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WO2021195089A1 - Protéines de fusion de l'antigène fc-coronavirus et acides nucléiques, vecteurs, compositions et procédés d'utilisation associés - Google Patents

Protéines de fusion de l'antigène fc-coronavirus et acides nucléiques, vecteurs, compositions et procédés d'utilisation associés Download PDF

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WO2021195089A1
WO2021195089A1 PCT/US2021/023695 US2021023695W WO2021195089A1 WO 2021195089 A1 WO2021195089 A1 WO 2021195089A1 US 2021023695 W US2021023695 W US 2021023695W WO 2021195089 A1 WO2021195089 A1 WO 2021195089A1
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Prior art keywords
coronavirus
protein
antigen fusion
fusion protein
antigen
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Inventor
Andreas Herrmann
Henry Hongjun Ji
Yanliang Zhang
Hui Xie
Wenzhong Guo
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Sorrento Therapeutics Inc
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Sorrento Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • TECHNICAL FIELD [0002] The present disclosure provides therapeutic agents comprising recombinant fusion proteins including an antibody Fc region coupled to a coronavirus antigen protein (herein referred to as a “Fc-coronavirus antigen fusion protein”), and nucleic acids (DNA or mRNA) encoding the Fc-coronavirus antigen fusion proteins, and expression vectors, compositions, and methods of use thereof for treating a coronavirus infection.
  • Fc-coronavirus antigen fusion protein an antibody Fc region coupled to a coronavirus antigen protein
  • nucleic acids DNA or mRNA
  • Coronaviruses is a group of viruses that causes diseases in birds, mammals and humans. The diseases include respiratory infections and enteric infections which can be mild or lethal.
  • Coronaviruses are viruses in the subfamily Orthocoronavirinae, in the family Coronaviridae, in the order Nidovirales.
  • the genus Coronavirus includes avian infectious bronchitis virus, bovine coronavirus, canine coronavirus, human coronavirus 299E, human coronavirus OC43, murine hepatitis virus, rat coronavirus, and porcine hemagglutinating encephalomyelitis virus.
  • the genus Torovirus includes Berne virus and Breda virus.
  • Coronaviruses are enveloped viruses having a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry.
  • coronavirus The genomic size of coronaviruses ranges from approximately 26 to 32 kilobases, which is believed to be the largest for an RNA virus.
  • the name “coronavirus” is derived from the Latin corona and the Greek korone (e.g., "garland” or “wreath”), meaning crown or halo.
  • the corona reference relates to the characteristic appearance of virions (the infective form of the virus) by electron microscopy, which have a fringe of large, bulbous surface projections creating an image reminiscent of a royal crown or of the solar corona. This morphology is created by the viral spike (S) peplomers, which are proteins that populate the surface of the virus and determine host tropism.
  • S viral spike
  • S spike
  • E envelope
  • M membrane
  • N nucleocapsid
  • SARS coronavirus a defined receptor-binding domain on S mediates the attachment of the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2).
  • ACE2 angiotensin-converting enzyme 2
  • Some coronaviruses also have a shorter spike-like protein called hemagglutinin esterase (HE).
  • HE hemagglutinin esterase
  • the recombinant Fc-coronavirus antigen fusion protein includes a coronavirus spike S1 protein or a fragment thereof; and an immunoglobulin Fc region or a fragment thereof; wherein the C-terminus of the coronavirus spike S1 protein or fragment thereof is linked to the N- terminus of the immunoglobulin Fc region or fragment thereof.
  • a recombinant Fc-coronavirus antigen fusion protein is described.
  • the recombinant Fc-coronavirus antigen fusion protein includes a coronavirus spike S1 protein or a fragment thereof; and an immunoglobulin Fc region or a fragment thereof; wherein the N-terminus of the coronavirus spike S1 protein or fragment thereof is linked to the C- terminus of the immunoglobulin Fc region or fragment thereof.
  • the coronavirus spike S1 protein may be derived from SARS-CoV-2, and may be the S1 protein of the “Wuhan” strain, the B.1.1.7 (“U.K.”) variant, the B.1.135 (“South Africa”) variant, the P.1 variant, the B.1.427/B.1.429 (“California”) variant, the B.1.526 (“New York”) variant, or an S1 protein of another strain or variant related to any of these, for example, having at least 95% identity to any of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.
  • coronavirus S1 protein of a fusion protein can have one or more amino acid changes with respect to a variant or isolate such as but not limited any disclosed herein, e.g., may have 98% or 99% identity to an S1 protein of a SARS-CoV-2 isolate or variant detected in an infected individual.
  • the coronavirus spike S1 protein may include an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1.
  • the Fc region may include an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3.
  • the recombinant Fc-coronavirus antigen fusion protein may include a signal peptide linked to the N-terminus of the Fc-coronavirus antigen fusion protein.
  • the signal peptide may include an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2.
  • the recombinant Fc-coronavirus antigen fusion protein may include an amino acid sequence having at least 95% sequence identity to SEQ ID NO:4.
  • a nucleic acid is described. The nucleic acid encodes any of the recombinant Fc-coronavirus antigen fusion proteins described herein.
  • the nucleic acid may be a DNA or an mRNA.
  • the nucleic acid may include a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:5.
  • an expression vector comprising a promoter operably linked to a nucleic acid described herein.
  • the expression vector may include a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:6.
  • compositions are described.
  • the compositions may include any of the recombinant Fc-coronavirus antigen fusion proteins, nucleic acids, or expression vectors described herein, and a pharmaceutically-acceptable excipient.
  • the composition may include nanoparticles comprising poly( ⁇ -amino esters) (PBAE) and a recombinant Fc-coronavirus antigen fusion protein, nucleic acid, or expression vector described herein.
  • PBAE poly( ⁇ -amino esters)
  • the composition may include liposomes or lipids, for example cationic lipids and a recombinant Fc-coronavirus nucleic acid, or expression vector described herein.
  • a host cell comprising an expression vector described herein is described.
  • a method of treating a coronavirus infection in a subject is described. The method includes administering to the subject an effective amount of a composition described herein.
  • a method of preventing a coronavirus infection in a subject includes administering to the subject an effective amount of a composition described herein.
  • the administering may include intravenous injection, intramuscular injection, or intradermal injection.
  • the administering may include electroporation.
  • the coronavirus infection may be a SARS-CoV-2 infection.
  • FIG.1A is a schematic of an example expression vector configured to express a Fc- Spike S1 fusion protein.
  • FIG.1B is an example DNA sequence (SEQ ID NO: 6) of the expression vector of FIG. 1A.
  • FIG.2A is an example amino acid sequence (SEQ ID NO:7) of SARS-Cov-2 spike protein with S1 and S2 subunits.
  • FIG.2B is an example amino acid sequence (SEQ ID NO:8) of SARS-Cov-2 spike protein S1 subunit.
  • FIG.3 is an example schematic showing PCR amplification of an expression vector configured to express a Fc-Spike S1 fusion protein to provide DNA vector for formulation.
  • FIG. 3 also shows the amino acid sequence of an exemplary Fc-Spike S1 fusion protein, which may be produced in a host cell from the Fc-Spike S1 fusion protein expression vector.
