are combined for use, no cell cytotoxicity is generated. From the above, it is shown that the combined use of both the Antibody B and Antibody D coronavirus neutralization antibodies has the strong synergistic effect without the antagonistic effect. Experimental results can be found in Table 7, Figure 21A and Figure 21B.
Table 7: Antibody B and Antibody D Combined BA. 2 Pseudovirus Neutralization Experiment Analysis Result

*Macsynergy uses raw data of three complex holes to calculate one synergy 
volume/Antagonism volume value.
A neutralizing assay was performed to confirm the synergetic effect, using pseudoviruses and then authentic viruses. Neutralization titers of antibodies were measured with a plaque reduction neutralization test (PRNT) using authentic SARS-CoV-2, Delta, and Omicron viruses. Briefly, Vero E6 cells (2.5 × 105) were seeded in each well of 24-well culture plates. The cells were incubated overnight at 37℃ with 5%CO2. On the following day, each antibody was serially diluted 5-fold in the culture medium with the highest concentration being 100 μg/ml. The diluted antibody was incubated with an equal volume including 600 PFU/ml SARS-CoV-2 at 37℃ for 1 h, after which the antibody-virus mixtures were inoculated onto preseeded Vero E6 cell monolayers in 24-well plates. After 1 h of infection, the inoculum was removed, and 100 μl of  overlay medium (DMEM supplemented with 0.8%methylcellulose, 2%FBS, and 1%P/S) was added to each well. After incubating the plates at 37℃ for 96 h, the plates were fixed with 8%paraformaldehyde and stained with 0.5%crystal violet. The plaques in each well were counted and normalized to the non-antibody-treated controls to calculate relative infectivity. PRNT50 values were calculated in GraphPad Prism 9.
The individual NAbs 55A8 (antibody D) and 58G6 (antibody B) could neutralize pseudotyped SARS-CoV-2 and the Delta, Omicron BA. 1 and BA. 2 variants, and Omicron BA.1+L452R (Figure 22A) as well as other SARS-CoV-2 variants (data not shown) . The combination of 58G6 and 55A8 showed clear synergetic effects against pseudotyped Omicron BA.1, BA. 1+L452R, and BA. 2 (Figure 22A) . Furthermore, the combination treatment showed synergetic effects against authentic Omicron BA. 1 (Figure 22B) . Notably, the IC50 for Omicron BA.1 (2.81 ng/mL) was much lower than the IC50 values for all other currently known NAbs. In comparison, the IC50 values of S30917 and LY-CoV140418 are 191.1 and 17.30 ng/mL, respectively. Therefore, the combination of 58G6 and 55A8 exhibited an enhanced neutralizing potency and breadth against SARS-CoV-2.
The Antibody B: Antibody D ratio was set as 1: 4 by researching the pseudovirus neutralization activities of two antibodies of the A8G6 in case of different proportioning preparations for the Omicron BA. 4/5 and Delta strains, so that the neutralization activity for the current popular strain BA. 4/5 is continuously maintained, and the neutralization activity for the Delta-like strains can be maintained.
(7) An A8G6 combined ratio determination-pseudovirus neutralization experiment is performed: in order to determine the combined ratio of two major active ingredients Antibody B and Antibody D in the A8G6 coronavirus neutralization antibody combined nasal spray, an Omicron (BA. 2, BA. 2.12.1, and BA. 5) and Delta coronavirus pseudovirus neutralization experiment is developed so as to determine the combined ratio of two antibodies in the A8G6. The pseudovirus neutralization experiment results show that when the coronavirus neutralization antibodies Antibody D and Antibody B are in use separately, IC50 for the coronavirus mutant strain Omicron (BA. 2) pseudovirus respectively is 20.4 ng/mL and 1951 ng/mL, which indicates that the neutralization activity of the Antibody D for the BA. 2 strain is higher than that of the Antibody B; and when the Antibody D and the Antibody B are combined for use (1: 1, 3: 2, and 2: 1) , the mean IC50 (mean±standard difference) for the BA. 2 pseudovirus respectively is 32.7±1.6 ng/mL, 39.3±2.8 ng/mL, and 27.9±1.1 ng/mL, which indicates that by combining two  antibodies for use, the BA. 2 neutralization activity is similar with that when the Antibody D is used separately can be maintained (Table 8; Figure 23A) .
Table 8: BA. 2 Pseudovirus Neutralization Experiment Results in different Antibody B: Antibody D combined ratios
The neutralization activities of the single antibodies and the combined antibodies are further researched, and the neutralization activity of the Antibody B for the BA. 2.12.1 strain is lower than that of the Antibody D, so two combinations with high Antibody D proportions are selected for research, i.e., Antibody D+Antibody B (3: 2 and 4: 1) . Results show that the IC50 for the BA. 2.12.1 pseudovirus in cases of separate use of the Antibody D, separate use of the Antibody B and combined use of the Antibody D and the Antibody B (3: 2 and 4: 1) respectively is 49.9 ng/mL, 811.3 ng/mL, 49.5 ng/mL, and 43.6 ng/mL, which indicates that the combined antibody can maintain the similar neutralization activity for the BA. 2.12.1 strain with that in case of separate use of the Antibody D (Table 9; Figure 23B) .
Table 9: BA. 2.12.1 Pseudovirus Neutralization Experiment Results in different Antibody B: Antibody D combined ratios