  • FIG.4A is an exemplary schematic showing an overview of preparation and administration of a nanoparticle formulation of an example Fc-coronavirus antigen fusion protein (“Fc-Spike S1 antigen”).
  • FIG.4B is an exemplary schematic showing an overview of preparation and administration of a nanoparticle formulation of an example Fc-coronavirus antigen fusion protein expression vector configured to express an Fc-Spike S1 fusion protein (“Fc-Spike S1 DNA construct”).
  • FIG.4C is an exemplary schematic showing an overview of preparation and administration of a nanoparticle formulation of an example mRNA encoding an Fc-coronavirus antigen fusion protein configured to be translated into an Fc-Spike S1 fusion protein (“Fc-Spike S1 mRNA construct”).
  • FIG.5 is an exemplary schematic showing a post-immunization response to SARS- CoV-2 in a subject administered with a Fc-coronavirus antigen fusion protein, a nucleic acid encoding the Fc-coronavirus antigen fusion protein, and/or an expression vector comprising a nucleic acid encoding a Fc-coronavirus antigen fusion protein, or a pharmaceutical formulation thereof.
  • FIG.6 is a schematic of an example method of producing a linearized double stranded DNA comprising a nucleic acid encoding an Fc-coronavirus antigen fusion protein, or a vector comprising a nucleic acid encoding an Fc-coronavirus antigen fusion protein.
  • FIG. 7A to FIG. 7D illustrates example production and secretion of S1-Fc-protein antigen by muscle cells.
  • linear dsDNA encoding S1-Fc was generated by PCR-based amplification and subjected to analytical DNA gel electrophoresis, assessing accurate size and excluding undesired intermediate product. (In FIG.
  • FIG.7A to FIG.7D is based on Fig.
  • FIG.8A to FIG.8C depicts example S1-Fc protein expression in murine biceps femoris and uptake by dendritic cells in vivo.
  • S1-Fc expression in murine muscle biceps femoris was assessed on day 47 by confocal microscopy and quantified. SD shown, T-test: ***) P ⁇ 0.001. Scale, 50 mm.
  • FIG.8B S1-protein produced in vivo by immunization with non-expiring S1-Fc encoding dsDNA, was internalized by dendritic cell.
  • a fluorescence profile illustrates S1 cellular uptake. Scale, 20 mm.
  • FIG.8A to FIG.8C is based on Supplemental Fig.1 of Herrmann et al., published on bioRxiv.org on June 30, 2020; doi: https://doi.org/10.1101/2020.06.29.178616.
  • FIG.9A is an example graph reporting recombinant rS1-Fc was expressed in CHO cells and produced rS1-Fc was analyzed by SEC HPLC to assess monomer purity and intrinsic oligomerization aggregation.
  • FIG.9B two independent batches of produced rS1-Fc were subjected to electrophoretic protein separation and visualized by Coomassie protein staining.
  • FIG.9A to FIG.9B is based on Supplemental Fig.2 of Herrmann et al., published on bioRxiv.org on June 30, 2020; doi: https://doi.org/10.1101/2020.06.29.178616.
  • FIG. 10G provides example data on the generation of functional rS1-Fc- protein antigen.
  • Recombinant rS1-Fc protein was purified by SEC HPLC and, as shown in FIG. 10A, subsequently subjected to Western blot analysis immunologically detecting and confirming human Fc (left) and SARS-CoV-2-S1-domain (right) migrating identically.
  • FIG. 10B shows that RAW 264.7 murine macrophage cells internalize rS1-Fc as assessed by flowcytometric analysis and FIG. 10C confirmed by confocal microscopy. Scale, 10 ⁇ m.
  • FIG. 10D provides ELISA data demonstrating that rS1-Fc binds to human ACE2.
  • FIG.10E and FIG.10F rS1-Fc homes to the inguinal lymph node within an hour upon administration into the biceps femoris as determined by longitudinal nIR imaging of the lymphatics. SD shown.
  • FIG. 10G rS1-Fc and/or S1 and/or processed S1 peptide was detected in splenic APCs 44 d after initial administration as assessed by flow cytometry.
  • FIG. 10A to FIG. 10G is based on Fig. 2 of Herrmann et al., published on bioRxiv.org on June 30, 2020; doi: https://doi.org/10.1101/2020.06.29.178616. [0040] FIG.11.
  • FIG. 12A to FIG. 12B report example data illustrating that the rS1-Fc is taken up by APCs.
  • splenic cell populations were exposed to rS1-Fc for time points as indicated and rS1-Fc cellular internalization by CD11c+ dendritic cells, F4/80+ macrophages and CD19+ B cells was assessed by flow cytometry.
  • FIG.12B inhibition of rS1-Fc cellular internalization was determined using flow cytometry by exposing splenic cell populations to rS1-Fc with or without CD16/CD32 Fc ⁇ R+ blocking antibody as indicated gating for S1-protein+ cell populations.
  • FIG. 12A to FIG.12B is based on Fig.3 of Herrmann et al., published on bioRxiv.org on June 30, 2020; doi: https://doi.org/10.1101/2020.06.29.178616. [0042]
  • FIG.13H report example data demonstrating that rS1-Fc immunization elicits early seroconversion, facilitating anti-S1-specific IgG production protecting against live SARS-CoV-2 challenge.
  • FIG.13A is a schematic representation of mice immunization and blood serum collection schedule.
  • FIG. 13B reports example dose-dependent and significant Th1 polarization upon high-dose non-expiring immunization with S1-Fc dsDNA as analyzed by fold- increased education of CD4 + IFN ⁇ + versus CD4 + IL-4 + T cells in vivo assessed by flow cytometry (right panel). Gating exemplary shown (left panels).
  • FIG.13C reports example S1-antigen-specific CD8 + T cell accumulation assessed by MHC-tetramer/S1-peptide flowcytometric analysis of cognate TCR expressed by CD8+ T cells.
  • FIG.13D reports example immunization-dose-dependent maturation of effector CD8+IFNg+ T cells was assessed by flow cytometry and quantified.
  • FIG. 13E reports example rapid seroconversion and production of S1-specific IgG antibody upon immunization with S1-Fc dsDNA doses as indicated, or, as in FIG. 13F, with rS1-Fc protein, assessed by ELISA.
  • FIG.13A to FIG.13H is based on Fig.4 of Herrmann et al., published on bioRxiv.org on June 30, 2020; doi: https://doi.org/10.1101/2020.06.29.178616. [0043]
  • FIG. 14D are graphs reporting example serum concentrations of anti-S1 antibody over time in mice after immunization with 50 ⁇ g (FIG.14A), 20 ⁇ g (FIG.14B), or 2 ⁇ g (FIG. 14C) of the ds DNA S1-Fc expression construct.
  • FIG. 14D is a graph reporting example serum concentrations of anti-S1 antibody over time in mice after immunization with 100 ⁇ g rS1-Fc protein.
  • FIG.15 is a graph reporting example serum concentrations of anti-S1 antibody in mice 14 days after immunization with 20 ⁇ g of either the linear ds DNA S1-Fc expression construct produced by PCR or the ds plasmid DNA that included the S1-Fc expression cassette.
  • FIG.16A to FIG.16C are graphs reporting example serum levels of S1 antibody in mice over time after immunization with the rS1-Fc protein using three different injection methods: intramuscular (FIG.16A), intradermal (FIG.16B), and epidermal (FIG.16C).
  • FIG.17A is a set of graphs reporting example quantification of labeled rS1-Fc and rS1 in the inguinal lymph node over time after intramuscular injection of the biceps femoris with labeled protein at three doses.
  • DETAILED DESCRIPTION [0047] Unless defined otherwise, technical and scientific terms used herein have meanings that are commonly understood by those of ordinary skill in the art unless defined otherwise. Generally, terminologies pertaining to techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art.
  • the term “and/or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other.
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system.
  • “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art.
  • “about” or “approximately” can mean a range of up to 10% (i.e., ⁇ 10%) or more depending on the limitations of the measurement system.
  • about 5 mg can include any number between 4.5 mg and 5.5 mg.
  • coronavirus infection refers to a human or animal that has cells that have been infected by a coronavirus. The infection can be established by performing a detection and/or viral titration from respiratory samples, or by assaying blood-circulating coronavirus-specific antibodies.
  • the detection in the individuals infected with coronavirus is made by conventional diagnostic methods, such as molecular biology (e.g., PCR or antigen detection), which are known to those skilled in the art.
  • the term “subject” as used herein refers to human and non-human animals, including vertebrates, mammals and non-mammals.
  • the subject can be human, non- human primates, simian, ape, murine (e.g., mice and rats), bovine, porcine, equine, canine, feline, caprine, lupine, ranine or piscine.
  • administering refers to the physical introduction of a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • the formulation is administered via a non-parenteral route, e.g., orally.
  • non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • treatment and “treating” refer to fighting the coronavirus infection in a human or animal subject.
  • treatment also refers to attenuating symptoms associated with the viral infection (e.g., respiratory syndrome, kidney failure, fever, and other symptoms relating to coronavirus infections).
  • effective amount refers to an amount of the therapeutic agent that when administered to a subject, is sufficient to affect a measurable improvement or prevention of a disease or disorder associated with coronavirus infection. For example, administering an effective dose sufficient to inhibit the proliferation and/or replication of the coronavirus, and/or the development of the viral infection within the subject.
  • Therapeutically effective amounts of the therapeutic agents provided herein, when used alone or in combination with an antiviral agent, will vary depending upon the relative activity of the therapeutic agent, and depending upon the subject and disease condition being treated, the weight and age and sex of the subject, the severity of the disease condition in the subject, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. In one embodiment, a therapeutically effective amount will depend on certain aspects of the subject to be treated and the disorder to be treated and may be ascertained by one skilled in the art using known techniques. In addition, as is known in the art, adjustments for age as well as the body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the disease may be necessary.
  • polypeptide and “protein” and other related terms used herein are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides may comprise natural and non-natural amino acids. Polypeptides include recombinant or chemically-synthesized forms. These terms encompass native and artificial proteins, protein fragments and polypeptide analogs (such as muteins, variants, chimeric proteins and fusion proteins) of a protein sequence as well as post-translationally, or otherwise covalently or non-covalently, modified proteins.
  • Polypeptides comprising amino acid sequences of an Fc- coronavirus antigen fusion protein or a derivative, mutein, or variant thereof , can be prepared using recombinant procedures are described herein.
  • the terms “nucleic acid”, “polynucleotide” and “oligonucleotide” and other related terms used herein are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically-synthesized forms.
  • Nucleic acids include DNA molecules (cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof.
  • Nucleic acid molecule can be single-stranded or double-stranded.
  • the nucleic acid molecules of the disclosure comprise a contiguous open reading frame encoding an Fc-coronavirus antigen fusion protein, or a derivative, mutein, or variant thereof.
  • nucleic acids comprise a one type of polynucleotides or a mixture of two or more different types of polynucleotides.
  • mutation refers to a change in a nucleic acid sequence or amino acid sequence that differs from a reference nucleic acid sequence or a reference amino acid sequence, respectively.
  • mutations includes a point mutation, insertion, deletion, amino acid substitution, inversion, rearrangement, splice, sequence fusion (e.g., gene fusion or RNA fusion), truncation, transversion, translocation, non- sense mutation, sequence repeat, single nucleotide polymorphism (SNP), or other genetic rearrangement.
  • SNP single nucleotide polymorphism
  • the term “recover” or “recovery” or “recovering”, and other related terms refers to obtaining a protein (e.g., an antibody or an antigen binding portion thereof), from a host cell culture medium or from host cell lysate or from the host cell membrane.
  • a Fc-coronavirus antigen fusion proteins described herein may be expressed by the host cell as a recombinant protein fused to a secretion signal peptide sequence which mediates secretion of the expressed protein.
  • the secreted protein can be recovered from the host cell medium.
  • the Fc-coronavirus antigen fusion protein is expressed by the host cell as a recombinant protein that lacks a secretion signal peptide sequence which can be recovered from the host cell lysate.
  • the Fc-coronavirus antigen fusion proteins described herein may be expressed by the host cell as a membrane-bound protein which can be recovered using a detergent to release the expressed protein from the host cell membrane.
  • the Fc-coronavirus antigen fusion proteins described herein can be subjected to procedures that remove cellular debris from the recovered protein.
  • the recovered Fc-coronavirus antigen fusion proteins can be subjected to chromatography, gel electrophoresis and/or dialysis.
  • the chromatography comprises any one or any combination or two or more procedures including affinity chromatography, hydroxyapatite chromatography, ion-exchange chromatography, reverse phase chromatography and/or chromatography on silica.
  • affinity chromatography comprises protein A or G (cell wall components from Staphylococcus aureus).
  • isolated refers to a protein (e.g., an antibody or an antigen binding portion thereof) or polynucleotide that is substantially free of other cellular material.
  • a protein may be rendered substantially free of naturally associated components (or components associated with a cellular expression system or chemical synthesis methods used to produce the antibody) by isolation, using protein purification techniques well known in the art.
  • isolated also refers in some embodiments to protein or polynucleotides that are substantially free of other molecules of the same species, for example other protein or polynucleotides having different amino acid or nucleotide sequences, respectively.
  • the purity of homogeneity of the desired molecule can be assayed using techniques well known in the art, including low resolution methods such as gel electrophoresis and high resolution methods such as HPLC or mass spectrophotometry.
  • an "antigen binding protein” and related terms used herein refers to a protein comprising a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen.
  • antigen binding proteins include antibodies, antibody fragments (e.g., an antigen binding portion of an antibody), antibody derivatives, and antibody analogs.
  • the antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives.
  • Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004, Biotechnol. Prog.20:639-654.
  • PAMs peptide antibody mimetics
  • An antigen binding protein can have, for example, the structure of an immunoglobulin.
  • an "immunoglobulin” refers to a tetrameric molecule composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy- terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Human light chains are classified as kappa or lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes).
  • the variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two antigen binding sites.
  • an antigen binding protein can be a synthetic molecule having a structure that differs from a tetrameric immunoglobulin molecule but still binds a target antigen or binds two or more target antigens.
  • a synthetic antigen binding protein can comprise antibody fragments, 1-6 or more polypeptide chains, asymmetrical assemblies of polypeptides, or other synthetic molecules.
  • the variable regions of immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein.