With the further development of epidemic, the BA. 5 strain has become a main epidemic strain, the neutralization activity of the combined antibody for the BA. 5 strain is studied, and results show that the mean IC50 (mean±standard difference) for the BA. 5 pseudovirus in cases of separate use of the Antibody D, separate use of the Antibody B and combined use of the Antibody D and the Antibody B (3: 2, 2: 1 and 4: 1) respectively is 221.8±42.1 ng/mL, > 100,000 ng/mL, 353.1±42.5 ng/mL, 408.5±92.3 ng/mL and 229.0±73.5 ng/mL (Table 10; Figure 23C) .
Table 10: BA. 5 Pseudovirus Neutralization Experiment Results in different Antibody B: Antibody D combined ratios
Meanwhile, in consideration of the strong pathogenicity of the Delta strain, the neutralization activity of the combined antibody for the Delta strain is researched. Results show that the mean IC50 for the Delta pseudovirus in cases of separate use of the Antibody D, separate use of the Antibody B and combined use of the Antibody D and the Antibody B (3: 2, 2: 1 and 4: 1)  respectively is 185.7±36.4 ng/mL, 0.89±0.06 ng/mL, 2.25±0.41 ng/mL, 2.14±0.79 ng/mL and 1.12±0.68 ng/mL (Table 11; Figure 23D) .
Table 11: Delta Pseudovirus Neutralization Experiment Results in different Antibody B: Antibody D combined ratios
From the above, the activities for the BA. 5 and Delta strains are taken as the main selection standards; the pseudovirus neutralization experiment results indicate that when the Antibody D and the Antibody B are combined for use in the ratio of 4: 1 among three ratios of 3: 2, 2: 1 and 4: 1, the IC50 is the highest; when the Antibody D and the Antibody B are combined for use in the ratio of 4: 1, the neutralization activity for the Omicron (BA. 5) strain is maintained unchanged relative to separate use of the Antibody D, and the neutralization activity for the Delta strain is maintained unchanged relative to separate use of the Antibody B so as to ensure that the combined antibody can keep the activities for two most important types of strains in current coronavirus varieties, and thus, the Antibody D: Antibody B ratio in the A8G6 preparation is determined as 4: 1.
(8) In-vitro neutralization activities of the A8G6 antibody for coronavirus wild strain and Delta and Omicron strain live viruses: by a plaque reduction neutralization test, in-vitro neutralization activities of the A8G6 and Antibody D for the wild-type, Delta and Omicron strain  live viruses are detected. Results show that: IC50 of the Antibody D for the wild-type, Delta and Omicron live viruses respectively is 3.03 ng/mL, 26.4 ng/mL and 8.24 ng/mL; and IC50 of the A8G6 for the wild-type, Delta and Omicron live viruses respectively is 1.28 ng/mL, 4.37 ng/mL, and 2.81 ng/mL. respectively. It is proved that the A8G6 and the Antibody D have high neutralization activities for the wild-type, Delta and Omicron live viruses. Test results can be found in Figure 24.
2.3 In-vivo Neutralization Activity
The following several in-vivo neutralization activity studies are performed on the coronavirus neutralization antibodies contained in the A8G6:
(1) Dose-effect evaluation of each antibody against a coronavirus Omicron strain that infects golden hamsters is performed:
Figure 25A-25C show the results of the protective efficacy of 55A8, 58G6 and 55A8/58G6 cocktails against Omicron in the hamster model. Syrian golden hamsters challenged with 104 PFU of Omicron were treated with 58G6, 55A8 or the two-antibody cocktail at 1 h pre-infection and 24 and 48 h post-infection. The turbinates, trachea and lungs were harvested on day 3 post-treatment and analyzed by viral titering (by PFU/g and qRT–PCR) . All the treatments led to robust viral clearance (Figures 25B and 25C) . Notably, the virus, detected by measurement of the RNA copy numbers and PFU in the upper respiratory tract (turbinate and trachea) and lower respiratory tract (lungs) , was completely or almost completely inhibited in the combination treatment group, while viral RNA or live virus could be detected in the other two groups that received 58G6 or 55A8 monotherapy. Taken together, these results showed that the combination of 58G6 and 55A8 could efficiently suppress the Omicron variant in vivo through a synergetic effect.
To refine the synergetic effect, more animal protection experiments were performed. To assess the protective efficacy of 55A8 against the Omicron variant, we intranasally challenged hamsters on day 0 with 104 PFU of Omicron BA. 1. At 1 h pre-infection and 24 and 48 h post-infection, groups of hamsters received a single intranasal (IN) treatment with 55A8 alone (Group 1, 1000 μg) or 55A8 in combination with 58G6 (Group 3, 500 μg for each antibody) . At 3, 24, and 48 h post-infection, groups of hamsters received a single IN treatment with 55A8 alone (Group 2, 1000 μg) or 55A8 in combination with 58G6 (Group 4, 500 μg for each antibody) (Figure 26A) . Another group (Group 5) was treated following the regimen for Group 1 but administered buffer instead of the antibody (Figure 26A) . We measured the effects of 55A8 alone or in combination with 58G6 on Omicron viral replication in clinically relevant tissues (i.e., the  nasal turbinates, trachea and lungs) , which were collected on day 3 post-infection. Omicron viral replication was detected by RT–qPCR and plaque assays.
As expected, hamsters treated with buffer had significant viral RNA copy numbers and viral titers in the turbinates and lungs. Pretreatment with 55A8 or the 2-cocktail at 1 h pre-infection followed by two-dose administration lowered the Omicron viral RNA copy number in the lungs by 4–5 logs. More notably, in the turbinates, pretreatment with the 55A8 and 58G6 mixture 1 h pre-infection followed by two-dose administration (Group 3) reduced the Omicron viral RNA copy number by six orders of magnitude, producing a copy number that was significantly lower than that in Group 1 (Figure 26B) . Consistently, the viral titers in Group 3 at 3 days post-infection (dpi) were significantly lower than those in the other groups or under the limit the detection (L.O.D. ) (Figure 26C) . The protective effects of 55A8 or the 2-cocktail dropped slightly when the treatment was administered at 3 h post-infection and repeated at both 24 and 48 h post-infection. These results indicate that 55A8 protects against IN infection with the Omicron variant and that combining 55A8 with 58G6 increases protective efficiency in hamsters. In fact, the animals treated with the combination of 58G6 and 55A8 maintained a higher weight increase than mock-infected controls (Group 5) (Figure 26D) . This confirmed that the 55A8/58G6 cocktail reduced Omicron viral replication and prevented disease symptoms without causing additional distress.
To determine the protective doses of the antibodies included in the combination therapy, we further experimented with four different doses (250, 100, 50, and 25 μg for each antibody, administered daily) as Groups 1, 2, 3 and 4, respectively; the antibodies were administered intranasally to evaluate protective efficacy and assess possible differences in protection (Figure 27A) . In addition, another group (Group 5) was administered buffer only as a control. All the tested doses provided protection (Figures 27B and 27C) . Surprisingly, in the 25 μg dose group (Group 4) , the viral RNA level and titer were generally similar to those in the other intervention groups. These data show that the combination of 58G6 and 55A8 confers protection even at the extremely low dose of 25 μg per antibody. Therefore, the 2-cocktail is a promising candidate against Omicron variant infection in vivo.
(2) Dose-effect evaluation of the A8G6 against the coronavirus Omicron strain that infects golden hamsters was performed: the A8G6 coronavirus neutralization antibody was applied in a nasal dripping manner to female golden hamsters (6 per group) , and the Omicron strain was used for animal challenge (1x104 PFU, 100 μL in total, and 50 μL per nostril) . G-1, G-2, G-3, and G-4 were respectively applied with 10 mg/kg, 4 mg/kg, 2 mg/kg, and 1 mg/kg of A8G6 antibody 3  hours before challenge, 1 day after challenge and 2 days after challenge; and G-5 is a control group. (see Figure 27A for Groups 1-5) body weight data was collected, trachea and lung tissue samples were collected, the virus RNA levels in the tissue samples were detected by qRT-PCR, and the virus titer was measured by a plaque assay method. Results are as follows:
Weight: after the golden hamsters are infected with the coronavirus Omicron strain for three days, the weights of the golden hamsters in the groups in which the doses of the A8G6 are 10 mg/kg, 4 mg/kg, 2 mg/kg and 1 mg/kg are respectively increased by 8.71±2.39%, 8.92±1.91%, 9.97±2.15%, and 7.83±1.86%, while the weight of the golden hamster in the control group is increased by 7.59±1.57%. Results can be found in Figure 27D.
Virus RNA level: the virus RNA loads Log10 (copies/g) in the trachea and the left and right lung tissues of the golden hamster in the 10 mg/kg nasal dripping group respectively are 3.11±1.28, 2.10±0.78, and 2.24±0.62; the virus RNA loads Log10 (copies/g) in the trachea and the left and right lung tissues of the golden hamster in the 4 mg/kg nasal dripping group respectively are 3.67±1.43, 3.00±0.82, and 2.75±0.57; the virus RNA loads Log10 (copies/g) in the trachea and the left and right lung tissues of the golden hamster in the 2 mg/kg nasal dripping group respectively are is 4.55±1.35, 3.83±1.54, and 3.64±1.45; the virus RNA loads Log10 (copies/g) in the trachea and the left and right lung tissues of the golden hamster in the 1 mg/kg nasal dripping group respectively are 3.74±1.67, 3.02±0.45, and 2.47±0.84; and the virus RNA loads Log10 (copies/g) in the trachea and the left and right lung tissues of the golden hamster in the control group respectively are 7.06±0.57, 7.96±0.65, and 7.20±1.33. After challenge, the virus RNA loads in the lung tissues of the golden hamsters in the nasal dripping administration groups are reduced by 3.56-5.86 Log10 values and are significantly lower than that in the control group. After challenge, the virus RNA loads in the tracheae of the golden hamsters in the nasal dripping administration groups are reduced by 2.51-3.95 Log10 values relative to the control group. It is obtained by statistical analysis that the P values of the experimental groups and the control group are less than 0.01 and have significant differences. The virus RNA loads in the trachea and the left and right lung tissues of the control group and the virus RNA loads in the tracheae and the left and right lung tissues of different dose administration groups are statistically analyzed; results show that data is in conformity with normal distribution and variance homogeneity analysis; and by using two-way analysis of variance (2-Way ANOVA) , the P values are less than 0.01 and have significant statistical differences. See Figure 27B.
Virus titer: after 3 days of challenge, all the golden hamsters were dissected, the tracheae and the left and right lung tissues were respectively taken to perform virus titer measurement. The  arithmetic means of the Log10 (PFU/g) virus titers in the trachea and the left and right lung tissues of the golden hamster in the control group respectively are 4.96±0.79, 6.24±1.04, and 6.25±0.55; the Log10 (PFU/g) virus titers in the tracheae and the left and right lung tissues in the 10 mg/kg, 4mg/kg and 1mg/kg nasal dripping groups are located at the detection limit (1.30) ; the means of the Log10 (PFU/g) virus titers in the trachea and the left and right lung tissues in the 2 mg/kg nasal dripping group respectively are 1.78±1.18, 1.71±1.00, and 1.82±1.27; and compared to the control group, the virus titers in the lung tissues in the nasal dripping groups are reduced by 4.43-4.95 Log10 values and the virus titers in the tracheae are reduced by 3.18-3.66 Log10 values. It is obtained by statistical analysis that the P values of the experimental groups and the control group are less than 0.0001 and have significant differences. The virus titers in the trachea and the left and right lung tissues of the control group and the virus titers in the tracheae and the left and right lung tissues of different dose administration groups are statistically analyzed, and results show that data is in conformity with variance homogeneity analysis; and normal distribution of the virus titer data is detected by the Kolmogorov-Smirnov test method, and results show that data of the experimental groups is not in conformity with normal distribution near or below the detection limit, and data of the control group is in conformity with normal distribution. By using two-way analysis of variance (2-Way ANOVA) , the P values are less than 0.0001 and have extremely significant statistical differences. Results can be found in Figure 27C.
Lung tissue pathology: the lung pathological damage of the Omicron infected golden hamsters is not obvious, and the administration groups and the control group are compared and there is no significant differences.
2.4 Immunological Characteristics
Coronavirus mutant strain B. 1.1.7 and B. 1.351 were used as cell models for researching whether the Antibody D mediates the Antibody-Dependent Enhancement (ADE) effect of the coronavirus infection, and results indicate that the Antibody D has no ADE effect. In combination with the in-vivo and in-vitro neutralization activity data of the Antibody D for the coronavirus live viruses, it is indicated that the Antibody D does not mediate the ADE effect of the coronavirus infection. Likewise, for the Antibody B, it was researched whether the Antibody B mediates the ADE effect of the coronavirus infection by using (i) coronavirus wild-type, D614G and D614G-based multiple mutant strain pseudoviruses and coronavirus mutant strains B. 1.1.7, BA. 1 and BA.2 as cell models and using various cell models such as (ii) coronavirus mutant strains B. 1.1.7, BA.2 and BA. 4 in Raji and Daudi. Results indicate that the Antibody B (i.e. 58G6) has no ADE effect. In combination with the in-vivo and in-vitro neutralization activity data of the Antibody B  for the coronavirus live viruses, it is indicated that the Antibody B does not mediate the ADE effect of the coronavirus infection.
3. Pharmacokinetics
Pharmacodynamics studies with respect to A8G6 neutralizing antibodies are mainly carried out with C57BL/6 mice and rhesus macaque for absorption and distribution tests. In the pharmacodynamics studies, studies on A8G6 in blood samples and tissue samples of mice and nasal swab samples of cynomolgus macaques are based on enzyme-linked immunosorbent assay (ELISA) .
3.1 Intravenous injection
The A8G6 coronavirus neutralizing antibody was administered by intravenous injection (1 mg/kg, 12 C57BL/6 mice, half male and half female) . The level of the A8G6 antibody in the blood was tested by ELISA, and the results showed that at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 168 h (7 d) , 336 h (14 d) , 504 h (21 d) and 672 h (28 d) after administration, the means of the A8G6 antibody in the blood of mice were 26, 453±2, 730 ng/mL, 24, 100±3, 023 ng/mL, 19,814±2, 532 ng/mL, 15, 627±2, 245 ng/mL, 13, 133±831 ng/mL, 11, 573±1, 305 ng/mL, 9,098±1, 094 ng/mL, 8, 808±799 ng/mL, 7, 592±1, 372 ng/mL, 6, 625±1, 049 ng/mL, 4, 448±1, 374 ng/mL, 2, 756±1, 149 ng/mL, and 2, 056±914 ng/mL respectively; the statistical half-life (24 h-672 h) of the A8G6 antibody in the blood of mice was 271h (11.3 d) ; statistically, the AUC0-672 h was 3315.5 μg·h/mL and the AUC (0-∞) was 4119.9 μg·h/mL; the CL was 0.243 mL/h/kg; the apparent volume of distribution (Vd) was 94.8 mL/kg. The above results indicate that A8G6 has a longer half-life in blood after intravenous injection, and mainly exists in blood and body fluids.
3.2 Distribution by intranasal administration
(1) Nasal dripping administration of A8G6 coronavirus neutralizing antibody in mice: this experiment employed 36 C57BL/6 mice (half male and half male) , of which 30 mice were in the experimental group (half male and half female) and the other 6 mice (half male and half female) were in the blank control group and not treated for the subsequent diluent of A8G6 standard curve samples. The concentration of A8G6 neutralizing antibody was 5 mg/mL; a dose of the A8G6 neutralizing antibody was administered by nasal dripping (each dose is instilled in each of the left and right nostrils by two drops (7.5 μL/drop each time) ) , and each dose is 30 μL or 150 ug in total. The level of A8G6 in nasal mucosa, blood, trachea, lung, and liver tissue of mice was tested by ELISA at 0.25 h, 1 h, 4 h, 8 h, and 24 h after the administration. The results showed that at 0.25 h, 1 h, 4 h, 8 h, and 24 h after the administration, the geometric means of A8G6 in serum were 13.7 ng/mL, 15.2 ng/mL, 23.4 ng/mL, 37.7 ng/mL and 26.1 ng/mL, respectively, and the  statistical AUC0-24h in serum was 0.809 μg·h/mL; at 0.25 h, 1 h, 4 h, and 8 h after the administration, the levels of A8G6 in nasal mucosa were 55737 ng/ml, 43710 ng/ml, 3083 ng/ml, and 1239 ng/ml, respectively; at 24 h after the administration, the level of A8G6 in LOB (Limit of blank, the highest limit of the blank sample) was 436 ng/mL or below, the statistical AUC0-24h of A8G6 in nasal mucosa was 166 μg·h/mL, and the half-life (0.25-24 h) of the A8G6 antibody in nasal mucosa of the mice was 2.71 h; the levels of A8G6 in the trachea were 254 ng/ml, 335 ng/ml, 83 ng/ml and 87.6 ng/ml, respectively and the level of A8G6 in LOB at 24 h after the administration was 53.3 ng/mL or below; the level of A8G6 in lung and liver tissue was near LOB. At 0.25 h, 1 h, 4 h, and 8 h after the administration, statistically, the levels of A8G6 in the blood was 0.02%, 0.03%, 0.76%, and 3.04%, respectively, and the levels of A8G6 in the trachea were 0.46%, 0.77%, 2.69%, and 7.07%of the antibody level in the nasal mucosa, respectively. It is indicated that by nasal dripping administration, the A8G6 antibody is predominantly distributed in the nasal mucosa and maintained at a high concentration for at least 8 h. Less A8G6 is distributed in the trachea, and the distribution of A8G6 in the lungs, liver and blood is extremely low. More details are shown in Figures 28A-28C.
(2) Pharmacokinetics of the drug in rhesus macaque after administration by nasal spray: 12 rhesus macaque were selected and divided into 2 groups, 4 females and 2 males per group. Administration by nasal spray: the drug was sprayed in the left nostril once and in the right nostril once as one dose. One dose of the drug was administered on D1. Three doses were administered on D5 at an interval of 4 h±5 min. Three doses were administered on D7 at an interval of 4 h±5 min. As shown in the results, in terms of tested nasal swab drug concentration, after administration with one dose by nasal spray, the concentration of drug in the nasal swab samples increased rapidly to a higher level with significant difference among animal individuals. At 0.25 h after the administration, the mean concentration was 50096±10263 ng/mL (36051-67343 ng/mL) and then slowly fluctuated down. The nasal swab drug concentration was slightly reversed and repeated because the data collected at 2 h, 4 h, 6 h after the administration were from different groups treated on different dates. At 24 h after the administration, the drug concentrations in nasal swabs was still maintained at a high level with a mean value of 7064±7625 ng/mL (357-20067 ng/mL) which decreased to about 14.1% (7064 vs 50096 ng/mL) of the mean value at 0.25 h after the administration. The drug concentration was consistently maintained at a high level in the nasal swab samples after administration by nasal spray with three doses per day, with significant difference among animal individuals. At 0.25 h after administration with the third dose, the drug concentration in nasal swabs reached the maximum with a mean value of 90870±37047 ng/mL (63052-160327 ng/mL) . The nasal swab drug concentration was slightly reversed and repeated  because the data collected at 2 h, 4 h, 6 h after the administration were from different groups treated on different dates. At 24 h after the administration, the drug concentrations in nasal swabs was still maintained at a high level with a mean value of 17882±22343 ng/mL (4058-59077 ng/mL) which decreased to about 19.7% (17882 vs 90870 ng/mL) of the mean value at 0.25 h after the administration. In terms of predominant PK parameters, according to the schemes of administration by nasal spray with one dose and three doses per day, the peak drug concentrations Cmax at the administration site were 50096 and 90870 ng/mL, respectively, both reached the peaks at Tmax of 0.25 h; the half lives t1/2 of the drug were 14.3 h and 12.4 h, respectively; AUC0- 24h was 380994 ·ng/mL and 872992·ng/mL, respectively; AUC0-∞ was 526542 and 1193712 ·ng/mL, respectively; MRT0-24h of the drug were 9.20 h and 8.59 h, respectively. The Cmax, AUC0-24h and AUC0-∞ after the administration with three doses per day were 1.81, 2.29 and 2.27 times higher than those after the administration with one dose per day, respectively. However, the administration with one dose and the administration with three doses were similar in terms of Tmax, t1/2 and MRT0-24h. It is indicated that the PK feature of the drug is not changed after the administration with three doses per day. In summary, after intranasal administration, the A8G6 neutralizing antibody is mainly distributed in the nasal mucosa, with low level in other tissues such as liver, lung, and blood. It is indicated that the concentration of the A8G6 antibody in nasal mucosa can maintain a predetermined target concentration which can meet the setting of the administration with three doses on the day, at 6-8 h after administration by nasal dripping.
4. Toxicology
Toxicological studies were conducted on the A8G6 coronavirus neutralizing antibody administered to CD-1 mice repeatedly by nasal dripping in non-GLP conditions and to cynomolgus macaques by single intravenous injection and repeated nasal spray. The research results showed that A8G6 did not cause serious irreversible abnormal reactions in the toxicological tests of different species and doses. The test results indicate that A8G6 was highly safe.
In addition, repeated nasal spray administration to rhesus macaque under GLP conditions was also performed, and animals in each test group did not visibly have a change in each index associated with the tested object. The results of pathological examination showed that no significant histopathological changes related to the administration of tested object were found in the necropsy tissues and organs of animals in each test group. The results of immunogenicity showed that A8G6 coronavirus neutralizing antibody combined with repeated nasal spray administration of a nasal spray caused weak immunogenicity in rhesus macaque (serum) after  repeated nasal spray administration, and basically no immunogenicity was observed at the administration site (i.e. nasal mucosa) . It is indicated that A8G6 has good safety.
5. Comprehensive evaluation and conclusions
In this example, according to the mechanism and characteristics of the A8G6 coronavirus neutralizing antibody nasal spray, the in vitro and in vivo pharmacodynamic effects of the A8G6 coronavirus neutralizing antibody were studied.
In terms of in vitro activity, from two aspects of neutralizing potency and binding ability, considering different epidemic strains, the corresponding RBD &Spike protein binding ability of WT, Delta, Omicron virus strains and the neutralizing potencies of various strains of pseudovirus and live virus were studied. The results of non-clinical pharmacodynamics studies showed that A8G6 coronavirus neutralizing antibody nasal spray effectively neutralized the coronavirus in the nasal cavity, thereby blocking the spread of the coronavirus in the body. Therefore, A8G6 has a function against coronavirus infection.
In terms of in vivo activity, it is contemplated that 1) the coronavirus can multiply in the lungs and other respiratory systems of golden hamsters; 2) infected golden hamsters lose weight, showing pathogenicity to the animals; and 3) the lungs of golden hamsters also show the same lesions seen in lungs of COVID-19 patients, so golden hamster was used as an animal model for in vivo live virus challenge. In the animal model of golden hamsters, the golden hamsters were administered with different doses of A8G6 by nasal dripping before/after inoculation with the Omicron strain, thereby evaluating the administration of A8G6 by nasal dripping in preventing and treating Omicron virus infection in golden hamsters.
Non-clinical pharmacodynamics studies found that A8G6 can effectively bind RBD proteins of different coronavirus strains, effectively neutralize different coronavirus pseudovirus strains Delta, Omicron (BA. 2, BA. 2.12.1, and BA. 5) , and live virus experiments also showed the superior virus neutralizing potency of A8G6.
In the in vivo pharmacodynamics studies using the golden hamster infection model, the administration of A8G6 by nasal dripping with four dosages (10 mg/kg, 4 mg/kg, 2 mg/kg and 1 mg/kg) , the titers of infectious virus in the lung and tracheal tissues were close to or below the limit of detection, and decreased by at least 4.43 and 3.18 Log10 values respectively as compared with those of the control group, indicating that the administration of A8G6 by nasal dripping can effectively reduce the infection of the coronavirus Omicron on the golden hamsters.
In order to investigate the pharmacokinetics of A8G6, single-dose intravenous injection to mice, administration to mice by nasal dripping with different doses, and administration to rhesus macaque by nasal spray were carried out to investigate the change of in vivo drug concentration of A8G6 over time under different routes of administration. Rhesus macaques were administered by nasal spray with 0.7 mg/dose of A8G6 each time, and the drug concentrations in nasal swab samples were tested at different time points. In addition, in the early toxicology test of A8G6 administered to CD-1 mice by nasal dripping, corresponding toxicokinetic studies were also carried out. In the early toxicology studies with cynomolgus macaques, the toxicokinetics under administration by nasal spray and intravenous injection were investigated. Toxicokinetics under the administration by nasal spray were investigated in toxicology studies with rhesus macaques under GLP conditions.
The A8G6 coronavirus neutralizing antibody nasal spray was administered to rhesus macaques for 7 days on every other day, 0.7 mg/dose, where one dose was administered on D1, one dose was administered on D3, three doses were administered on D5 at an interval of 4 h±5 min, and three doses were administered on D7 at an interval of 4 h±5 min. Nasal swab samples were taken during the administration period. The results showed that in the schemes of the daily nasal spray with one dose and three doses, the change trends of the drug concentration in the nasal swabs were basically the same, and the drug concentrations in the nasal swabs both reached the maximum at 0.25 h after administration, and the trends of drug CL in the nasal swabs were basically the same at 24 h after administration, which reduced to about 14.1%to 19.7%of the CL at 0.25 h after administration. Compared with the scheme of daily nasal spray with one dose, the scheme of daily nasal spray with three doses significantly increased the drug concentration. At the same time point, the mean drug concentration after administration with three doses was 1.4 to 4.1 times that after administration with one dose. More details are shown in Figure 29. According to the schemes of administration by nasal spray with one dose and three doses per day, the peak drug concentrations Cmax at the administration site were 50096 and 90870 ng/mL, respectively, both reached the peaks at Tmax of 0.25 h; the half lives t1/2 of the drug were 14.3 h and 12.4 h, respectively; AUC0-24h was 380994 ·ng/mL and 872992·ng/mL, respectively; AUC0-∞ was 526542 and 1193712 ng/mL, respectively; MRT0-24h of the drug was 9.20 h and 8.59 h, respectively. The Cmax, AUC0-24h and AUC0-∞ after the administration with three doses per day were 1.81, 2.29 and 2.27 times higher than those after the administration with one dose per day, respectively. However, the administration with one dose and the administration with three doses were similar in terms of Tmax, t1/2 and MRT0-24h. It is indicated that the PK feature of the drug is not changed after the administration with three doses per day.
In the in vivo challenge experiment of live virus (Omicron) in golden hamsters, the administration of A8G6 by nasal dripping with four dosages (10 mg/kg, 4 mg/kg, 2 mg/kg and 1 mg/kg) can effectively prevent the infection of the coronavirus Omicron strain on the golden hamsters. In PK experiments with rhesus macaques, at 8 h after administration by nasal spray with three doses of 5 mg/mL A8G6 at an interval of 4 h±5 min, the concentration of A8G6 was 39029±20390 ng/ml, which effectively neutralized the current epidemic virus strains. In addition, the concentration of A8G6 in the serum was extremely low, indicating that the AUC of A8G6 nasal spray administration system is low. It is therefore contemplated that the dosage of 5 mg/mL is selected as a clinically planned dosage, which can not only effectively prevent the current epidemic strains, but also greatly reduce the risk of losing neutralizing activity against future variants. In a toxicological experiment performed at this concentration, the animals in all groups did not exhibit abnormal alterations related to the drug.
Administration frequency: the results of non-clinical pharmacodynamics and pharmacokinetics tests show that A8G6 coronavirus neutralizing antibody nasal spray can stay in the nasal cavity for a certain period of time after nasal spray administration, and maintain a high concentration. Therefore, clinically, 3 doses of nasal spray per day can be taken as needed. The time of continuous administration can be considered to be not more than 14 days.
EXAMPLE 8: Summary of clinical experiments for the mixture (A8G6) of antibody B and antibody D
Overview of background: Antibody B and Antibody D, as the two active components of the coronavirus neutralizing antibody A8G6, were screened from the peripheral blood lymphocytes of convalescent patients with coronavirus pneumonia and 209 coronavirus-specific antibodies were isolated from them. The screened Antibody B is a super antibody that has a strong and effective neutralizing effect on the popular British strains, Indian strains, South African strains and Indian Delta strains, and has high affinity, high neutralizing potency, and broad-spectrum mutant activity against the coronavirus, and supports superior-activity antibody structure analysis. Antibody D is a super antibody with high affinity and high neutralizing potency against the coronavirus Omicron (BA. 1 and BA. 2) strains. The binding regions of Antibody B and Antibody D to RBD do not overlap with each other, and there is no competitive relationship between Antibody B and Antibody D. On the contrary, Antibody B and Antibody D have a certain synergistic effect on each coronavirus strain. The cocktail therapy with two Antibody B and Antibody D synergistic antibodies can be effective in broadly neutralizing SARS-CoV-2 and its variants, including Omicron strains BA. 1 and BA. 2. Among the currently reported neutralizing  antibodies against coronavirus in the world, the A8G6 antibody is one of the best in terms of affinity and neutralizing potency against the coronavirus.
The Antibody B neutralizing antibody and the Antibody D neutralizing antibody are the active components in A8G6 coronavirus neutralizing antibody nasal spray. The Antibody B neutralizing antibody and the Antibody D neutralizing antibody were isolated and purified from the culture supernatant of stably expressing cell lines. The clinical preparation of A8G6 contains a total antibody level of 5 mg/mL, where the mass ratio of the Antibody D neutralizing antibody to the Antibody B neutralizing antibody is 4: 1. Excipients in the clinical preparation can be found in Table 12 below:
Table 12: Overview of excipients in the A8G6 neutralizing antibody nasal spray