  • An antigen binding protein may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently.
  • the CDRs permit the antigen binding protein to specifically bind to a particular antigen of interest.
  • the assignment of amino acids to each domain is in accordance with the definitions of Kabat et al. in Sequences of Proteins of Immunological Interest, 5 th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no.91-3242, 1991.
  • IMGT.RTM international ImMunoGeneTics information system
  • Lefranc et al Dev. Comp. Immunol.29:185-203; 2005
  • AHo Hegger and Pluckthun, J. Mol. Biol.309(3):657-670; 2001
  • Chothia Al-Lazikani et al., 1997 Journal of Molecular Biology 273:927-948; Contact (Maccallum et al., 1996 Journal of Molecular Biology 262:732-745, and Aho (Honegger and Pluckthun 2001 Journal of Molecular Biology 309:657- 670.
  • an "antibody” and “antibodies” and related terms used herein refers to an intact immunoglobulin or to an antigen binding portion thereof (or an antigen binding fragment thereof) that binds specifically to an antigen.
  • Antigen binding portions or the antigen binding fragment may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding portions include, inter alia, Fab, Fab', F(ab') 2 , Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
  • Antibodies include recombinantly produced antibodies and antigen binding portions.
  • Antibodies include non-human, chimeric, humanized and fully human antibodies.
  • Antibodies include monospecific, multispecific (e.g., bispecific, trispecific and higher order specificities).
  • Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, heavy chain dimers. Antibodies include F(ab’)2 fragments, Fab’ fragments and Fab fragments. Antibodies include single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragment (scFv), camelized antibodies, affibodies, disulfide- linked Fvs (sdFv), anti-idiotypic antibodies (anti-Id), minibodies. Antibodies include monoclonal and polyclonal populations.
  • a “neutralizing antibody” and related terms refers to an antibody that is capable of specifically binding to the neutralizing epitope of its target antigen (e.g., coronavirus spike protein) and substantially inhibiting or eliminating the biological activity of the target antigen (e.g., coronavirus spike protein).
  • the neutralizing antibody can reduce the biological activity of the target antigen by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher levels of reduced biological activity.
  • an “antigen binding domain,” “antigen binding region,” or “antigen binding site” and other related terms used herein refer to a portion of an antigen binding protein that contains amino acid residues (or other moieties) that interact with an antigen and contribute to the antigen binding protein's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least part of at least one of its CDR domains.
  • telomere binding refers to non-covalent or covalent preferential binding to an antigen relative to other molecules or moieties (e.g., an antibody specifically binds to a particular antigen relative to other available antigens).
  • an antibody specifically binds to a target antigen if it binds to the antigen with a dissociation constant KD of 10 -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or less.
  • a dissociation constant (K D ) can be measured using a BIACORE surface plasmon resonance (SPR) assay.
  • SPR surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).
  • An "epitope" and related terms as used herein refers to a portion of an antigen that is bound by an antigen binding protein (e.g., by an antibody or an antigen binding portion thereof).
  • An epitope can comprise portions of two or more antigens that are bound by an antigen binding protein.
  • An epitope can comprise non-contiguous portions of an antigen or of two or more antigens (e.g., amino acid residues that are not contiguous in an antigen’s primary sequence but that, in the context of the antigen’s tertiary and quaternary structure, are near enough to each other to be bound by an antigen binding protein).
  • the variable regions, particularly the CDRs, of an antibody interact with the epitope.
  • An "antibody fragment”, “antibody portion”, “antigen-binding fragment of an antibody”, or “antigen-binding portion of an antibody” and other related terms used herein refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; Fd; and Fv fragments, as well as dAb; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.
  • Antigen binding portions of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer antigen binding properties to the antibody fragment.
  • Antigen-binding fragments that bind a coronavirus spike protein (S-protein) are described herein.
  • the terms “Fab”, “Fab fragment” and other related terms refers to a monovalent fragment comprising a variable light chain region (VL), constant light chain region (CL), variable heavy chain region (VH), and first constant region (CH1).
  • a Fab is capable of binding an antigen.
  • An F(ab') 2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • a F(Ab’)2 has antigen binding capability.
  • An Fd fragment comprises VH and CH1 regions.
  • An Fv fragment comprises VL and VH regions.
  • An Fv can bind an antigen.
  • a dAb fragment has a V H domain, a V L domain, or an antigen-binding fragment of a V H or VL domain (U.S. Patents 6,846,634 and 6,696,245; U.S.
  • Fab fragments comprising antigen binding portions from an antibody that binds a coronavirus spike protein (S-protein) are described herein.
  • S-protein coronavirus spike protein
  • the term “hinge” refers to a hinge region comprising any one or any combination of two or more regions comprising an upper, core or lower hinge sequences from an IgG1, IgG2, IgG3 or IgG4 immunoglobulin molecule.
  • the hinge region comprises an IgG1 upper hinge sequence EPKSCDKTHT.
  • the hinge region comprises an IgG1 core hinge sequence CPXC, wherein X is P, R or S. In one embodiment, the hinge region comprises a lower hinge/CH2 sequence PAPELLGGP. In one embodiment, the hinge is joined to an Fc region (CH2) having the amino acid sequence SVFLFPPKPKDT. In one embodiment, the hinge region includes the amino acid sequence of an upper, core and lower hinge and comprises EPKSCDKTHTCPPCPAP ELLGGP. In one embodiment, the hinge region comprises one, two, three or more cysteines that can form at least one, two, three or more interchain disulfide bonds.
  • a single-chain antibody is an antibody in which a V L and a V H region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain.
  • a linker e.g., a synthetic sequence of amino acid residues
  • the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).
  • Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises V H and V L domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994, Structure 2:1121-23). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites.
  • polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites.
  • tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.
  • the term “human antibody” refers to antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (e.g., a fully human antibody).
  • a “humanized” antibody refers to an antibody having a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non- human species antibody, when it is administered to a human subject.
  • certain amino acids in the framework and constant domains of the heavy and/or light chains of the non- human species antibody are mutated to produce the humanized antibody.
  • the constant domain(s) from a human antibody are fused to the variable domain(s) of a non- human species.
  • one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the likely immunogenicity of the non- human antibody when it is administered to a human subject, wherein the changed amino acid residues either are not critical for immunospecific binding of the antibody to its antigen, or the changes to the amino acid sequence that are made are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non- human antibody to the antigen. Examples of how to make humanized antibodies may be found in U.S. Pat. Nos.6,054,297, 5,886,152 and 5,877,293.
  • chimeric antibody refers to an antibody that contains one or more regions from a first antibody and one or more regions from one or more other antibodies.
  • one or more of the CDRs are derived from a human antibody.
  • all of the CDRs are derived from a human antibody.
  • the CDRs from more than one human antibody are mixed and matched in a chimeric antibody.
  • a chimeric antibody may comprise a CDR1 from the light chain of a first human antibody, a CDR2 and a CDR3 from the light chain of a second human antibody, and the CDRs from the heavy chain from a third antibody.
  • the CDRs originate from different species such as human and mouse, or human and rabbit, or human and goat.
  • the framework regions may be derived from one of the same antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody.