According to the medication requirements, the label volume was set at 4.5 mL to ensure that the nasal spray can be sprayed at least 42 times in total.
Validation of biological analysis methods: Two methods were developed to test the concentrations of A8G6 neutralizing antibody in human serum and nasal swabs. In the first method, the ELISA was performed to test the total anti-RBD antibody concentration. Since most of the subjects have been vaccinated against coronavirus, there are high and variable concentrations of anti-RBD baseline antibodies in the serum, so the first method is not suitable for testing the concentration of A8G6 in serum samples. Because the concentration of the anti-RBD baseline antibodies in the nasal cavity is very low, the first approach is more suitable for testing the concentration of A8G6 in the nasal cavity. The second method is also developed and can specifically test the concentration of Antibody B in A8G6. This method is used to test the concentration of Antibody B in serum to estimate the concentration of A8G6.
Summary of clinical sample testing and analysis: A total of 360 serum samples were provided in this IIT study and tested by the first method (anti-RBD ELISA) . The results showed that during the entire test period, the concentration of the coronavirus antibody in the blood of the subjects did not change significantly before and after administration, but there were significant differences among the subject individuals. There were 3 subjects who had not got the coronavirus vaccine (1c10, 1d02, 4009) and the concentration of coronavirus antibodies in the blood was significantly lower than that of other subjects. It is speculated that the current testing and analysis method may not be able to effectively distinguish the A8G6 coronavirus neutralizing antibody from the antibody produced after vaccination with the coronavirus vaccine. The huge difference  in antibodies between individuals may be related to individual factors, the type of the vaccine injected, and the time of injection. When the second Antibody B-specific assay was used to test serum samples which were samples mainly from Cohorts 3 and 4 after multiple-day administration, the results showed that the concentrations of Antibody B in all samples were below the lower limit of detection (0.5 ng/mL) , indicating that A8G6 enters the blood at an extremely low level. A total of 1032 nasal swab samples were provided in this IIT study and the samples met the acceptance criteria. The test results showed that the baseline antibody concentration in the nasal cavities of the subjects was less than 10 ng/mL, which was not affected by the vaccination status of the coronavirus vaccine. After administration of the A8G6 antibody by nasal spray, the antibody concentration in the nasal cavity increased significantly. The results indicate that antibodies produced by vaccination itself have a low concentration in the nasal cavity, and the current analysis method can effectively test the concentration of A8G6 in the nasal cavity.
Summary of Study Results: See Table 13 below for an overview of the study.
Table 13: IIT Study Overview
Before starting this IIT study, the A8G6 neutralization antibody was subjected to a series of in-vitro and preclinical studies to support an administration duration for dosages in clinical studies. The IIT study is a randomized, double-blind, placebo-parallel controlled clinical study for safety, tolerance, and pharmacokinetics of the A8G6 neutralization antibody nasal spray in 108 healthy subjects (male or female) . The primary objective thereof is to evaluate the safety and tolerance of the A8G6 neutralization antibody nasal spray in healthy subjects, and the secondary  objective thereof is to evaluate drug concentrations in serums and by nasal swab testing of the A8G6 neutralization antibody nasal spray in healthy subjects. After screening participants for qualified subjects, they are assigned to Cohort 1, Cohort 2, Cohort 3 and Cohort 4 and subjected to blood and nasal swab tests for drug concentration testing at corresponding time points from 1 day before administration to 3±1 days after final administration (7±2 days for Cohort 4) . Cohort 1 is subjected to a safety check at Day 2 and 3±1 day after final administration; for Cohort 2, at Day 4 and 3±1 day after final administration; for Cohort 3, at Day 8 and 3±1 day after final administration; and for Cohort 4, at Day17 and 7±2 day after final administration. Administration dosages and frequencies are as following: a test group: the A8G6 neutralization antibody nasal spray; specification: 5 mg/mL, 6 mL/vial; intranasal administration, the nasal spray is sprayed once (or twice) in each of left and right nostrils as a single dosage, 140 μL (or 280 μL) /dosage; and a placebo group: only an excipient; specification: 0 mg/mL, 6 mL/vial, intranasal administration, the excipient is sprayed once (or twice) in each of left and right nostrils as a single dosage, 140 μL (or 280 μL) /dosage. Dosages administered to different cohorts are as follows:
Cohort 1a (12 subjects) : intranasal administration, once for each of left and right nostrils as a single dosage, 140 μL/dosage, 1 dosage per day for a total of 1 day; Cohort 1b (12 subjects) : intranasal administration, once for each of left and right nostrils as a single dosage, 140 μL/dosage, 2 dosage per day with a total of 1 day; Cohort 1c (12 subjects) : intranasal administration, once for each of left and right nostrils and repeating spaying once for each of left and right nostrils as a single dosage, 280 μL/dosage, 1 dosage per day with a total of 1 day; and Cohort 1d (12 subjects) : intranasal administration, once for each of left and right nostrils as a single dosage, 140 μL/dosage, 4 dosages per day at an interval of 4 h for a total of 1 day; and Cohort 2 (12 subjects) : intranasal administration, once for each of left and right nostrils as a single dosage, 140 μL/dosage, 4 dosages per day at an interval of 4 h for 3 consecutive days; Cohort 3 (12 subjects) : intranasal administration, once for each of left and right nostrils as a single dosage, 140 μL/dosage, 4 dosages per day at an interval of 4 h for 7 consecutive days; and Cohort 4 (36 subjects) : intranasal administration, once for each of left and right nostrils as a single dosage, 140 μL/dosage, 4 dosages per day at an interval of 4 h for 14 consecutive days.
Primary clinical pharmacological features: the results of the IIT clinical study present the following pharmacokinetics features.
The A8G6 neutralization antibody concentration in the nasal cavity can be maintained above Omicron BA. 4/5 neutralization concentration IC90 (5 ug/mL) 4-8 h after administration of 1-2 dosages per day, but not completely above 5 ug/mL at 12 h after administration. The Omicron  BA.4/5 neutralization concentration IC90 (5 ug/mL) is maintained at 4-12 h after administration for 4 dosages per day. In particular, the mean concentration after 12 h, 13-22 ug/mL, is much higher than IC90, 5 ug/mL, which could be drug accumulation in the nasal cavity after multiple administration in a day. From the results, it can be seen that single-dosage administration can achieve a target concentration coverage for 8 h. Hence, 3 dosages per day with a 8-h interval between each dosage can achieve the target concentration coverage for 24h of a day. A BA. 4/5 pseudovirus neutralization experimental study is performed for nasal swab samples of A8G6 IIT. From the inhibition rates, nasal swab samples are collected at 4h after administration after 4 dosages per day, and could achieve an inhibition rate more than 90%for BA. 4/5 pseudovirus after 1: 10 dilution. The inhibition rates basically correspond to A8G6 concentrations in the nasal cavity. Inhibition rates for BA. 4/5 pseudovirus of some samples are above 80%at 8 h, 12 h and 24 h after nasal spray. The pseudovirus neutralization experiment confirms that A8G6 can maintain the useful effect against coronavirus variant BA. 4/5 in the nasal cavity for a longer time.
At 24 h after final administration, drug concentrations by nasal swab testing in subjects are decreased significantly, and drug concentrations by nasal swab testing in most subjects are below a detection limit at 3±1 days after final administration. Drug concentrations by nasal swab testing in some subjects in Cohort 4 is below the detection limit at 48 h after administration.
Blood drug concentrations of all 108 subjects in the study are not increased significantly after administration, while the Antibody B coronavirus antibody concentrations in blood in subjects in Cohorts 3 and 4 are basically below the lower detection limit before and after administration. Considering that the administration route for test drugs is nasal spray, it is presumed that a main component thereof, the A8G6 coronavirus neutralization antibody, and components thereof rarely enters or do not enter the bloodstream.
In summary, in IIT, the pharmacokinetic study after nasal administration provides a basis for further optimizing a nasal administration frequency and an dosing interval, safety data in human bodies and next phase I clinical trials.
Clinical pharmacology experimental design: In the IIT study, 108 healthy volunteers, aged 18-65 years (including critical values) , were selected as healthy subjects, with a mean height of 164.16±7.89 cm, a mean weight of 58.10±11.13 kg and a mean BMI of 21.42±2.76 kg/m2. The sex ratio is appropriate. Clinical dosages are selected based primarily on results of pharmacogenetic and pharmacodynamic studies in the previous example on preclinical mice, cynomolgus macaques and rhesus macaque. The combination nasal spray of the A8G6 coronavirus neutralization antibody has 2 active ingredients, a Antibody B neutralization antibody  and a Antibody D neutralization antibody. Antibody B and Antibody D have high binding and neutralizing abilities with wild strain S and mutant strains Alpha, Beta, Indian, Delta, Gamma, Kappa, Lambda, and D614G of the coronavirus. The pseudovirus neutralizing capacity testing is performed on Antibody D and Antibody B mixed in different ratios, and results show that a mixed antibody of Antibody D and Antibody B with a ratio of 4: 1 (a mass ratio) is the most active against BA.4/5 pseudovirus with an IC50 value of 407 ng/mL (IC90 = 5073 ng/mL) . The pseudo-viral neutralization experimental data after combination administration of Antibody B and Antibody D are analyzed by analyzing template MacSynergy Ⅱ, to determine a standard of a combined effect thereof under the experimental system according to a Bliss independence model, and possible synergistic and antagonistic effects of the combination of the two drugs are analyzed. Results show that combination of two coronavirus neutralizing antibodies, Antibody B and Antibody D, has a strong synergistic effect without antagonistic effect. The mean IC50 values (a mean±standard deviation) of coronavirus neutralization antibody, A8G6, against coronavirus mutant strains Delta, Omicron (B. 1.1.529, BA. 2, BA. 2.12.1 and BA. 5) pseudoviruses are 6.4±1.0 ng/mL, 13.3±0.4 ng/mL, 13.2±4.9 ng/mL, 36.3±6.1 ng/mL and 407.6±92.1 ng/mL, respectively, indicating that A8G6 can effectively neutralize the above coronavirus mutant strains. In addition, IC50 values of A8G6 against wild-type, Delta and Omicron (BA. 1) live viruses are 1.28 ng/mL, 4.37 ng/mL and 2.81 ng/mL, respectively. The antibody combination shows very good synergistic effects. In-vivo live virus infection tests on golden hamsters show that nasal dripping administration of A8G6 at four dosage concentrations (10 mg/mL, 4 mg/mL, 2 mg/mL and 1 mg/mL) results in titers of infectious virus in both lung and tracheal tissues close to or below the detection limit, with decrease of at least 4.43 and 3.18 Log10 values, respectively, compared to the control, indicating that nasal dripping administration of A8G6 can effectively reduce infection of golden hamsters by the coronavirus Omicron. In conjunction with dosage concentration selections of Antibody B, 2 mg/mL and 5 mg/mL are selected as clinical candidates for effective dosing formulation concentrations of A8G6. In the next pharmacokinetic study, the pharmacokinetics in rhesus macaque after intranasal administration was studied by using a higher one of the two candidate concentrations, 5 mg/mL, to provide a basis for clinical administration concentrations and dosages. The rhesus macaque was administered with A8G6 by nasal spray, 1 dosage of 140 uL, 5 mg/mL (0.7 mg/dosage) , and the nasal swab testing concentration is 11457 ± 14054 ng/ml 8-24 h after administration, twice as high as the antibody concentration required to neutralize the coronavirus Omicron BA. 4/5 IC90. The pharmacokinetic study result further confirms that 5 mg/mL is a feasible clinical dosing concentration. The toxicological test result shows that there is no toxic reaction and death after administration to mice via nasal dripping for  2 weeks in each administration group with 5 mg/mL Antibody D, 10 mg/mL Antibody D, 20 mg/mL Antibody D and 5 mg/mL A8G6 and 10 mg/mL A8G6; there is no toxic reaction and death for cynomolgus macaques administered with Antibody D via intravenous injection at 1 mg/kg; there is no toxic reaction and death for cynomolgus macaques administered via nasal spray with A8G6 ( (2.8+2.8) mg/dosage) and Antibody D (2.8 mg/dosage) groups for 2 weeks; and there is no toxic reaction and death for rhesus macaque in the Antibody D group (24 mg/mL) , combined low-dose group (2 mg/mL) , combined medium-dose group (10 mg/mL) , combined high-dose group (30 mg/mL) after nasal spray administration for 14 d. In the tissue cross-reactivity between monkeys and humans, there is no identical or similar antigenic determinant binding to Antibody B and Antibody D antibodies in normal monkey tissue microarrays or in related tissue cells in normal human tissue microarrays. The test result indicates that A8G6 has a high safety.
Considering the neutralizing activity of the combined antibodies against different strains, structural features binding to Spike, yields of two antibodies, analytical assay of mixed antibodies, preclinical safety, and PK characteristics of the nasal spray antibody, the final total antibody concentration of the combined formulation is determined as 5 mg/ml.
In this clinical trial, an A8G6 content is 0.7 mg/dosage (140 μL, 5 mg/mL A8G6) , and the highest dosage is four dosages per day for 14 days, a cumulative total of 39.2 mg A8G6 (7.84 mg Antibody B + 31.36 mg Antibody D) , or about 0.65 mg/kg (with Antibody B 0.16 mg/kg, and Antibody D 0.49 mg/mL) . After nasal dripping to mice or cynomolgus macaques, it is shown that the amount of the Antibody B antibody entering the bloodstream is less than 1/100. Even the full dosage antibody (Antibody B + Antibody D) is introduced into the bloodstream, the safe margins of A8G6 with respect to mice and cynomolgus macaques are greater than 300 and 20-fold, respectively. Therefore, 5 mg/mL A8G6 at 140 μL per nasal spray for 14 days has a sufficient safe margin.
Summary of each clinical study: the IIT study was carried out on four cohorts, as shown in Table 14.



In Cohort 1a, the mean blood neutralization antibody concentration is 2075.9±3048.7 ng/mL during the baseline period, 2406.4±3740.5 ng/mL at the second day of the trial, and 2436.3±3656.4 ng/mL during the follow-up period. Although antibody concentrations are varied widely among subjects, throughout the trial, the blood antibody concentrations are not varied much and tended to be stable. The antibody concentrations by nasal swab testing after administration are significantly different between the test groups and the control group, and the antibody concentrations by nasal swab testing of the test group is decreased gradually over time after administration. The mean value of the neutralization antibody concentrations by nasal swab testing is 3.54±1.15 ng/mL during the baseline period; the mean neutralization antibody concentration in left nostril is 54534±22696 ng/mL at 15 min after administration on D1; the mean neutralization antibody concentration in left nostril is 65062±36064 ng/mL at 1 h after administration on D1; the mean neutralization antibody concentration in right nostril is 29131±21036 ng/mL at 4 h after administration on D1; the mean neutralization antibody concentration in right nostril is 14227±8486.2 ng/mL at 8 h after administration on D1; the mean neutralization antibody concentrations in left and right nostrils are 984.55±1539.9 ng/mL and 1734.5±2596.3, respectively at 24 h after administration on D1; and during the follow-up period, the mean neutralization antibody concentrations in left and right nostrils are 138.52±178.43 ng/mL and 768.51±1898.6 ng/mL, respectively.
In Cohort 1b, the mean blood neutralization antibody concentration is 833.90±1283.6 ng/mL during the baseline period, 925.47±1440.7 ng/mL at the second day of the trial, and 970.30±1584.5 ng/mL during the follow-up period. Although antibody concentrations are varied widely among subjects, throughout the trial, the antibody concentrations in the blood are not varied much and tended to be stable. The mean value of the neutralization antibody concentrations by nasal swab testing is 23.12 ng/mL during the baseline period; during the trial period, the mean neutralization antibody concentration in left nostril is 29008±24144 ng/mL at 15 min before administration of a second dosage on D1; the mean neutralization antibody concentration in left nostril is 149822±97301 ng/mL at 1 h after administration of the second dosage on D1; the mean neutralization antibody concentration in right nostril is 29823±25443 ng/mL at 4 h after administration of the second dosage on D1; the mean neutralization antibody concentration in right nostril is 40142±70821 ng/mL at 8 h after administration of the second dosage on D1; the mean neutralization antibody concentrations in left and right nostrils are 1282.5±2326.4 ng/mL and 524.89±766.92, respectively at 24 h after administration of the first  dosage; and during the follow-up period, the mean neutralization antibody concentrations in left and right nostrils are 76.14±131.73 ng/mL and 32.66±44.43 ng/mL, respectively.
In Cohort 1c, the mean blood neutralization antibody concentration is 642.78±745.23 ng/mL during the baseline period, 594.53±737.58 ng/mL at the second day of the trial, and 574.35±642.12 ng/mL during the follow-up period. Although antibody concentrations are varied widely among subjects, throughout the trial, the antibody concentrations in the blood are not varied much and tended to be stable. The mean value of the neutralization antibody concentrations by nasal swab testing is 7.34 ng/mL during the baseline period; during the trial period, the mean neutralization antibody concentration in left nostril is 9388.5±5591.2 ng/mL at 15 min after administration on D1; the mean neutralization antibody concentration in left nostril is 5857.5±4232.6 ng/mL at 1 h after administration on D1; the mean neutralization antibody concentration in right nostril is 5578.1±3683.3 ng/mL at 4 h after administration on D1; the mean neutralization antibody concentration in right nostril is 2763.2±2986.2 ng/mL at 8 h after administration on D1; the mean neutralization antibody concentrations in left and right nostrils are 355.59±501.31 ng/mL and 373.58±682.39 ng/mL, respectively at 24 h after administration; and during the follow-up period, the mean neutralization antibody concentrations in left and right nostrils are 4.94±4.58 ng/mL and 7.81±7.15 ng/mL, respectively.
In Cohort 1d, the mean blood neutralization antibody concentration is 790.09±852.05 ng/mL during the baseline period, 681.38±751.58 ng/mL at the second day of the trial, and 669.56±723.34 ng/mL during the follow-up period. Although antibody concentrations are varied widely among subjects, throughout the trial, the antibody concentrations in the blood are not varied much and tended to be stable. The mean value of the neutralization antibody concentrations by nasal swab testing is 4.63±4.01 ng/mL during the baseline period; the mean neutralization antibody concentration in left nostril is 41901±42219 ng/mL at 15 min before administration of a fourth dosage on D1; the mean neutralization antibody concentration in left nostril is 85004±49356 ng/mL at 1 h after administration of a fourth dosage on D1; the mean neutralization antibody concentration in right nostril is 115448±39702 ng/mL at 4 h after administration of a fourth dosage on D1; the mean neutralization antibody concentration in right nostril is 22184±19545 ng/mL at 8 h after administration of a fourth dosage on D1; the mean neutralization antibody concentrations in left and right nostrils are 22894±29619 ng/mL and 13242±13701 ng/mL, respectively at 24 h after administration of a fourth dosage on D1;  and during the follow-up period, the mean neutralization antibody concentrations in left and right nostrils are 1194.6±1892.3 ng/mL and 775.76±1411.5 ng/mL, respectively.
In Cohort 2, the mean blood neutralization antibody concentration is 492.48±725.80 ng/mL during the baseline period, 479.64±696.08 ng/mL at the fourth day of the trial, and 500.21±742.42 ng/mL during the follow-up period. Although antibody concentrations are varied widely among subjects, throughout the trial, the antibody concentrations in the blood are not varied much and tended to be stable. The mean value of the neutralization antibody concentrations by nasal swab testing is below the lower detection limit during the baseline period; the mean neutralization antibody concentration in left nostril is 32159±15305 ng/mL at 0.5 h before administration of a second dosage on D1; the mean neutralization antibody concentration in right nostril is 62697±47550 ng/mL at 0.5 h before administration of a fourth dosage on D3; the mean neutralization antibody concentration in left nostril is 20455±39514 ng/mL at 12 h after final administration on D4; the mean neutralization antibody concentration in right nostril is 17307±26128 ng/mL at 24 h after final administration on D4; and the mean neutralization antibody concentrations in left and right nostrils are 189.22±416.92 ng/mL and 1174.7±1669.2 ng/mL during the follow-up period.
In Cohort 3, the mean blood neutralization antibody concentration is 633.75±807.66 ng/mL during the baseline period, 591.95±729.15 ng/mL at 12 h after final administration during the trial, and 588.58±670.01 ng/mL during the follow-up period. Although antibody concentrations are varied widely among subjects, throughout the trial, the antibody concentrations in the blood are not varied much and tended to be stable. The mean value of the neutralization antibody concentrations by nasal swab testing is 7.91 ng/mL during the baseline period; the mean neutralization antibody concentration in left nostril is 30805±18892 ng/mL 0.5 h after administration of a second dosage on D1; the mean neutralization antibody concentration in right nostril is 19444±12761 ng/mL at 0.5 h before administration of a fourth dosage on D7; the mean neutralization antibody concentration in left nostril is 13507±7636.7 ng/mL at 12 h after final administration; the mean neutralization antibody concentration in right nostril is 5715.1±5630.7 ng/mL at 24 h after final administration; and the mean neutralization antibody concentrations in left and right nostrils are 474.62±624.14 ng/mL and 233.01±368.99 ng/mL during the follow-up period.
In Cohort 4, the mean blood neutralization antibody concentration is 1385.7±2775.5 ng/mL during the baseline period, 1255.8±2517.3 ng/mL at 12 h after final administration during the trial, 1278.9±2620.9 ng/mL in 3 days after final administration and 1276.2±2650.5  ng/mL during the follow-up period. Although antibody concentrations are varied widely among subjects, throughout the trial, the antibody concentrations in the blood are not varied much and tended to be stable. The mean value of the neutralization antibody concentration by nasal swab testing is 3.42 ng/mL during the baseline period; the mean neutralization antibody concentration in left nostril is 36138±23212 ng/mL at 0.5 h before administration of the second dosage on D1; the mean neutralization antibody concentration in left nostril is 53615±81154 ng/mL at 0.5 h before administration of the second dose on D8; the mean neutralization antibody concentration in left nostril is 54037±53645 ng/mL at 15 min before administration of the fourth dosage on D14; the mean neutralization antibody concentration in left nostril is 72717±80364 ng/mL at 1 h after administration of the fourth dosage on D14; the means neutralization antibody concentration in right nostril is 28702±25733 ng/mL 4 h after administration of the fourth dosage on D14; the means neutralization antibody concentration in right nostril is 30625±73799 ng/mL at 12 h after final administration; the mean neutralization antibody concentration in left nostril is 12065±39525 ng/mL on D16; the mean values of the neutralization antibody concentrations in left and right nostrils is 2332.4±6804.5 ng/mL and 1578.1±4757.1 ng/mL, respectively on D17; and the mean values of the neutralization antibody concentrations in left and right nostrils during the follow-up period are 2841.2±7987.6 ng/mL and 1109.4±3455.1 ng/mL, respectively.
The following are conclusions regarding drug concentrations.
A large amount of neutralization antibodies could be detected by nasal swab testing after the subjects are administered, presuming that coronavirus neutralization antibody A8G6, a main component of the test drug, could be effectively adhered to the nasal cavity. And, the drug concentration by nasal swab testing is decreased gradually over time after subjects stop receiving drugs; the drug concentration by nasal swab testing is decreased significantly at 24 h after final administration, the drug concentration by nasal swab testing in most subjects is below a detection limit in 3±1 days after final administration, and in some subjects in Cohort 4, the drug concentrations by nasal swab testing are below the detection limit at 48 h after administration. In Cohort 1a, the mean drug concentration by nasal swab testing in subjects is highest at 1 h after administration, presumably due to individual differences among subjects (nasal drug concentrations are much higher in subject 1a08 than in other subjects at 1 h after administration) . It is presumed that the test drug is mainly adhered to the nasal region and that the drug concentrations in nasal regions is gradually decreased over time.
Based on data of drug concentrations by nasal swab testing for subjects in Cohort 1, it can be seen that the time to maintain neutralization antibody concentrations in nasal cavities is increased with a dosing frequency: subjects in Cohort 1d (receiving 4 dosages per day at 140 μL/dosage) have significantly higher drug concentrations by nasal swab testing at 24 h after administration than subjects in Cohort 1a (receiving 1 dosage per day at 140 μL/dosage) , Cohort 1b (receiving 2 dosages per day at 140 μL/dosage) , and Cohort 1c (receiving 1 dosage per day, 280 μL/dosage) . It is presumed that there is a cumulative effect for drug concentrations at nasal sites after administration, with drug concentrations by nasal swab testing maintained for a longer after with multiple administration than single administration or single high-dosage administration.
EXAMPLE 9. FORMULATION DEVELOPMENT FOR Antibody B
We conducted studies to determine the buffer system, emulsifier, humectant, cryoprotectant, and antibiotics for the intranasal formulation of Antibody B, including:
1) using high throughput screening system UNcle to study the melting temperature (Tm) of the antibody in phosphate buffer (PB) or histidine system; the temperature of the onset of heat induced denaturing (Tonset) ; and the temperature of the onset of the aggregation (Tagg) . (The higher these temperature values, the more stable the antibody is in the buffer system, which was then used to determine the buffer system and pH;
2) using a formulation comprising the cryoprotectant trehalose and the emulsifier TWEEN 80 (Polysorbate 80; PS80) to select the humectant and the controlled release agent in mouse/rat animal studies of intranasal administration;
3) adjusting the concentration of trehalose based on osmotic pressure requirements;
4) determining the concentration of the antibiotics based on antibacterial efficacy and stability.
Based on the following studies, the intranasal formulation of Antibody B was determined to be: 5mg/ml Antibody B antibody, 20mM phosphate buffer, 4% (w/v) trehalose, 1.7% (w/v) glycerin, 0.1% (w/v) hydroxypropyl methylcellulose (HPMC) , 0.01% (w/v) benzalkonium chloride, 0.02% (w/v) PS80, pH 6.0. We then evaluated the stability of the formulation using 2～8℃ test, 25/40℃ accelerated test, and 300rpm vibrational test.
Determination of pH and buffer system:
We compared two buffer systems: (i) 20mM phosphate buffer (PB) , pH 6.0±0.2; and (ii) 20mM histidine, pH5.5±0.2, as shown in Table 15. The phosphate buffer system was selected based on the result of Tm, Tonset, and Tagg.
Table 15: Analysis of Buffer Systems
Determination of the humectant and the controlled release agent:
To enhance the retention of the antibody in the nasal cavity, we used a basal formulation comprising 20mM PB (pH 6.0) , 8%trehalose, 0.02%PS80 to study the effect of eight additives for rat intranasal administration; and used the same basal formulation to study the effect of four additives for mouse intranasal administration.
Rat Studies:
Using the basal formulation comprising 20mM PB (pH 6.0) , 8%trehalose, and 0.02%PS80, we tested the effect of eight additives: polyethylene glycol peg (PEG) 400 (PEG400) , PEG4000, Polyvinylpyrrolidone K30 (PVP-K30) , D-sorbitol, Glycerin, Mannitol, HPMC, and hydroxyethyl cellulose (HEC) on the retention of the Antibody B antibody in rat nasal cavity. Each rat was intranasally administered 25 μL/nasal cavity (total 50 μL) , and the nasal mucosal samples were collected 4 hours after administration and the amounts of antibodies in the samples were determined by ELISA. The different formulations were shown in Table 16. As shown in Figure 30A, the results demonstrate that the formulations containing glycerin or HPMC retained more antibodies in the nasal cavity as compared to other formulations.
Table 16: Rat Study Design of Formulations

Mouse studies:
Similar to the rat studies above, using the basal formulation comprising 20mM PB (pH 6.0) , 8%trehalose, and 0.02%PS80, we tested the effect of four additives: PEG400, Glycerin, Mannitol, and HPMC on the retention of the Antibody B antibody in mouse nasal cavity. Each rat was intranasally administered two dose of 7.5 μL/nasal cavity (total 30 μL) , and the nasal mucosal samples were collected 6 hours after administration and the amount of antibodies in the samples were determined by ELISA. The different formulations were shown in Table 17. As shown in Figure 30B, the results demonstrated that the formulations containing glycerin or HPMC retained more antibodies in the nasal cavity as compared to other formulations.
Table 17: Mouse Study Design of Formulations
Based on these studies, we have selected HPMC as the controlled release agent and added glycerin as a humectant, which can improve the humidity of the nasal cavity/mucosa after antibody administration.
Optimization of Cryoprotectant/Osmotic Pressure Regulator
To ensure proper osmotic pressure and antibody stability of the formulation, we tested the concentration of trehalose as the cryoprotectant/osmotic pressure regulator. As shown in Table 18, the Tm, Tonset, and Tagg of the antibody were very similar at both formulations comprising 4%or 8% (w/v) of trehalose. Because the higher 8%concentration of trehalose  resulted in higher overall osmotic pressure of the formulation (520 mOsm/kg) , which is more likely to cause discomfort in the nasal cavity, the trehalose concentration was set as 4%in the formulation, resulting in an overall osmotic pressure of 391 mOsm/kg.
Table 18: Tm/Tonset/Tagg of Antibody and Overall Osmotic Pressure under Different Concentrations of trehalose
Determination of Antibiotic Concentration:
Selection of the antibiotic: an initial study was carried out to select among benzyl alcohol, chlorobutanol, and benzalkonium chloride as the antibiotic for the formulation. Both benzyl alcohol and chlorobutanol were ruled out due to their pungent odors. On the other hand, benzalkonium chloride is an FDA-approved antibiotic that is water soluble and insensitive to pH, making it an appropriate antibiotic in a weakly acidic condition. Thus, benzalkonium chloride was selected for subsequent study.
Concentration of the antibiotic: we tested the antibody stability and inhibition of bacteria growth in formulations containing different concentrations of the antibiotic benzalkonium chloride. Other than the concentration of benzalkonium chloride, other components have the same concentration in the formulation. Table 19 shows the antibody stability study design under 5℃, 25℃, 40℃, or 300rpm shaking conditions. The results are shown in Table 20.
Table 19: Antibody Stability Study Design for Formulations Comprising Different Concentrations of Antibiotic
X= Appearance; size exclusion chromatography (SEC) ; isoelectric focusing capillary (CE) (reducing/non-reducing) ; protein concentration; pH; Tm/Tagg.
Y= Activity.