  • a portion of the heavy and/or light chain is identical with, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with, homologous to, or derived from an antibody (-ies) from another species or belonging to another antibody class or subclass.
  • variant polypeptides and variants of polypeptides refers to a polypeptide comprising an amino acid sequence with one or more amino acid residues inserted into, deleted from and/or substituted into the amino acid sequence relative to a reference polypeptide sequence.
  • Polypeptide variants include fusion proteins.
  • a variant polynucleotide comprises a nucleotide sequence with one or more nucleotides inserted into, deleted from and/or substituted into the nucleotide sequence relative to another polynucleotide sequence.
  • Polynucleotide variants include fusion polynucleotides.
  • the term “derivative” of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, e.g., via conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.
  • the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below.
  • Fc or “Fc region” as used herein refers to the portion of an antibody heavy chain constant region beginning in or after the hinge region and ending at the C-terminus of the heavy chain.
  • the Fc region comprises at least a portion of the CH and CH3 regions and may, or may not, include a portion of the hinge region.
  • Two polypeptide chains each carrying a half Fc region can dimerize to form a full Fc domain.
  • An Fc domain can bind Fc cell surface receptors and some proteins of the immune complement system.
  • An Fc domain exhibits effector function, including any one or any combination of two or more activities including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), opsonization and/or cell binding.
  • An Fc domain can bind an Fc receptor, including Fc ⁇ RI (e.g., CD64), Fc ⁇ RII (e.g, CD32) and/or Fc ⁇ RIII (e.g., CD16a).
  • Fc ⁇ RI e.g., CD64
  • Fc ⁇ RII e.g, CD32
  • Fc ⁇ RIII e.g., CD16a
  • ADCP antibody-dependent phagocytosis
  • labeled antibody or related terms as used herein refers to antibodies and their antigen binding portions thereof that are unlabeled or joined to a detectable label or moiety for detection, wherein the detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin, streptavidin or protein A.
  • the “percent identity” or “percent homology” and related terms used herein refers to a quantitative measurement of the similarity between two polypeptide or between two polynucleotide sequences.
  • the percent identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions that are shared between the two polypeptide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polypeptide sequences.
  • the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at aligned positions that are shared between the two polynucleotide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polynucleotide sequences.
  • a comparison of the sequences and determination of the percent identity between two polypeptide sequences, or between two polynucleotide sequences, may be accomplished using a mathematical algorithm.
  • the "percent identity” or “percent homology" of two polypeptide or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters.
  • the amino acid sequence of a Fc-coronavirus antigen fusion protein may be similar but not identical to any of the amino acid sequences of the polypeptides that make up any of the Fc-coronavirus antigen fusion proteins described herein.
  • the similarities between an Fc-coronavirus antigen fusion proteins and the Fc-coronavirus antigen fusion proteins described herein can be at least 95%, or at or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, to any of the polypeptides that make up any of the Fc-coronavirus antigen fusion proteins described herein.
  • the amino acid substitutions comprise one or more conservative amino acid substitutions.
  • a "conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol.24: 307-331, herein incorporated by reference in its entirety.
  • 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 are cysteine and methionine.
  • Antibodies can be obtained from sources such as serum or plasma that contain immunoglobulins having varied antigenic specificity.
  • antibodies can be enriched for a particular antigenic specificity.
  • Such enriched preparations of antibodies usually are made of less than about 10% antibody having specific binding activity for the particular antigen. Subjecting these preparations to several rounds of affinity purification can increase the proportion of antibody having specific binding activity for the antigen.
  • Antibodies prepared in this manner are often referred to as "monospecific.” Monospecific antibody preparations can be made up of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% antibody having specific binding activity for the particular antigen.
  • Antibodies can be produced using recombinant nucleic acid technology as described below.
  • a "vector" and related terms used herein refers to a nucleic acid molecule (e.g., DNA or RNA) which can be operably linked to foreign genetic material (e.g., nucleic acid transgene).
  • Vectors can be used as a vehicle to introduce foreign genetic material into a cell (e.g., host cell).
  • Vectors can include at least one restriction endonuclease recognition sequence for insertion of the transgene into the vector.
  • Vectors can include at least one gene sequence that confers antibiotic resistance or a selectable characteristic to aid in selection of host cells that harbor a vector- transgene construct.
  • Vectors can be single-stranded or double-stranded nucleic acid molecules.
  • Vectors can be linear or circular nucleic acid molecules.
  • vector refers to a linear or circular double stranded extrachromosomal DNA molecule which can be linked to a transgene, and is capable of replicating in a host cell, and transcribing and/or translating the transgene.
  • a viral vector typically contains viral RNA or DNA backbone sequences which can be linked to the transgene. The viral backbone sequences can be modified to disable infection but retain insertion of the viral backbone and the co-linked transgene into a host cell genome.
  • examples of viral vectors include retroviral, lentiviral, adenoviral, adeno-associated, baculoviral, papovaviral, vaccinia viral, herpes simplex viral and Epstein Barr viral vectors.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • An "expression vector” is a type of vector that can contain one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers. Expression vectors can include ribosomal binding sites and/or polyadenylation sites.
  • Regulatory sequences direct transcription, or transcription and translation, of a transgene, such as a DNA or RNA transgene encoding a Fc-coronavirus antigen fusion protein described herein, linked to the expression vector which is transduced into a host cell.
  • the regulatory sequence(s) can control the level, timing and/or location of expression of the transgene.
  • the regulatory sequence can, for example, exert its effects directly on the transgene, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the nucleic acid).
  • Regulatory sequences can be part of a vector.
  • a transgene is “operably linked” to a vector when there is linkage between the transgene and the vector to permit functioning or expression of the transgene sequences contained in the vector.
  • a transgene is "operably linked" to a regulatory sequence when the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the transgene.
  • transfected or “transformed” or “transduced” or other related terms used herein refer to a process by which exogenous nucleic acid (e.g., transgene) is transferred or introduced into a host cell.
  • a “transfected” or “transformed” or “transduced” host cell is one which has been transfected, transformed or transduced with exogenous nucleic acid (transgene).
  • the host cell includes the primary subject cell and its progeny.
  • host cell or “or a population of host cells” or related terms as used herein refer to a cell (or a population thereof or a plurality of a host cell) into which foreign (exogenous or transgene) nucleic acids have been introduced.
  • the foreign nucleic acids can include an expression vector operably linked to a transgene, and the host cell can be used to express the nucleic acid and/or polypeptide encoded by the transgene.
  • a host cell (or a population thereof) can be a cultured cell or can be extracted from a subject.
  • the host cell (or a population thereof) includes the primary subject cell and its progeny without any regard for the number of passages.
  • Progeny cells may or may not harbor identical genetic material compared to the parent cell.
  • Host cells encompass progeny cells.
  • a host cell describes any cell (including its progeny) that has been modified, transfected, transduced, transformed, and/or manipulated in any way to express an antibody, as disclosed herein.
  • the host cell (or population thereof) can be introduced with an expression vector operably linked to a nucleic acid encoding the desired antibody, or an antigen binding portion thereof, described herein.
  • Host cells and populations thereof can harbor an expression vector that is stably integrated into the host’s genome or can harbor an extrachromosomal expression vector.
  • host cells and populations thereof can harbor an extrachromosomal vector that is present after several cell divisions or is present transiently and is lost after several cell divisions.
  • a host cell can be a prokaryote, for example, E.
  • coli or it can be a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), a mammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma.
  • a single-celled eukaryote e.g., a yeast or other fungus
  • a plant cell e.g., a tobacco or tomato plant cell
  • a mammalian cell e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell
  • a hybridoma e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse
  • a host cell can be introduced with an expression vector operably linked to a nucleic acid encoding a desired antibody thereby generating a transfected/transformed host cell which is cultured under conditions suitable for expression of the antibody by the transfected/transformed host cell, and optionally recovering the antibody from the transfected/transformed host cells (e.g., recovery from host cell lysate) or recovery from the culture medium.
  • host cells comprise non-human cells including CHO, BHK, NS0, SP2/0, and YB2/0.
  • host cells comprise human cells including HEK293, HT-1080, Huh-7 and PER.C6.
  • host cells examples include the COS- 7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23: 175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines which grow in serum- free media (see Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DX-B 11, which is deficient in DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci.
  • COS- 7 line of monkey kidney cells ATCC CRL 1651
  • L cells C127 cells
  • 3T3 cells ATCC CCL 163
  • CHO Chinese hamster ovary
  • HeLa cells include lymphoid cells such as Y0, NS0 or Sp20.
  • a host cell is a mammalian host cell, but is not a human host cell.
  • a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell.
  • the phrase “transgenic host cell” or “recombinant host cell” can be used to denote a host cell that has been transformed or transfected with a nucleic acid to be expressed.
  • a host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid.
  • a host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell may be a cell comprised in a multicellular organism, such as a human.
  • a host cell may be a cell of a human subject, such as a muscle cell (myocyte) and/or a dendritic cell of a human subject.
  • a host cell may permit expression of a transgene in vivo, such as in a human subject.
  • Polypeptides of the present disclosure can be produced using any methods known in the art.
  • the polypeptides are produced by recombinant nucleic acid methods by inserting a nucleic acid sequence (e.g., DNA) encoding the polypeptide into a recombinant expression vector which is introduced into a host cell and expressed by the host cell under conditions promoting expression.
  • nucleic acid e.g., DNA
  • encoding the polypeptide is operably linked to an expression vector carrying one or more suitable transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes.
  • suitable transcriptional or translational regulatory elements include a transcriptional promoter (e.g.
  • a Jet promoter SEQ ID NO:12
  • an optional operator sequence to control transcription a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the initiation and termination of transcription and translation (e.g. Kozak sequences; SV40 transcription termination sequence, etc.).
  • the expression vector can include an origin or replication that confers replication capabilities in the host cell.
  • the expression vector can include a gene that confers selection to facilitate recognition of transgenic host cells (e.g., transformants).
  • the recombinant DNA can also encode any type of protein tag sequence that may be useful for purifying the protein.
  • protein tags include but are not limited to a histidine tag, a FLAG tag, a myc tag, an HA tag, or a GST tag.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts can be found in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).
  • the expression vector construct can be introduced into the host cell using a method appropriate for the host cell.
  • Suitable host cells include prokaryotes, yeast, mammalian cells, or bacterial cells.
  • Suitable bacteria include gram negative or gram positive organisms, for example, E. coli or Bacillus spp. Yeast, preferably from the Saccharomyces species, such as S.
  • cerevisiae may also be used for production of polypeptides.
  • Various mammalian or insect cell culture systems can also be employed to express recombinant proteins.
  • Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, (Bio/Technology, 6:47, 1988).
  • suitable mammalian host cell lines include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines.
  • Purified polypeptides are prepared by culturing suitable host/vector systems to express the recombinant proteins.
  • polypeptides disclosed herein For many applications, the small size of many of the polypeptides disclosed herein would make expression in E. coli as the preferred method for expression.
  • An mRNA or protein expressed from an expression vector may be then purified from culture media or cell extracts.
  • Polypeptides disclosed herein can be produced using cell-translation systems. For such purposes the nucleic acids encoding the polypeptides must be modified to allow in vitro transcription to produce mRNA and to allow cell-free translation of the mRNA in the particular cell-free system being utilized (eukaryotic such as a mammalian or yeast cell-free translation system or prokaryotic such as a bacterial cell-free translation system.
  • Nucleic acids encoding any of the various polypeptides disclosed herein may be synthesized chemically. Codon usage may be selected so as to improve expression in a cell. Such codon usage will depend on the cell type selected. Specialized codon usage patterns have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See for example: Mayfield et al., Proc. Natl. Acad. Sci. USA.2003100(2):438-42; Sinclair et al. Protein Expr. Purif.2002 (1):96-105; Connell N D. Curr. Opin. Biotechnol.2001 12(5):446-9; Makrides et al. Microbiol.
  • Polypeptides described herein can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications to the protein can also be produced by chemical synthesis. [00109] Polypeptides described herein can be purified by isolation/purification methods for proteins generally known in the field of protein chemistry.
  • Non-limiting examples include extraction, recrystallization, salting out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reversed-phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution or any combinations of these.
  • polypeptides may be exchanged into different buffers and/or concentrated by any of a variety of methods known to the art, including, but not limited to, filtration and dialysis.
  • polypeptides described herein are preferably at least 65% pure, at least 75% pure, at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% pure. Regardless of the exact numerical value of the purity, the polypeptide is sufficiently pure for use as a pharmaceutical product.
  • the polypeptides herein can further comprise post- translational modifications. Exemplary post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation or addition of a polypeptide side chain or of a hydrophobic group.
  • the modified polypeptides may contain non-amino acid elements, such as lipids, poly- or mono-saccharide, and phosphates.
  • a preferred form of glycosylation is sialylation, which conjugates one or more sialic acid moieties to the polypeptide.
  • Sialic acid moieties improve solubility and serum half-life while also reducing the possible immunogenicity of the protein. See Raju et al. Biochemistry.200131; 40(30):8868-76.
  • the polypeptides described herein can be modified to become soluble polypeptides which comprises linking the polypeptides to non-proteinaceous polymers.
  • the non-proteinaceous polymer comprises polyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylenes, in the manner as set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • PEG is a water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol.3, pages 138-161).
  • PEG is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented by the formula: X— O(CH 2 CH 2 O) n —CH 2 CH 2 OH (1), where n is 20 to 2300 and X is H or a terminal modification, e.g., a C1-4 alkyl.
  • the PEG terminates on one end with hydroxy or methoxy, i.e., X is H or CH3 (“methoxy PEG”).
  • a PEG can contain further chemical groups which are necessary for binding reactions; which results from the chemical synthesis of the molecule; or which is a spacer for optimal distance of parts of the molecule.
  • PEG can consist of one or more PEG side-chains which are linked together.
  • PEGs with more than one PEG chain are called multiarmed or branched PEGs.
  • Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol.
  • a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide.
  • Branched PEG are described in, for example, EP-A 0473084 and U.S. Pat. No. 5,932,462.
  • PEGs includes two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini et al., Bioconjugate Chem.6 (1995) 62-69).
  • the serum clearance rate of PEG-modified polypeptide may be modulated (e.g., increased or decreased) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative to the clearance rate of the unmodified antibodies and antigen binding proteins binding polypeptides.