The bacteriostatic efficacy test demonstrated that both concentrations of the antibiotic benzalkonium chloride meets the requirement of bacterial inhibition. In addition, the antibody stability was also similar under both concentrations of the antibiotic and within acceptable ranges. Thus, the concentration of the antibiotic benzalkonium chloride was determined to be 0.01%in the formulation..
In addition, the formulation tested in the antibody stability study is also the final formulation used in the pharmaceutical composition of Antibody B. The results in the Tables above demonstrated that this formulation could maintain the antibody stability under long-term stability test and accelerated stability test.
EXAMPLE 10. FORMULATION DEVELOPMENT FOR A8G6
The development of A8G6 formulation was based on the results and selection of the buffer system, emulsifier, humectant, cryoprotectant, and antibiotics tested for the Antibody B formulation in the last Example. During the development of A8G6 formulation, we studied the final concentration and relative ratio of Antibody D and Antibody B, the effect of antibiotic concentration on the antibody stability, and the compatibility of the formulation with the primary packaging material. Based on the results below, the formulation of A8G6 was determined to be 1 mg/mL Antibody B antibody, 4 mg/mL Antibody D antibody, 20 mM PB, 4% (w/v) trehalose, 2%(w/v) glycerin, 0.1% (w/v) HPMC, 0.01% (w/v) benzalkonium chloride, 0.02% (w/v) PS80, pH 6.0.
Determination of the Concentrations of the Active Ingredients
Based on the activity against BA. 5 and Delta strain neutralization study, the best IC50 of Antibody D and Antibody B combination was achieved at a 4: 1 ratio among three different ratios (3: 2, 2: 1, and 4: 1) . Thus, the ratio of Antibody D antibody to Antibody B antibody in A8G6 formulation was determined to be 4: 1, and the other ingredients of the A8G6 formulation was determined to be 20 mM phosphate buffer, 4% (w/v) trehalose, 2% (w/v) glycerin (increased by 0.3%as compared to the glyceri%in Antibody B formulation to improve the antibody retention rate in the nasal cavity) , 0.1% (w/v) HPMC, 0.01% (w/v) benzalkonium chloride, 0.02% (w/v) PS80, pH 6.0. Two concentrations of total amount of antibodies, 5 mg/mL and 2 mg/mL respectively, underwent stability test according to the study design in Table 23.
Table 23: Study Design to Determine Total Antibody Concentration


X= Appearance; size exclusion chromatography (SEC) ; isoelectric focusing capillary (CE)
(reducing/non-reducing) ; protein concentration; pH; cation-exchange chromatography (CEX) ; Osmotic 
Pressure.
Y= binding affinity
Z= DSD/SP/PG, bacteria inhibition efficacy
NA=not assayed
The results are shown in Table 24, which show that , at 3-month time point of 5℃ storage condition, the formulation comprising 5 mg/mL antibodies had a little lesser stability decrease as compared to the formulation comprising 2 mg/mL antibodies; at 4-week time point of 25℃storage condition, the sum of main SEC peak for the formulation comprising 2 mg/mL antibodies decreased to 95.6%, whereas that of the formulation comprising 5 mg/mL antibodies was maintained at 99.0%, demonstrating that the 2 mg/mL antibody concentration resulted in faster SEC purity decrease at the earlier stage. For the purity measured by CEX, both 25℃ and 40℃accelerated conditions yielded similar results for both formulations, whereas at 3-month time point of 5℃ storage, the formulation comprising 2mg/mL antibody had about 14.3%decrease of CEX main peak, while the the formulation comprising 5mg/mL antibody maintained the CEX main peak. Taken together, the formulation comprising 5mg/mL antibody displayed superior stability as analyzed by both SEC and CEX, and thus 5mg/mL total antibody concentration (4mg/mL Antibody D antibody, 1mg/mL Antibody B antibody) was used for subsequent formulation development.

Determination of Antibiotic Concentration in A8G6 Formulation
According to the results in Table 24, 0.01%benzalkonium chloride in the A8G6 formulation did not significantly affect the antibody stability. Based on the bacteriostatic efficacy test according to Chinese Pharmacopoeia (see results in Table 25) , when the A8G6 formulation contained 0.01%benzalkonium chloride, the bacterial inhibition efficacy met the requirements according to Chinese Pharmacopoeia (for bacteria, lg decrease ≥2 after 2-day, lg decrease ≥ 3 after 7-day, and the increase amount of bacteria is no more than 0.5 lg after 28-day as compared to the last time point; for fungi, lg decrease ≥ 2 after 14-day lg, and the increase amount of bacteria is no more than 0.5 lg after 28-day as compared to the last time point) . Based on these results, the 0.01%benzalkonium chloride concentration was determined to meet the formulation requirement.
Table 25: Bacteriostatic Efficacy Test

“lg decrease” indicates the differences between the colony lg number and the corresponding 
colony lg number in 1 mL (g) of the testing sample
NA: not assayed
Verification of A8G6 Formulation
This study tested the stability of representative batches of Antibody D antibodies and Antibody B antibodies in the indicated formulation to verify the stability of the formulation. All samples were invertedly stored under various test conditions to examine the compatibility with the packaging materials to ensure the suitability for clinical applications. The study design is shown in Table 26.
Table 26: Verification of A8G6 Formulation

X: appearance, pH, concentration, SEC, CEX, CE (NR&R) ; Y: binding affinity
The results are shown in Table 27. For the representative A8G6 formulation samples, all the measured parameters were stable for 4 weeks under 5℃ or for 1-week under shaking condition. And 4-week after the 25℃ accelerated test, only the purity measurement under CEX was decreased by about 10%but the activity was not significantly affected (remaining within 70%～130%range) . Thus, the formulation of 1 mg/mL Antibody B antibody, 4 mg/mL Antibody D, 20mM PB, 4% (w/v) trehalose, 2% (w/v) glycerin, 0.1% (w/v) HPMC, 0.01% (w/v) benzalkonium chloride, 0.02% (w/v) PS80, pH 6.0 can meet clinical study needs and are fully compatible with the packaging material.
Table 27: Verification of A8G6 Formulation

A: indicating colorless to pale yellow liquid; clear to slight opalescent; no visible substance
B: indicating colorless to pale yellow liquid; clear to slight opalescent; with small amount of visible 
substance with <2mm length.
We also studied the droplet size distribution (DSD) of the A8G6 formulation. 10 vials of the A8G6 formulation were tested using Symapatec H3976 to determine the DSD at 3cm or 6 cm distance, collecting ≤ 10 μm diameter, and the diameter to which 10% (D10) , 50% (D50) , 90%(D90) of the droplets were less than and the half width ( (D90-D10) /D50) (SPAN) , using the average of the 10 samples as the final results shown in Table 28. The results indicate that the DSD of the A8G6 formulation meeting the expectation. (Most droplets had >10 μm diameter) .
Table 28: Results of the Droplet Laser Test