  • the PEG-modified antibodies and antigen binding proteins may have a half-life (t 1/2 ) which is enhanced relative to the half-life of the unmodified polypeptide.
  • the half-life of PEG-modified polypeptide may be enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by 1000% relative to the half-life of the unmodified antibodies and antigen binding proteins.
  • the protein half-life is determined in vitro, such as in a buffered saline solution or in serum.
  • the protein half-life is an in vivo half-life, such as the half-life of the protein in the serum or other bodily fluid of an animal.
  • the present disclosure provides therapeutic compositions comprising any of the Fc- coronavirus antigen fusion proteins, nucleic acids encoding the Fc-coronavirus antigen fusion proteins, and/or expression vectors comprising the Fc-coronavirus antigen fusion proteins described herein in an admixture with a pharmaceutically-acceptable excipient.
  • An excipient encompasses carriers, stabilizers and excipients.
  • Excipients of pharmaceutically acceptable excipients includes for example inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Additional examples include buffering agents, stabilizing agents, preservatives, non-ionic detergents, anti-oxidants and isotonifiers. [00116] Therapeutic compositions and methods for preparing them are well known in the art and are found, for example, in “Remington: The Science and Practice of Pharmacy” (20th ed., ed. A. R.
  • compositions can be formulated for parenteral administration may, and can for example, contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the Fc-coronavirus antigen fusion proteins, nucleic acids and/or vectors described herein.
  • Nanoparticulate formulations may be used to control the biodistribution of the Fc-coronavirus antigen fusion proteins, nucleic acids and/or vectors described herein.
  • poly( ⁇ -amino esters) PBAE
  • Methods of providing suitable nanoparticle formulations using PBAE are known in the art (e.g. Xinyi Jiang, et al.
  • Nanoparticle engineered TRAIL-overexpressing adipose-derived stem cells target and eradicate glioblastoma via intracranial delivery; Jiang-Feng Wang et al. (2011) Academic Journal of Second Military Medical University 31(5):473-476, Polymers poly( ⁇ -amino esters) nanoparticles for gene delivery).
  • Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • the concentration of the Fc-coronavirus antigen fusion protein, nucleic acids and/or vectors described herein in the formulation varies depending upon a number of factors, including the dosage of the Fc-coronavirus antigen fusion proteins, nucleic acids and/or vectors described herein to be administered, and the route of administration.
  • Any of the Fc-coronavirus antigen fusion proteins, nucleic acids and/or vectors described herein may be optionally administered as a pharmaceutically acceptable salt, such as non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry.
  • acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like.
  • Metal complexes include zinc, iron, and the like.
  • the antibody (or antigen binding portions thereof) is formulated in the presence of sodium acetate to increase thermal stability.
  • any of the Fc-coronavirus antigen fusion proteins, nucleic acids and/or vectors described herein may be formulated for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.
  • subject refers to human and non-human animals, including vertebrates, mammals and non-mammals.
  • the subject can be human, non- human primates, simian, ape, murine (e.g., mice and rats), bovine, porcine, equine, canine, feline, caprine, lupine, ranine or piscine.
  • administering refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • the formulation is administered via a non-parenteral route, e.g., orally.
  • non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • the terms "effective amount”, “therapeutically effective amount” or “effective dose” or related terms may be used interchangeably and refer to an amount of Fc-coronavirus antigen fusion proteins, nucleic acids and/or vectors described herein that when administered to a subject, is sufficient to effect a measurable improvement or prevention of a disease associated with coronavirus infection.
  • Therapeutically effective amounts of a Fc-coronavirus antigen fusion protein, nucleic acids and/or vectors described herein, when used alone or in combination, will vary depending upon the relative activity of the Fc-coronavirus antigen fusion proteins, nucleic acids and/or vectors described herein and depending upon the subject and disease condition being treated, the weight and age and sex of the subject, the severity of the disease condition and symptoms in the subject, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. [00125] In some embodiments, a therapeutically effective amount will depend on certain aspects of the subject to be treated and the disorder to be treated and may be ascertained by one skilled in the art using known techniques.
  • the Fc-coronavirus antigen fusion proteins, nucleic acids and/or vectors described herein may be administered at about 0.01 g/kg to about 50 mg/kg per day, 0.01 mg/kg to about 30 mg/kg per day, or 0.1 mg/kg to about 20 mg/kg per day.
  • the Fc-coronavirus antigen fusion proteins, nucleic acids and/or vectors described herein may be administered daily (e.g., once, twice, three times, or four times daily) or preferably less frequently (e.g., weekly, every two weeks, every three weeks, monthly, or quarterly).
  • the present disclosure provides methods for treating a subject having a coronavirus infection.
  • the coronavirus infection may comprise acute respiratory distress syndrome (ARDS), or Severe Acute Respiratory Syndrome (SARS), or Middle East Respiratory Syndrome (MERS), or SARS-CoV, or MERS-CoV, or COVID-19 (infection with the 2019 novel coronavirus known as 2019-nCoV or SARS-CoV-2).
  • ARDS acute respiratory distress syndrome
  • SARS Severe Acute Respiratory Syndrome
  • MERS-CoV Middle East Respiratory Syndrome
  • COVID-19 infection with the 2019 novel coronavirus known as 2019-nCoV or SARS-CoV-2
  • Fc-coronavirus antigen fusion proteins [00127] The present disclosure provides Fc-coronavirus antigen fusion proteins.
  • the coronavirus antigen of the Fc-coronavirus antigen fusion protein may comprise a coronavirus protein such as a coronavirus spike (S) protein, a coronavirus membrane (M) protein, and/or a coronavirus envelope (E) protein.
  • the coronavirus antigen of the Fc-coronavirus antigen fusion protein may comprise an Fc region or a fragment thereof linked directly or indirectly to the coronavirus protein.
  • the Fc- coronavirus antigen fusion protein may comprise an Fc region linked directly or indirectly to the N-terminus or the C-terminus of the coronavirus protein or fragment thereof.
  • the Fc-coronavirus antigen fusion protein may comprise a signal peptide linked directly or indirectly to the N-terminus or the C-terminus of the coronavirus protein or fragment thereof.
  • the Fc-coronavirus antigen fusion protein is a soluble polypeptide (e.g., a secreted polypeptide).
  • Fc-Spike S1 fusion proteins the coronavirus antigen protein of the Fc-coronavirus antigen fusion protein may comprise a coronavirus spike (S) protein, such as a coronavirus spike S1 protein or a fragment thereof.
  • the coronavirus spike S1 protein may be a spike S1 protein derived from SARS-CoV-2 (e.g., GenBank MN908947.3). In some embodiments, the coronavirus spike S1 protein may be amino acid residues 17-685 of a spike S1 protein derived from SARS-CoV-2.
  • a spike S1 protein derived from SARS-CoV-2 may comprise an amino acid sequence as follows: [00130]
  • the S1 protein may be derived from the B.1.1.7 (B.1.1.7, 501Y.V1, 20I) SARS-CoV-2 variant, and may have the following sequence: ( Q ) [00131]
  • the S1 protein may be derived from the B.1.351 (B.1.351, 20H) SARS-CoV-2 variant, and may have the following sequence:
  • the S1 protein may be derived from the P.1 (501Y.V3, 20J) variant, and may have the following sequence: [00133]
  • the S1 moiety of a fusion protein provided herein can have the sequence of any other variant of a SARS-CoV-2 S1 protein, for example, may have the sequence of the S1 protein of a variant that may arise in a population or regional locale, and may have, for example, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to any of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, for example at least about 98% or 99% amino acid sequence identity to any of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.