1. Single dose: only conducted measurement to one dose
Further Numbered Embodiments
Further embodiments of the present disclosure are provided in the numbered embodiments below
Embodiment 1. A pharmaceutical composition, comprising:
an active ingredient or a precursor thereof;
no more than about 40% (w/v) of a cryoprotectant;
no more than about 30% (w/v) of a humectant;
no more than about 5% (w/v) of an emulsifier;
no more than about 10% (w/v) of a controlled release agent; and
a buffer.
Embodiment 2. The pharmaceutical composition of Embodiment 1, wherein the pharmaceutical composition is suitable for mucosal administration to a human.
Embodiment 3. The pharmaceutical composition of Embodiment 1 or 2, wherein the pharmaceutical composition is suitable for intranasal administration to a human.
Embodiment 4. The pharmaceutical composition of any one of Embodiments 1-3, wherein the cryoprotectant is selected from the group consisting of trehalose, glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol, and sucrose, or a combination thereof.
Embodiment 5. The pharmaceutical composition of Embodiment 4, wherein the cryoprotectant in the pharmaceutical composition is:
no more than 40% (w/v) trehalose;
no more than 40% (w/v) glycerol;
no more than 10% (w/v) dimethyl sulfoxide;
no more than 40% (w/v) ethylene glycol;
no more than 20% (w/v) polyethylene glycol; or
no more than 0.5 M sucrose.
Embodiment 6. The pharmaceutical composition of Embodiment 4, wherein the cryoprotectant in the pharmaceutical composition is:
about 2%-about 8% (w/v) trehalose;
about 1%-about 20% (w/v) glycerol;
about 1%-about 10% (w/v) dimethyl sulfoxide;
about 4%-about 30% (w/v) ethylene glycol;
about 5%-about 15% (w/v) polyethylene glycol; or
about 0.1 M -about 0.4 M sucrose.
Embodiment 7. The pharmaceutical composition of any one of Embodiments 1-6, wherein the cryoprotectant is trehalose.
Embodiment 8. The pharmaceutical composition of any one of Embodiments 1-7, comprising about 0.3% (w/v) to about 12% (w/v) of the cryoprotectant.
Embodiment 9. The pharmaceutical composition of any one of Embodiments 1-7, comprising about 2% (w/v) to about 8% (w/v) of the cryoprotectant.
Embodiment 10. The pharmaceutical composition of any one of Embodiments 1-7, comprising about 4% (w/v) of the cryoprotectant.
Embodiment 11. The pharmaceutical composition of any one of Embodiments 1-10, wherein the humectant is selected from the group consisting of glycerin, mannitol, polyethylene glycol 400, polyethylene glycol 4000, D-sorbitol, chitosan, xylitol, and sodium hyaluronate, or a combination thereof.
Embodiment 12. The pharmaceutical composition of Embodiment 11, wherein the humectant in the pharmaceutical composition is:
no more than 30% (w/v) glycerin;
no more than 7% (w/v) mannitol;
no more than 20% (w/v) polyethylene glycol 400;
about 5%-about 15% (w/v) polyethylene glycol 4000;
about 3%-about 15% (w/v) D-sorbitol;
about 0.01%-about 3% (w/v) chitosan;
no more than 10% (w/v) xylitol; or
about 0.1%-about 2.0% (w/v) sodium hyaluronate.
Embodiment 13. The pharmaceutical composition of Embodiment 11, wherein the humectant in the pharmaceutical composition is:
about 2%-about 6% (w/v) glycerin;
about 3%-about 5% (w/v) mannitol;
about 10%-about 18% (w/v) polyethylene glycol 400;
about 5%-about 10% (w/v) polyethylene glycol 4000;
about 3%-about 6% (w/v) D-sorbitol;
about 0.01%-about 1% (w/v) chitosan;
about 1%-about 10% (w/v) xylitol; or
about 0.1%-about 1.0% (w/v) sodium hyaluronate.
Embodiment 14. The pharmaceutical composition of any one of Embodiments 1-13, wherein the humectant is glycerin.
Embodiment 15. The pharmaceutical composition of any one of Embodiments 1-14, comprising about 0.2% (w/v) to about 10% (w/v) of the humectant.
Embodiment 16. The pharmaceutical composition of any one of Embodiments 1-14, comprising about 1% (w/v) to about 4% (w/v) of the humectant.
Embodiment 17. The pharmaceutical composition of Embodiment 16, comprising about 1.7% (w/v) to about 2% (w/v) of the humectant.
Embodiment 18. The pharmaceutical composition of any one of Embodiments 1-17, wherein the emulsifier is selected from the group consisting of TWEEN 80, polysorbate 20, lecithin, sorbitan esters, mono-and/or diglycerides, and sodium stearoyl lactylate, or a combination thereof.
Embodiment 19. The pharmaceutical composition of any one of Embodiments 1-18, wherein the emulsifier is TWEEN 80.
Embodiment 20. The pharmaceutical composition of any one of Embodiments 1-19, comprising about 0.002% (w/v) to about 0.1% (w/v) of the emulsifier.
Embodiment 21. The pharmaceutical composition of any one of Embodiments 1-19, comprising about 0.005% (w/v) to about 2% (w/v) of the emulsifier.
Embodiment 22. The pharmaceutical composition of Embodiment 21, comprising about 0.005% (w/v) to about 0.04% (w/v) of the emulsifier.
Embodiment 23. The pharmaceutical composition of Embodiment 21, comprising about 0.01% (w/v) to about 0.02% (w/v) of the emulsifier.
Embodiment 24. The pharmaceutical composition of any one of Embodiments 1-23, wherein the controlled release agent is selected from the group consisting of hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone K30, or a combination thereof.
Embodiment 25. The pharmaceutical composition of any one of Embodiments 1-24, wherein the controlled release agent is hydroxypropyl methylcellulose (HPMC) .
Embodiment 26. The pharmaceutical composition of any one of Embodiments 1-25, comprising about 0.01% (w/v) to about 5% (w/v) , or about 0.01% (w/v) to about 1% (w/v) , of the controlled release agent.
Embodiment 27. The pharmaceutical composition of any one of Embodiments 1-25, comprising about 0.004% (w/v) to about 0.4% (w/v) of the controlled release agent.
Embodiment 28. The pharmaceutical composition of any one of Embodiments 1-25, comprising about 0.01% (w/v) to about 0.2% (w/v) of the controlled release agent.
Embodiment 29. The pharmaceutical composition of Embodiment 28, comprising about 0.02% (w/v) , or about 0.1% (w/v) , of the controlled release agent.
Embodiment 30. The pharmaceutical composition of any one of Embodiments 1-24, wherein the controlled release agent is polyvinylpyrrolidone K30.
Embodiment 31. The pharmaceutical composition of any one of Embodiments 1-30, comprising about 2% (w/v) to about 10% (w/v) , or about 3% (w/v) to about 6% (w/v) , of the controlled release agent.
Embodiment 32. The pharmaceutical composition of any one of Embodiments 1-31, wherein the buffer is selected from the group consisting of a phosphate buffer, a tris buffer, and a glycine buffer.
Embodiment 33. The pharmaceutical composition of Embodiment 32, wherein the buffer in the pharmaceutical composition is:
about 10 mM -about 100 mM phosphate;
about 10 mM -about 100 mM tris; or
about 0.1 M -about 0.5 M glycine.
Embodiment 34. The pharmaceutical composition of Embodiment 32, wherein the buffer in the pharmaceutical composition is:
about 10 mM -about 40 mM phosphate;
about 10 mM -about 40 mM tris; or
about 0.1 M -about 0.2 M glycine.
Embodiment 35. The pharmaceutical composition of any one of Embodiments 1-34, wherein the buffer is a phosphate buffer.
Embodiment 36. The pharmaceutical composition of any one of Embodiments 1-35, wherein the buffer in the pharmaceutical composition is about 20 mM phosphate or tris.
Embodiment 37. The pharmaceutical composition of any one of Embodiments 1-36, wherein the buffer has a pH of about 4-8, or about 5-7.
Embodiment 38. The pharmaceutical composition of any one of Embodiments 1-36, wherein the buffer has a pH of about 5.5-6.5.
Embodiment 39. The pharmaceutical composition of Embodiment 38, wherein the buffer has a pH of about 6.0.
Embodiment 40. The pharmaceutical composition of any one of Embodiments 1-39, comprising an antibiotic.
Embodiment 41. The pharmaceutical composition of Embodiment 40, wherein the antibiotic comprises benzalkonium chloride, benzyl alcohol, chlorobutanol, or a combination thereof.
Embodiment 42. The pharmaceutical composition of Embodiment 41, wherein the antibiotic in the pharmaceutical composition is:
about 0.002%-about 0.02% (w/v) benzalkonium chloride;
no more than 3.0% (v/v) benzyl alcohol; or
about 0.5%-about 2.0% (w/v) chlorobutanol.
Embodiment 43. The pharmaceutical composition of Embodiment 41, wherein the antibiotic in the pharmaceutical composition is:
about 0.005%-about 0.02% (w/v) benzalkonium chloride;
about 1.0%-about 3.0% (v/v) benzyl alcohol; or
about 1.0%-2.0% (w/v) chlorobutanol.
Embodiment 44. The pharmaceutical composition of any one of Embodiments 40-43, wherein the antibiotic is benzalkonium chloride.
Embodiment 45. The pharmaceutical composition of any one of Embodiments 40-44, comprising about 0.002% (w/v) to about 0.1% (w/v) of the antibiotic.
Embodiment 46. The pharmaceutical composition of Embodiment 45, comprising about 0.005% (w/v) to about 0.02% (w/v) , or about 0.01% (w/v) , of the antibiotic.
Embodiment 47. The pharmaceutical composition of any one of Embodiments 1-46, wherein the mean retention time of the active ingredient or a precursor thereof in a nasal cavity of a primate is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, or at least 12 hours, after intranasal administration of the pharmaceutical composition to the primate.
Embodiment 48. The pharmaceutical composition of Embodiment 47, wherein the primate is a rhesus macaque.
Embodiment 49. The pharmaceutical composition of any one of Embodiments 1-48, comprising:
about 0.3% (w/v) to about 12% (w/v) of a cryoprotectant;
about 0.2% (w/v) to about 10% (w/v) of a humectant;
about 0.002% (w/v) to about 0.1% (w/v) of an emulsifier; and
about 0.004% (w/v) to about 0.4% (w/v) of a controlled release agent.
Embodiment 50. The pharmaceutical composition of any one of Embodiments 1-48, comprising:
about 2%-about 8% (w/v) of the cryoprotectant;
about 1%–about 4% (w/v) of the humectant;
about 0.005%-about 0.04% (w/v) of the emulsifier;
about 0.01%-about 0.2% (w/v) of the controlled release agent; and
the buffer has a pH of about 5.5-6.5.
Embodiment 51. The pharmaceutical composition of any one of Embodiments 1-48, comprising:
about 4% (w/v) of the cryoprotectant;
about 1.7%–about 2% (w/v) of the humectant;
about 0.01%-about 0.02% (w/v) of the emulsifier;
about 0.02%-about 0.1% (w/v) of the controlled release agent; and
the buffer has a pH of about 6.0.
Embodiment 52. The pharmaceutical composition of any one of Embodiments 1-48, comprising:
about 4% (w/v) of the cryoprotectant;
about 2% (w/v) of the humectant;
about 0.01% (w/v) of the emulsifier;
about 0.02% (w/v) of the controlled release agent; and
the buffer has a pH of about 6.0.
Embodiment 53. The pharmaceutical composition of any one of Embodiments 1-48, comprising:
about 4% (w/v) of the cryoprotectant;
about 2% (w/v) of the humectant;
about 0.02% (w/v) of the emulsifier;
about 0.1% (w/v) of the controlled release agent; and
the buffer has a pH of about 6.0.
Embodiment 54. The pharmaceutical composition of any one of Embodiments 1-48, comprising:
about 4% (w/v) of the cryoprotectant;
about 1.7% (w/v) of the humectant;
about 0.02% (w/v) of the emulsifier;
about 0.1% (w/v) of the controlled release agent; and
the buffer has a pH of about 6.0.
Embodiment 55. The pharmaceutical composition of any one of Embodiments 49-54, wherein the buffer comprises about 20 mM phosphate salt, optionally wherein the phosphate salt is a sodium phosphate salt.
Embodiment 56. The pharmaceutical composition of any one of Embodiments 49-55, wherein the cryoprotectant is trehalose, the humectant is glycerin, the emulsifier is TWEEN 80, the controlled release agent is HPMC.
Embodiment 57. The pharmaceutical composition of any one of Embodiments 49-56, comprising about 0.01% (w/v) benzalkonium chloride.
Embodiment 58. The pharmaceutical composition of any one of Embodiments 1-57, wherein the active ingredient comprises a polypeptide.
Embodiment 59. The pharmaceutical composition of Embodiment 58, wherein the active ingredient comprises an antibody or an antigen binding fragment thereof.
Embodiment 60. The pharmaceutical composition of any one of Embodiments 1-59, wherein the active ingredient has a concentration of about 0.1-100 mg/ml, about 0.5-50 mg/ml, or about 1-30 mg/ml.
Embodiment 61. The pharmaceutical composition of Embodiment 60, wherein the active ingredient has a concentration of about 5 mg/ml.
Embodiment 62. The pharmaceutical composition of any one of Embodiments 59-61, wherein the antibody or antigen fragment thereof binds to a coronavirus.
Embodiment 63. The pharmaceutical composition of Embodiment 62, wherein the antibody or antigen fragment thereof binds to SARS-CoV-2.
Embodiment 64. The pharmaceutical composition of any one of Embodiments 59-63, comprising one or more of:
(a) an antibody A or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 13, a HCDR2 comprising SEQ ID NO: 14, and a HCDR3  comprising SEQ ID NO: 15 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 10, a LCDR2 comprising SEQ ID NO: 11, and a LCDR3 comprising SEQ ID NO: 12;
(b) an antibody A-1 or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 4, a HCDR2 comprising SEQ ID NO: 5, and a HCDR3 comprising SEQ ID NO: 6 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 1, a LCDR2 comprising SEQ ID NO: 2, and a LCDR3 comprising SEQ ID NO: 3;
(c) an antibody B or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 23, a HCDR2 comprising SEQ ID NO: 24, and a HCDR3 comprising SEQ ID NO: 25 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, and a LCDR3 comprising SEQ ID NO: 22;
(d) an antibody C or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 33, a HCDR2 comprising SEQ ID NO: 34, and a HCDR3 comprising SEQ ID NO: 35 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 30, a LCDR2 comprising SEQ ID NO: 31, and a LCDR3 comprising SEQ ID NO: 32;
(e) an antibody D/E or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 43, a HCDR2 comprising SEQ ID NO: 44, and a HCDR3 comprising SEQ ID NO: 45 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 40, a LCDR2 comprising SEQ ID NO: 41, and a LCDR3 comprising SEQ ID NO: 42;and
(f) an antibody F or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 63, a HCDR2 comprising SEQ ID NO: 64, and a HCDR3 comprising SEQ ID NO: 65 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62.
Embodiment 65. The pharmaceutical composition of Embodiment 64, wherein:
(a) the antibody A or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 18 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 16 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
(b) the antibody A-1 or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 8 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 7 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
(c) the antibody B or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 28 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 26 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
(d) the antibody C or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 38 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 36 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
(e) the antibody D/E or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 48 or 52 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 46 or 50 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; and/or
(f) the antibody F or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 68 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 66 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
Embodiment 66. The pharmaceutical composition of Embodiment 64 or 65, wherein(e) the antibody D/E or antigen binding fragment comprises:
a heavy chain constant region (HCR) comprising or consisting of SEQ ID NO: 49 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a light chain constant region (LCR) comprising or  consisting of SEQ ID NO: 47 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or
a HCR comprising or consisting of SEQ ID NO: 53 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a LCR comprising or consisting of SEQ ID NO: 51 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or
a combination thereof.
Embodiment 67. The pharmaceutical composition of any one of Embodiments 64-66, comprising at least two of the antibodies or antigen binding fragments.
Embodiment 68. The pharmaceutical composition of Embodiment 67, comprising (c) the antibody B or antigen binding fragment thereof and (e) the antibody D/E or antigen binding fragment thereof.
Embodiment 69. The pharmaceutical composition of Embodiment 68, wherein the ratio of (c) the antibody B or antigen binding fragment thereof to (e) the antibody D/E or antigen binding fragment thereof is between about 1: 2 and about 1: 8.
Embodiment 70. The pharmaceutical composition of Embodiment 68, wherein the ratio of (c) the antibody B or antigen binding fragment thereof to (e) the antibody D/E or antigen binding fragment thereof is about 1: 4.
Embodiment 71. The pharmaceutical composition of any one of Embodiments 64-70, comprising (d) the antibody C or antigen binding fragment thereof.
Embodiment 72. The pharmaceutical composition of any one of Embodiments 64-71, comprising (f) the antibody F or antigen binding fragment thereof.
Embodiment 73. The pharmaceutical composition of any one of Embodiments 64-72, wherein the antibody is an IgG, an IgM, or an IgA.
Embodiment 74. The pharmaceutical composition of any one of Embodiments 1-73, wherein the pharmaceutical composition is administered via a nasal administration device.
Embodiment 75. A nasal or mucosal administration device comprising the pharmaceutical composition of any one of Embodiments 1-74.
Embodiment 76. A kit comprising the pharmaceutical composition of any one of Embodiments 1-74 and a nasal or mucosal administration device.
Embodiment 77. The nasal administration device of Embodiment 75 or the kit of Embodiment 76, wherein the nasal administration device is a metered dose nasal pump device or a unit dose nasal pump device.
Embodiment 78. An antibody or antigen binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) , a heavy chain complementarity determining region 2 (HCDR2) and a heavy chain complementarity determining region 3 (HCDR3) , wherein:
the HCDR1 comprises the amino acid sequence of GFTFSGSA (SEQ ID NO: 13) ;
the HCDR2 comprises the amino acid sequence of IVVGSGNT (SEQ ID NO: 14) ; and
the HCDR3 comprises the amino acid sequence of AAPYCSSTSCRDGFDI (SEQ ID NO: 15) .
Embodiment 79. The antibody or antigen binding fragment of Embodiment 78, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 18, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
Embodiment 80. The antibody or antigen binding fragment of Embodiment 78 or 79, comprising a light chain variable domain (VL) comprising a light chain complementarity determining region 1 (LCDR1) , a light chain complementarity determining region 2 (LCDR2) and a light chain complementarity determining region 3 (LCDR3) , wherein:
the LCDR1 comprises the amino acid sequence of QSVRSSY (SEQ ID NO: 10) ;
the LCDR2 comprises the amino acid sequence of GAS (SEQ ID NO: 11) ; and
the LCDR3 comprises the amino acid sequence of QQYGRSPWT (SEQ ID NO: 12) .
Embodiment 81. The antibody or antigen binding fragment of any one of Embodiments 78-80, wherein the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
Embodiment 82. An antibody or antigen binding fragment thereof, comprising:
(i) a VH comprising a HCDR1 comprising SEQ ID NO: 23, a HCDR2 comprising SEQ ID NO: 24, and a HCDR3 comprising SEQ ID NO: 25 and a VL comprising a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, and a LCDR3 comprising SEQ ID NO: 22;
(ii) a VH comprising a HCDR1 comprising SEQ ID NO: 33, a HCDR2 comprising SEQ ID NO: 34, and a HCDR3 comprising SEQ ID NO: 35 and a VL comprising a LCDR1 comprising SEQ ID NO: 30, a LCDR2 comprising SEQ ID NO: 31, and a LCDR3 comprising SEQ ID NO: 32;
(iii) a VH comprising a HCDR1 comprising SEQ ID NO: 43, a HCDR2 comprising SEQ ID NO: 44, and a HCDR3 comprising SEQ ID NO: 45 and a VL comprising a LCDR1 comprising SEQ ID NO: 40, a LCDR2 comprising SEQ ID NO: 41, and a LCDR3 comprising SEQ ID NO: 42; or
(iv) a VH comprising a HCDR1 comprising SEQ ID NO: 63, a HCDR2 comprising SEQ ID NO: 64, and a HCDR3 comprising SEQ ID NO: 65 and a VL comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62; .
Embodiment 83. The antibody or antigen binding fragment of any one of Embodiments 78-82, wherein the antibody or antigen binding fragment is for intranasal administration.
Embodiment 84. The antibody or antigen binding fragment of any one of Embodiments 78-83, wherein the antibody or antigen binding fragment is capable of binding to a SARS-CoV-2 spike protein, optionally wherein the SARS-CoV-2 spike proteins comprises the amino acid sequence set forth in SEQ ID NO: 106, SEQ ID NO: 108, or SEQ ID NO: 110.
Embodiment 85. The antibody or antigen binding fragment of any one of Embodiments 78-84, wherein the antibody is an IgG, an IgM, or an IgA.
Embodiment 86. A polynucleotide encoding the antibody or antigen binding fragment of any one of Embodiments 78-85.
Embodiment 87. A vector comprising the polynucleotide of Embodiment 86.
Embodiment 88. A composition comprising the antibody or antigen binding fragment of any one of Embodiments 78-85, the polynucleotide of Embodiment 86, or the vector of Embodiment 87.
Embodiment 89. A cell comprising the polynucleotide of Embodiment 86 or the vector of Embodiment 87.
Embodiment 90. A method of producing the antibody or antigen binding fragment of any one of Embodiments 78-85, comprising culturing a cell comprising a vector encoding the antibody or antigen binding fragment thereof and harvesting the antibody or antigen binding fragment from the culture medium of the cell.
Embodiment 91. A method of treating or preventing a SARS-CoV-2 infection in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen binding fragment of any one of Embodiments 78-85.
Embodiment 92. The method of Embodiment 91, wherein the antibody or antigen binding fragment is administered intranasally, subcutaneously, intravenously, or intramuscularly.
Embodiment 93. The method of Embodiment 92, wherein the antibody or antigen binding fragment is administered intranasally.
Embodiment 94. A combination of antibodies or antigen binding fragments thereof, comprising at least two of the following antibodies or antigen binding fragments thereof:
(a) an antibody A or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 13, a HCDR2 comprising SEQ ID NO: 14, and a HCDR3 comprising SEQ ID NO: 15 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 10, a LCDR2 comprising SEQ ID NO: 11, and a LCDR3 comprising SEQ ID NO: 12;
(b) an antibody A-1 or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 4, a HCDR2 comprising SEQ ID NO: 5, and a HCDR3 comprising SEQ ID NO: 6 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 1, a LCDR2 comprising SEQ ID NO: 2, and a LCDR3 comprising SEQ ID NO: 3;
(c) an antibody B or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 23, a HCDR2 comprising SEQ ID NO: 24, and a HCDR3  comprising SEQ ID NO: 25 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, and a LCDR3 comprising SEQ ID NO: 22;
(d) an antibody C or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 33, a HCDR2 comprising SEQ ID NO: 34, and a HCDR3 comprising SEQ ID NO: 35 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 30, a LCDR2 comprising SEQ ID NO: 31, and a LCDR3 comprising SEQ ID NO: 32;
(e) an antibody D/E or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 43, a HCDR2 comprising SEQ ID NO: 44, and a HCDR3 comprising SEQ ID NO: 45 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 40, a LCDR2 comprising SEQ ID NO: 41, and a LCDR3 comprising SEQ ID NO: 42;and
(f) an antibody F or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 63, a HCDR2 comprising SEQ ID NO: 64, and a HCDR3 comprising SEQ ID NO: 65 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62.
Embodiment 95. The combination of antibodies or antigen binding fragments of Embodiment 94, wherein:
(a) the antibody A or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 18 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 16 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
(b) the antibody A-1 or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 8 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 7 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
(c) the antibody B or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 28 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising  or consisting of SEQ ID NO: 26 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
(d) the antibody C or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 38 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 36 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
(e) the antibody D/E or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 48 or 52 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 46 or 50 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; and/or
(f) the antibody F or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 68 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 66 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
Embodiment 96. The combination of antibodies or antigen binding fragments of Embodiment 94 or 95, wherein (e) the antibody D/E or antigen binding fragment comprises:
a heavy chain constant region (HCR) comprising or consisting of SEQ ID NO: 49 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a light chain constant region (LCR) comprising or consisting of SEQ ID NO: 47 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or
a HCR comprising or consisting of SEQ ID NO: 53 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a LCR comprising or consisting of SEQ ID NO: 51 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or
a combination thereof.
Embodiment 97. The combination of antibodies or antigen binding fragments of any one of Embodiments 94-96, comprising (c) the antibody B or antigen binding fragment thereof and (e) the antibody D/E or antigen binding fragment thereof.
Embodiment 98. The combination of antibodies or antigen binding fragments of Embodiment 97, wherein the ratio of (c) the antibody B or antigen binding fragment thereof to (e) the antibody D/E or antigen binding fragment thereof is between about 1: 2 and about 1: 8.
Embodiment 99. The combination of antibodies or antigen binding fragments of Embodiment 98, wherein the ratio of (c) the antibody B or antigen binding fragment thereof to (e) the antibody D/E or antigen binding fragment thereof is about 1: 4.
Embodiment 100. The combination of antibodies or antigen binding fragments of any one of Embodiments 94-99, comprising (d) the antibody C or antigen binding fragment thereof.
Embodiment 101. The combination of antibodies or antigen binding fragments of any one of Embodiments 94-100, comprising (f) the antibody F or antigen binding fragment thereof.
Embodiment 102. The combination of antibodies or antigen binding fragments of Embodiment 94-101, wherein the combination displays a synergistic effect against COVID-19 Omicron BA. 2, BA. 2.12.1, or BA. 5 strain.
Embodiment 103. A method of delivering an active ingredient or a precursor thereof to a nasal cavity or a mucosa of a subject, comprising administering to the subject the pharmaceutical composition of any one of Embodiments 1-74.
Embodiment 104. A method of preventing a disease in a subject in need thereof, comprising administering the pharmaceutical composition of any one of Embodiments 1-74, the antibody or antigen binding fragment of any one of Embodiments 78-85, or the combination of any one of Embodiments 94-102, to the subject.
Embodiment 105. A method of treating a disease in a subject in need thereof, comprising administering the pharmaceutical composition of any one of Embodiments 1-74, the antibody or antigen binding fragment of any one of Embodiments 78-85, or the combination of any one of Embodiments 94-102, to the subject.
Embodiment 106. The method of Embodiment 104 or 105, wherein the pharmaceutical composition, or the antibody or antigen binding fragment or the combination, is administered to a mucosa of the subject.
Embodiment 107. The method of any one of Embodiments 104-106, where the pharmaceutical composition, or the antibody or antigen binding fragment or the combination, is administered intranasally to the subject.
Embodiment 108. The method of any one of Embodiments 103-107, wherein about 30-150 μl per administration of the pharmaceutical composition is delivered to a nasal cavity of the subject.
Embodiment 109. The method of Embodiment 106, wherein about 70-100 μl per administration of the pharmaceutical composition is delivered to the nasal cavity of the subject.
Embodiment 110. The method of any one of Embodiments 103-109, wherein about 0.15 mg to about 0.75 mg of the active ingredient or a precursor thereof is delivered to the nasal cavity per administration.
Embodiment 111. The method of any one of Embodiments 103-110, wherein the pharmaceutical composition, the antibody or antigen binding fragment, or the combination, is delivered to the subject about 1-6 times per day.
Embodiment 112. The method of Embodiment 111, wherein the pharmaceutical composition, the antibody or antigen binding fragment, or the combination, is delivered to the subject about 2-3 times per day.
Embodiment 113. The method of any one of Embodiments 103-112, wherein the pharmaceutical composition, the antibody or antigen binding fragment thereof, or the combination, is delivered to the subject for about 7-14 days.
Embodiment 114. The method of any one of Embodiments 104-113, wherein administering the pharmaceutical composition, the antibody or antigen binding fragment thereof, or the combination, starts prior to the onset of any symptom of the disease.
Embodiment 115. The method of any one of Embodiments 104-113, wherein administering the pharmaceutical composition, the antibody or antigen binding fragment thereof, or the combination, starts after the onset of at least one symptom of the disease.
Embodiment 116. The method of any one of Embodiments 114-115, wherein the symptom is a respiratory symptom.
Embodiment 117. The method of any one of Embodiments 104-116, wherein the disease comprises a respiratory symptom.
Embodiment 118. The method of Embodiment 116 or 117, wherein the respiratory symptom is allergy, nasal congestion, nasal infection, or a combination thereof.
Embodiment 119. The method of any one of Embodiments 104-118, wherein the disease is a respiratory infection caused by a virus, a bacterial, a fungus, or a combination thereof.
Embodiment 120. The method of any one of Embodiments 104-119, wherein the disease is a viral infection.
Embodiment 121. The method of Embodiment 120, wherein the disease is a chronic viral infection.
Embodiment 122. The method of Embodiment 120, wherein the disease is an acute viral infection.
Embodiment 123. The method of Embodiment 120, wherein the disease is a coronavirus infection.
Embodiment 124. The method of Embodiment 123, wherein the disease is SARS-CoV-2 infection.
Embodiment 125. The method of any one of Embodiments 104-124, wherein the concentration of the antibody or antigen binding fragment thereof in a nasal cavity of the subject is at least 100-fold higher than the concentration of the antibody or antigen binding fragment thereof in the blood sample of the subject within 24 hours after the administration as measured by ELISA.
All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GeneID entries) , patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants to relate to  each and every individual publication, database entry (e.g., Genbank sequences or GeneID entries) , patent application, or patent identified even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

Claims (125)

  1. A pharmaceutical composition, comprising:
    an active ingredient or a precursor thereof;
    no more than about 40% (w/v) of a cryoprotectant;
    no more than about 30% (w/v) of a humectant;
    no more than about 5% (w/v) of an emulsifier;
    no more than about 10% (w/v) of a controlled release agent; and
    a buffer.
  2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is suitable for mucosal administration to a human.
  3. The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutical composition is suitable for intranasal administration to a human.
  4. The pharmaceutical composition of any one of claims 1-3, wherein the cryoprotectant is selected from the group consisting of trehalose, glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol, and sucrose, or a combination thereof.
  5. The pharmaceutical composition of claim 4, wherein the cryoprotectant in the pharmaceutical composition is:
    no more than 40% (w/v) trehalose;
    no more than 40% (w/v) glycerol;
    no more than 10% (w/v) dimethyl sulfoxide;
    no more than 40% (w/v) ethylene glycol;
    no more than 20% (w/v) polyethylene glycol; or
    no more than 0.5 M sucrose.
  6. The pharmaceutical composition of claim 4, wherein the cryoprotectant in the pharmaceutical composition is:
    about 2%-about 8% (w/v) trehalose;
    about 1%-about 20% (w/v) glycerol;
    about 1%-about 10% (w/v) dimethyl sulfoxide;
    about 4%-about 30% (w/v) ethylene glycol;
    about 5%-about 15% (w/v) polyethylene glycol; or
    about 0.1 M -about 0.4 M sucrose.
  7. The pharmaceutical composition of any one of claims 1-6, wherein the cryoprotectant is trehalose.
  8. The pharmaceutical composition of any one of claims 1-7, comprising about 0.3% (w/v) to about 12% (w/v) of the cryoprotectant.
  9. The pharmaceutical composition of any one of claims 1-7, comprising about 2% (w/v) to about 8% (w/v) of the cryoprotectant.
  10. The pharmaceutical composition of any one of claims 1-7, comprising about 4% (w/v) of the cryoprotectant.
  11. The pharmaceutical composition of any one of claims 1-10, wherein the humectant is selected from the group consisting of glycerin, mannitol, polyethylene glycol 400, polyethylene glycol 4000, D-sorbitol, chitosan, xylitol, and sodium hyaluronate, or a combination thereof.
  12. The pharmaceutical composition of claim 11, wherein the humectant in the pharmaceutical composition is:
    no more than 30% (w/v) glycerin;
    no more than 7% (w/v) mannitol;
    no more than 20% (w/v) polyethylene glycol 400;
    about 5%-about 15% (w/v) polyethylene glycol 4000;
    about 3%-about 15% (w/v) D-sorbitol;
    about 0.01%-about 3% (w/v) chitosan;
    no more than 10% (w/v) xylitol; or
    about 0.1%-about 2.0% (w/v) sodium hyaluronate.
  13. The pharmaceutical composition of claim 11, wherein the humectant in the pharmaceutical composition is:
    about 2%-about 6% (w/v) glycerin;
    about 3%-about 5% (w/v) mannitol;
    about 10%-about 18% (w/v) polyethylene glycol 400;
    about 5%-about 10% (w/v) polyethylene glycol 4000;
    about 3%-about 6% (w/v) D-sorbitol;
    about 0.01%-about 1% (w/v) chitosan;
    about 1%-about 10% (w/v) xylitol; or
    about 0.1%-about 1.0% (w/v) sodium hyaluronate.
  14. The pharmaceutical composition of any one of claims 1-13, wherein the humectant is glycerin.
  15. The pharmaceutical composition of any one of claims 1-14, comprising about 0.2% (w/v) to about 10% (w/v) of the humectant.
  16. The pharmaceutical composition of any one of claims 1-14, comprising about 1% (w/v) to about 4% (w/v) of the humectant.
  17. The pharmaceutical composition of claim 16, comprising about 1.7% (w/v) to about 2% (w/v) of the humectant.
  18. The pharmaceutical composition of any one of claims 1-17, wherein the emulsifier is selected from the group consisting of TWEEN 80, polysorbate 20, lecithin, sorbitan esters, mono-and/or diglycerides, and sodium stearoyl lactylate, or a combination thereof.
  19. The pharmaceutical composition of any one of claims 1-18, wherein the emulsifier is TWEEN 80.
  20. The pharmaceutical composition of any one of claims 1-19, comprising about 0.002% (w/v) to about 0.1% (w/v) of the emulsifier.
  21. The pharmaceutical composition of any one of claims 1-19, comprising about 0.005% (w/v) to about 2% (w/v) of the emulsifier.
  22. The pharmaceutical composition of claim 21, comprising about 0.005% (w/v) to about 0.04% (w/v) of the emulsifier.
  23. The pharmaceutical composition of claim 21, comprising about 0.01% (w/v) to about 0.02% (w/v) of the emulsifier.
  24. The pharmaceutical composition of any one of claims 1-23, wherein the controlled release agent is selected from the group consisting of hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone K30, or a combination thereof.
  25. The pharmaceutical composition of any one of claims 1-24, wherein the controlled release agent is hydroxypropyl methylcellulose (HPMC) .
  26. The pharmaceutical composition of any one of claims 1-25, comprising about 0.01% (w/v) to about 5% (w/v) , or about 0.01% (w/v) to about 1% (w/v) , of the controlled release agent.
  27. The pharmaceutical composition of any one of claims 1-25, comprising about 0.004% (w/v) to about 0.4% (w/v) of the controlled release agent.
  28. The pharmaceutical composition of any one of claims 1-25, comprising about 0.01% (w/v) to about 0.2% (w/v) of the controlled release agent.
  29. The pharmaceutical composition of claim 28, comprising about 0.02% (w/v) , or about 0.1% (w/v) , of the controlled release agent.
  30. The pharmaceutical composition of any one of claims 1-24, wherein the controlled release agent is polyvinylpyrrolidone K30.
  31. The pharmaceutical composition of any one of claims 1-30, comprising about 2% (w/v) to about 10% (w/v) , or about 3% (w/v) to about 6% (w/v) , of the controlled release agent.
  32. The pharmaceutical composition of any one of claims 1-31, wherein the buffer is selected from the group consisting of a phosphate buffer, a tris buffer, and a glycine buffer.
  33. The pharmaceutical composition of claim 32, wherein the buffer in the pharmaceutical composition is:
    about 10 mM -about 100 mM phosphate;
    about 10 mM -about 100 mM tris; or
    about 0.1 M -about 0.5 M glycine.
  34. The pharmaceutical composition of claim 32, wherein the buffer in the pharmaceutical composition is:
    about 10 mM -about 40 mM phosphate;
    about 10 mM -about 40 mM tris; or
    about 0.1 M -about 0.2 M glycine.
  35. The pharmaceutical composition of any one of claims 1-34, wherein the buffer is a phosphate buffer.
  36. The pharmaceutical composition of any one of claims 1-35, wherein the buffer in the pharmaceutical composition is about 20 mM phosphate or tris.
  37. The pharmaceutical composition of any one of claims 1-36, wherein the buffer has a pH of about 4-8, or about 5-7.
  38. The pharmaceutical composition of any one of claims 1-36, wherein the buffer has a pH of about 5.5-6.5.
  39. The pharmaceutical composition of claim 38, wherein the buffer has a pH of about 6.0.
  40. The pharmaceutical composition of any one of claims 1-39, comprising an antibiotic.
  41. The pharmaceutical composition of claim 40, wherein the antibiotic comprises benzalkonium chloride, benzyl alcohol, chlorobutanol, or a combination thereof.
  42. The pharmaceutical composition of claim 41, wherein the antibiotic in the pharmaceutical composition is:
    about 0.002%-about 0.02% (w/v) benzalkonium chloride;
    no more than 3.0% (v/v) benzyl alcohol; or
    about 0.5%-about 2.0% (w/v) chlorobutanol.
  43. The pharmaceutical composition of claim 41, wherein the antibiotic in the pharmaceutical composition is:
    about 0.005%-about 0.02% (w/v) benzalkonium chloride;
    about 1.0%-about 3.0% (v/v) benzyl alcohol; or
    about 1.0%-2.0% (w/v) chlorobutanol.
  44. The pharmaceutical composition of any one of claims 40-43, wherein the antibiotic is benzalkonium chloride.
  45. The pharmaceutical composition of any one of claims 40-44, comprising about 0.002% (w/v) to about 0.1% (w/v) of the antibiotic.
  46. The pharmaceutical composition of claim 45, comprising about 0.005% (w/v) to about 0.02% (w/v) , or about 0.01% (w/v) , of the antibiotic.
  47. The pharmaceutical composition of any one of claims 1-46, wherein the mean retention time of the active ingredient or a precursor thereof in a nasal cavity of a primate is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, or at least 12 hours, after intranasal administration of the pharmaceutical composition to the primate.
  48. The pharmaceutical composition of claim 47, wherein the primate is a rhesus macaque.
  49. The pharmaceutical composition of any one of claims 1-48, comprising:
    about 0.3% (w/v) to about 12% (w/v) of a cryoprotectant;
    about 0.2% (w/v) to about 10% (w/v) of a humectant;
    about 0.002% (w/v) to about 0.1% (w/v) of an emulsifier; and
    about 0.004% (w/v) to about 0.4% (w/v) of a controlled release agent.
  50. The pharmaceutical composition of any one of claims 1-48, comprising:
    about 2%-about 8% (w/v) of the cryoprotectant;
    about 1%–about 4% (w/v) of the humectant;
    about 0.005%-about 0.04% (w/v) of the emulsifier;
    about 0.01%-about 0.2% (w/v) of the controlled release agent; and
    the buffer has a pH of about 5.5-6.5.
  51. The pharmaceutical composition of any one of claims 1-48, comprising:
    about 4% (w/v) of the cryoprotectant;
    about 1.7%–about 2% (w/v) of the humectant;
    about 0.01%-about 0.02% (w/v) of the emulsifier;
    about 0.02%-about 0.1% (w/v) of the controlled release agent; and
    the buffer has a pH of about 6.0.
  52. The pharmaceutical composition of any one of claims 1-48, comprising:
    about 4% (w/v) of the cryoprotectant;
    about 2% (w/v) of the humectant;
    about 0.01% (w/v) of the emulsifier;
    about 0.02% (w/v) of the controlled release agent; and
    the buffer has a pH of about 6.0.
  53. The pharmaceutical composition of any one of claims 1-48, comprising:
    about 4% (w/v) of the cryoprotectant;
    about 2% (w/v) of the humectant;
    about 0.02% (w/v) of the emulsifier;
    about 0.1% (w/v) of the controlled release agent; and
    the buffer has a pH of about 6.0.
  54. The pharmaceutical composition of any one of claims 1-48, comprising:
    about 4% (w/v) of the cryoprotectant;
    about 1.7% (w/v) of the humectant;
    about 0.02% (w/v) of the emulsifier;
    about 0.1% (w/v) of the controlled release agent; and
    the buffer has a pH of about 6.0.
  55. The pharmaceutical composition of any one of claims 49-54, wherein the buffer comprises about 20 mM phosphate salt, optionally wherein the phosphate salt is a sodium phosphate salt.
  56. The pharmaceutical composition of any one of claims 49-55, wherein the cryoprotectant is trehalose, the humectant is glycerin, the emulsifier is TWEEN 80, the controlled release agent is HPMC.
  57. The pharmaceutical composition of any one of claims 49-56, comprising about 0.01% (w/v) benzalkonium chloride.
  58. The pharmaceutical composition of any one of claims 1-57, wherein the active ingredient comprises a polypeptide.
  59. The pharmaceutical composition of claim 58, wherein the active ingredient comprises an antibody or an antigen binding fragment thereof.
  60. The pharmaceutical composition of any one of claims 1-59, wherein the active ingredient has a concentration of about 0.1-100 mg/ml, about 0.5-50 mg/ml, or about 1-30 mg/ml.
  61. The pharmaceutical composition of claim 60, wherein the active ingredient has a concentration of about 5 mg/ml.
  62. The pharmaceutical composition of any one of claims 59-61, wherein the antibody or antigen fragment thereof binds to a coronavirus.
  63. The pharmaceutical composition of claim 62, wherein the antibody or antigen fragment thereof binds to SARS-CoV-2.
  64. The pharmaceutical composition of any one of claims 59-63, comprising one or more of:
    (a) an antibody A or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 13, a HCDR2 comprising SEQ ID NO: 14, and a HCDR3 comprising SEQ ID NO: 15 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 10, a LCDR2 comprising SEQ ID NO: 11, and a LCDR3 comprising SEQ ID NO: 12;
    (b) an antibody A-1 or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 4, a HCDR2 comprising SEQ ID NO: 5, and a HCDR3 comprising SEQ ID NO: 6 and (ii) a VL comprising a LCDR1 comprising  SEQ ID NO: 1, a LCDR2 comprising SEQ ID NO: 2, and a LCDR3 comprising SEQ ID NO: 3;
    (c) an antibody B or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 23, a HCDR2 comprising SEQ ID NO: 24, and a HCDR3 comprising SEQ ID NO: 25 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, and a LCDR3 comprising SEQ ID NO: 22;
    (d) an antibody C or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 33, a HCDR2 comprising SEQ ID NO: 34, and a HCDR3 comprising SEQ ID NO: 35 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 30, a LCDR2 comprising SEQ ID NO: 31, and a LCDR3 comprising SEQ ID NO: 32;
    (e) an antibody D/E or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 43, a HCDR2 comprising SEQ ID NO: 44, and a HCDR3 comprising SEQ ID NO: 45 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 40, a LCDR2 comprising SEQ ID NO: 41, and a LCDR3 comprising SEQ ID NO: 42; and
    (f) an antibody F or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 63, a HCDR2 comprising SEQ ID NO: 64, and a HCDR3 comprising SEQ ID NO: 65 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62.
  65. The pharmaceutical composition of claim 64, wherein:
    (a) the antibody A or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 18 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 16 or a sequence having at least 70%, at  least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
    (b) the antibody A-1 or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 8 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 7 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
    (c) the antibody B or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 28 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 26 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
    (d) the antibody C or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 38 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 36 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
    (e) the antibody D/E or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 48 or 52 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 46 or 50 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; and/or
    (f) the antibody F or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 68 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a  VL comprising or consisting of SEQ ID NO: 66 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
  66. The pharmaceutical composition of claim 64 or 65, wherein (e) the antibody D/E or antigen binding fragment comprises:
    a heavy chain constant region (HCR) comprising or consisting of SEQ ID NO: 49 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a light chain constant region (LCR) comprising or consisting of SEQ ID NO: 47 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or
    a HCR comprising or consisting of SEQ ID NO: 53 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a LCR comprising or consisting of SEQ ID NO: 51 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or
    a combination thereof.
  67. The pharmaceutical composition of any one of claims 64-66, comprising at least two of the antibodies or antigen binding fragments.
  68. The pharmaceutical composition of claim 67, comprising (c) the antibody B or antigen binding fragment thereof and (e) the antibody D/E or antigen binding fragment thereof.
  69. The pharmaceutical composition of claim 68, wherein the ratio of (c) the antibody B or antigen binding fragment thereof to (e) the antibody D/E or antigen binding fragment thereof is between about 1: 2 and about 1: 8.
  70. The pharmaceutical composition of claim 68, wherein the ratio of (c) the antibody B or antigen binding fragment thereof to (e) the antibody D/E or antigen binding fragment thereof is about 1: 4.
  71. The pharmaceutical composition of any one of claims 64-70, comprising (d) the antibody C or antigen binding fragment thereof.
  72. The pharmaceutical composition of any one of claims 64-71, comprising (f) the antibody F or antigen binding fragment thereof.
  73. The pharmaceutical composition of any one of claims 64-72, wherein the antibody is an IgG, an IgM, or an IgA.
  74. The pharmaceutical composition of any one of claims 1-73, wherein the pharmaceutical composition is administered via a nasal administration device.
  75. A nasal or mucosal administration device comprising the pharmaceutical composition of any one of claims 1-74.
  76. A kit comprising the pharmaceutical composition of any one of claims 1-74 and a nasal or mucosal administration device.
  77. The nasal administration device of claim 75 or the kit of claim 76, wherein the nasal administration device is a metered dose nasal pump device or a unit dose nasal pump device.
  78. An antibody or antigen binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) , a heavy chain complementarity determining region 2 (HCDR2) and a heavy chain complementarity determining region 3 (HCDR3) , wherein:
    the HCDR1 comprises the amino acid sequence of GFTFSGSA (SEQ ID NO: 13) ;
    the HCDR2 comprises the amino acid sequence of IVVGSGNT (SEQ ID NO: 14) ; and
    the HCDR3 comprises the amino acid sequence of AAPYCSSTSCRDGFDI (SEQ ID NO: 15) .
  79. The antibody or antigen binding fragment of claim 78, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 18, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
  80. The antibody or antigen binding fragment of claim 78 or 79, comprising a light chain variable domain (VL) comprising a light chain complementarity determining region 1 (LCDR1) , a light chain complementarity determining region 2 (LCDR2) and a light chain complementarity determining region 3 (LCDR3) , wherein:
    the LCDR1 comprises the amino acid sequence of QSVRSSY (SEQ ID NO: 10) ;
    the LCDR2 comprises the amino acid sequence of GAS (SEQ ID NO: 11) ; and
    the LCDR3 comprises the amino acid sequence of QQYGRSPWT (SEQ ID NO: 12) .
  81. The antibody or antigen binding fragment of any one of claims 78-80, wherein the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
  82. An antibody or antigen binding fragment thereof, comprising:
    (i) a VH comprising a HCDR1 comprising SEQ ID NO: 23, a HCDR2 comprising SEQ ID NO: 24, and a HCDR3 comprising SEQ ID NO: 25 and a VL comprising a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, and a LCDR3 comprising SEQ ID NO: 22;
    (ii) a VH comprising a HCDR1 comprising SEQ ID NO: 33, a HCDR2 comprising SEQ ID NO: 34, and a HCDR3 comprising SEQ ID NO: 35 and a VL comprising a LCDR1 comprising SEQ ID NO: 30, a LCDR2 comprising SEQ ID NO: 31, and a LCDR3 comprising SEQ ID NO: 32;
    (iii) a VH comprising a HCDR1 comprising SEQ ID NO: 43, a HCDR2 comprising SEQ ID NO: 44, and a HCDR3 comprising SEQ ID NO: 45 and a VL comprising a LCDR1 comprising SEQ ID NO: 40, a LCDR2 comprising SEQ ID NO: 41, and a LCDR3 comprising SEQ ID NO: 42; or
    (iv) a VH comprising a HCDR1 comprising SEQ ID NO: 63, a HCDR2 comprising SEQ ID NO: 64, and a HCDR3 comprising SEQ ID NO: 65 and a VL comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62;.
  83. The antibody or antigen binding fragment of any one of claims 78-82, wherein the antibody or antigen binding fragment is for intranasal administration.
  84. The antibody or antigen binding fragment of any one of claims 78-83, wherein the antibody or antigen binding fragment is capable of binding to a SARS-CoV-2 spike protein, optionally wherein the SARS-CoV-2 spike proteins comprises the amino acid sequence set forth in SEQ ID NO: 106, SEQ ID NO: 108, or SEQ ID NO: 110.
  85. The antibody or antigen binding fragment of any one of claims 78-84, wherein the antibody is an IgG, an IgM, or an IgA.
  86. A polynucleotide encoding the antibody or antigen binding fragment of any one of claims 78-85.
  87. A vector comprising the polynucleotide of claim 86.
  88. A composition comprising the antibody or antigen binding fragment of any one of claims 78-85, the polynucleotide of claim 86, or the vector of claim 87.
  89. A cell comprising the polynucleotide of claim 86 or the vector of claim 87.
  90. A method of producing the antibody or antigen binding fragment of any one of claims 78-85, comprising culturing a cell comprising a vector encoding the antibody or antigen binding fragment thereof and harvesting the antibody or antigen binding fragment from the culture medium of the cell.
  91. A method of treating or preventing a SARS-CoV-2 infection in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen binding fragment of any one of claims 78-85.
  92. The method of claim 91, wherein the antibody or antigen binding fragment is administered intranasally, subcutaneously, intravenously, or intramuscularly.
  93. The method of claim 92, wherein the antibody or antigen binding fragment is administered intranasally.
  94. A combination of antibodies or antigen binding fragments thereof, comprising at least two of the following antibodies or antigen binding fragments thereof:
    (a) an antibody A or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 13, a HCDR2 comprising SEQ ID NO: 14, and a HCDR3 comprising SEQ ID NO: 15 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 10, a LCDR2 comprising SEQ ID NO: 11, and a LCDR3 comprising SEQ ID NO: 12;
    (b) an antibody A-1 or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 4, a HCDR2 comprising SEQ ID NO: 5, and a HCDR3 comprising SEQ ID NO: 6 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 1, a LCDR2 comprising SEQ ID NO: 2, and a LCDR3 comprising SEQ ID NO: 3;
    (c) an antibody B or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 23, a HCDR2 comprising SEQ ID NO: 24, and a HCDR3 comprising SEQ ID NO: 25 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, and a LCDR3 comprising SEQ ID NO: 22;
    (d) an antibody C or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 33, a HCDR2 comprising SEQ ID NO: 34, and a HCDR3 comprising SEQ ID NO: 35 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 30, a LCDR2 comprising SEQ ID NO: 31, and a LCDR3 comprising SEQ ID NO: 32;
    (e) an antibody D/E or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 43, a HCDR2 comprising SEQ ID NO: 44, and a HCDR3 comprising SEQ ID NO: 45 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 40, a LCDR2 comprising SEQ ID NO: 41, and a LCDR3 comprising SEQ ID NO: 42; and
    (f) an antibody F or antigen binding fragment thereof comprising (i) a VH comprising a HCDR1 comprising SEQ ID NO: 63, a HCDR2 comprising SEQ ID NO: 64, and a HCDR3 comprising SEQ ID NO: 65 and (ii) a VL comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62.
  95. The combination of antibodies or antigen binding fragments of claim 94, wherein:
    (a) the antibody A or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 18 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 16 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
    (b) the antibody A-1 or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 8 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 7 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
    (c) the antibody B or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 28 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 26 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
    (d) the antibody C or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 38 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 36 or a sequence having at least 70%, at  least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto;
    (e) the antibody D/E or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 48 or 52 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 46 or 50 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; and/or
    (f) the antibody F or antigen binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO: 68 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a VL comprising or consisting of SEQ ID NO: 66 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto.
  96. The combination of antibodies or antigen binding fragments of claim 94 or 95, wherein (e) the antibody D/E or antigen binding fragment comprises:
    a heavy chain constant region (HCR) comprising or consisting of SEQ ID NO: 49 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a light chain constant region (LCR) comprising or consisting of SEQ ID NO: 47 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or
    a HCR comprising or consisting of SEQ ID NO: 53 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto and a LCR comprising or consisting of SEQ ID NO: 51 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity thereto; or
    a combination thereof.
  97. The combination of antibodies or antigen binding fragments of any one of claims 94-96, comprising (c) the antibody B or antigen binding fragment thereof and (e) the antibody D/E or antigen binding fragment thereof.
  98. The combination of antibodies or antigen binding fragments of claim 97, wherein the ratio of (c) the antibody B or antigen binding fragment thereof to (e) the antibody D/E or antigen binding fragment thereof is between about 1: 2 and about 1: 8.
  99. The combination of antibodies or antigen binding fragments of claim 98, wherein the ratio of (c) the antibody B or antigen binding fragment thereof to (e) the antibody D/E or antigen binding fragment thereof is about 1: 4.
  100. The combination of antibodies or antigen binding fragments of any one of claims 94-99, comprising (d) the antibody C or antigen binding fragment thereof.
  101. The combination of antibodies or antigen binding fragments of any one of claims 94-100, comprising (f) the antibody F or antigen binding fragment thereof.
  102. The combination of antibodies or antigen binding fragments of claim 94-101, wherein the combination displays a synergistic effect against COVID-19 Omicron BA. 2, BA. 2.12.1, or BA. 5 strain.
  103. A method of delivering an active ingredient or a precursor thereof to a nasal cavity or a mucosa of a subject, comprising administering to the subject the pharmaceutical composition of any one of claims 1-74.
  104. A method of preventing a disease in a subject in need thereof, comprising administering the pharmaceutical composition of any one of claims 1-74, the antibody or antigen binding fragment of any one of claims 78-85, or the combination of any one of claims 94-102, to the subject.
  105. A method of treating a disease in a subject in need thereof, comprising administering the pharmaceutical composition of any one of claims 1-74, the antibody or antigen binding fragment of any one of claims 78-85, or the combination of any one of claims 94-102, to the subject.
  106. The method of claim 104 or 105, wherein the pharmaceutical composition, or the antibody or antigen binding fragment or the combination, is administered to a mucosa of the subject.
  107. The method of any one of claims 104-106, where the pharmaceutical composition, or the antibody or antigen binding fragment or the combination, is administered intranasally to the subject.
  108. The method of any one of claims 103-107, wherein about 30-150 μl per administration of the pharmaceutical composition is delivered to a nasal cavity of the subject.
  109. The method of claim 106, wherein about 70-100 μl per administration of the pharmaceutical composition is delivered to the nasal cavity of the subject.
  110. The method of any one of claims 103-109, wherein about 0.15 mg to about 0.75 mg of the active ingredient or a precursor thereof is delivered to the nasal cavity per administration.
  111. The method of any one of claims 103-110, wherein the pharmaceutical composition, the antibody or antigen binding fragment, or the combination, is delivered to the subject about 1-6 times per day.
  112. The method of claim 111, wherein the pharmaceutical composition, the antibody or antigen binding fragment, or the combination, is delivered to the subject about 2-3 times per day.
  113. The method of any one of claims 103-112, wherein the pharmaceutical composition, the antibody or antigen binding fragment thereof, or the combination, is delivered to the subject for about 7-14 days.
  114. The method of any one of claims 104-113, wherein administering the pharmaceutical composition, the antibody or antigen binding fragment thereof, or the combination, starts prior to the onset of any symptom of the disease.
  115. The method of any one of claims 104-113, wherein administering the pharmaceutical composition, the antibody or antigen binding fragment thereof, or the combination, starts after the onset of at least one symptom of the disease.
  116. The method of any one of claims 114-115, wherein the symptom is a respiratory symptom.
  117. The method of any one of claims 104-116, wherein the disease comprises a respiratory symptom.
  118. The method of claim 116 or 117, wherein the respiratory symptom is allergy, nasal congestion, nasal infection, or a combination thereof.
  119. The method of any one of claims 104-118, wherein the disease is a respiratory infection caused by a virus, a bacterial, a fungus, or a combination thereof.
  120. The method of any one of claims 104-119, wherein the disease is a viral infection.
  121. The method of claim 120, wherein the disease is a chronic viral infection.
  122. The method of claim 120, wherein the disease is an acute viral infection.
  123. The method of claim 120, wherein the disease is a coronavirus infection.
  124. The method of claim 123, wherein the disease is SARS-CoV-2 infection.
  125. The method of any one of claims 104-124, wherein the concentration of the antibody or antigen binding fragment thereof in a nasal cavity of the subject is at least 100-fold higher than the concentration of the antibody or antigen binding fragment thereof in the blood sample of the subject within 24 hours after the administration as measured by ELISA.
PCT/CN2023/077013 2022-02-18 2023-02-18 Intranasal formulations and anti-sars-cov-2-spike protein antibodies Ceased WO2023155902A1 (en)

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