  • the coronavirus antigen protein of the Fc-coronavirus antigen fusion protein may comprise a signal peptide linked to the N-terminus of the coronavirus antigen protein or fragment thereof.
  • the signal peptide sequence is not limiting to the fusion proteins of the invention, and any signal peptide sequence that directs secretion of the fusion protein from mammalian cells can be suitable for including in the fusion protein, for example, at the N- terminus of the fusion protein.
  • the signal peptide may comprise an amino acid sequence as follows: MEWSWVFLFFLSVTTGVHS (SEQ ID NO:2).
  • the Fc-coronavirus antigen fusion protein may have an Fc region linked to the C-terminus of the coronavirus antigen protein or fragment thereof.
  • the Fc region can have, for example, the Fc region sequence of a human IgG, or a sequence at least 95% identical thereto, and preferably at least 97%, at least 98%, or at least 99% amino acid sequence identity to a human IgG Fc region.
  • the Fc region of the Fc-coronavirus antigen fusion protein may comprise an amino acid sequence as follows: KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO:3).
  • an Fc-coronavirus antigen fusion protein may be a Fc-Spike S1 fusion protein, for example having the amino acid sequence: SEQ ID NO: 4 comprise a signal peptide, amino acids 20-689 of SEQ ID NO:4 comprise amino acids 17-685 of a spike S1 protein derived from SARS-CoV-2, and amino acids 690-915 (in bold) of SEQ ID NO:4 comprise an Fc region.
  • the mature produced S1-Fc fusion protein has the amino acid sequence:
  • the coronavirus spike S1 protein may be a full-length spike S1 polypeptide, such as a full-length spike polypeptide of a SARS-CoV-2 coronavirus, or it may be a derivative of or a fragment of or a subunit of a coronavirus spike S1 polypeptide.
  • the present disclosure provides nucleic acids encoding Fc- coronavirus antigen fusion proteins, such as any disclosed herein.
  • the nucleic acids include without limitation DNA or mRNA.
  • the present disclosure provides nucleic acids encoding Fc-Spike S1 fusion proteins.
  • a nucleic acid encoding the exemplary amino acid sequence of Fc-Spike S1 fusion protein of SEQ ID NO:4 may have the DNA sequence:
  • nucleic acid sequences comprising all possible codons for the amino acid sequence of SEQ ID NO: 4 is also contemplated in the present disclosure.
  • an mRNA sequence has uracil (u) substituted for thymine (t).
  • the present disclosure provides an mRNA encoding the exemplary amino acid sequence of Fc-Spike S1 fusion protein of SEQ ID NO:4 may have the nucleotide sequence of SEQ ID NO: 5, wherein uracil (u) is substituted for thymine (t).
  • the present disclosure provides expression vectors configured to express Fc-coronavirus antigen fusion proteins and/or mRNAs encoding the Fc-coronavirus antigen fusion proteins.
  • the present disclosure provides expression vectors configured to express Fc-Spike S1 fusion proteins, and/or mRNAs encoding the Fc-Spike S1 fusion proteins.
  • the expression vectors comprise at least one promoter operably linked to a nucleic acid which encodes the Fc-coronavirus antigen fusion protein.
  • a promoter used in an expression vector can be, for example, a JeT promoter, a CMV promoter, an EF1alpha promoter, an RSV promoter, an SV40 promoter, a CAG promoter, or a beta-actin promoter.
  • Additional promoters that may be considered for use in nucleic acid constructs for delivery to muscle include, without limitation, a desmin promoter, a skeletal alpha-actin (ASKA) promoter, a troponin I (TNNI2) promoter, a muscle creatine kinase (MCK) promoter, a truncated MCK (tMCK) promoter, a myosin heavy chain (MHC) promoter, a hybrid a-myosin heavy chain enhancer-/MCK enhancer-promoter (MHCK7) promoter, a C5-12 promoter, an MCK promoter, a tMCK promoter, or an MHCK7 promoter.
  • ASKA skeletal alpha-actin
  • TNNI2 troponin I
  • MCK muscle creatine kinase
  • tMCK truncated MCK
  • MHC myosin heavy chain
  • MHCK7 promoter a hybrid a-myosin heavy chain enhancer-/
  • FIG.1A is a schematic of an example expression vector configured to express a Fc-Spike S1 fusion protein.
  • the exemplary expression vector of FIG.1A comprises a Jet promoter for regulation of transcription initiation in host cells, a Kozak sequence for regulation of protein translation initiation from the transcribed mRNA, and a nucleic acid coding sequence encoding a fusion protein comprising a SARS-CoV-2 spike S1 protein fragment including amino acids 17-685 of the SARS-CoV-2 spike S1 protein and having a signal peptide linked to the N-terminus of the SARS-CoV-2 spike S1 protein fragment and an Fc region linked to the C-terminus of the SARS-CoV-2 spike S1 protein fragment.
  • FIG.1B is an example DNA sequence (SEQ ID NO: 6) of the expression vector of FIG.1A.
  • the sequence of the Jet promoter is shown in uppercase letters, the Kozak sequence is shown in uppercase underlined, the coding sequence of the fusion protein comprising the SARS-CoV-2 spike S1 protein fragment having a signal peptide linked to the N- terminus of the SARS-CoV-2 spike S1 protein fragment and an Fc region linked to the C- terminus of the SARS-CoV-2 spike S1 protein fragment is shown in lowercase, wherein the signal peptide DNA sequence is shown lowercase underlined, and the Fc region is shown in lowercase bold.
  • FIG.2A is an example amino acid sequence (SEQ ID NO:7) of SARS-Cov-2 spike protein with S1 and S2 subunits, wherein the S1 subunit is underlined.
  • FIG.2B is an example amino acid sequence (SEQ ID NO:8) of SARS-Cov-2 spike protein S1 subunit.
  • the present disclosure provides methods of providing Fc- coronavirus antigen fusion proteins, nucleic acids encoding the Fc-coronavirus antigen fusion proteins, and expression vectors configured to express the Fc-coronavirus antigen fusion proteins mRNAs and polypeptides.
  • Fc-coronavirus antigen fusion protein expression vectors may be provided in sufficient quantities for formulating for administration.
  • the Fc-coronavirus antigen fusion protein expression vectors may be comprised in a plasmid suitable for propagation in cell culture, such as a bacterial cell culture.
  • the plasmid may comprise restriction endonuclease sites flanking the Fc- coronavirus antigen fusion protein expression vector to allow recovery of a linear double-stranded DNA comprising the Fc-coronavirus antigen fusion protein expression vector.
  • Fc-coronavirus antigen fusion protein expression vectors may be amplified using PCR.
  • primers designed to bind to regions flanking the expression vector sequence may be used to produce an amplified PCR product comprising the Fc-coronavirus antigen fusion protein expression vector.
  • the amplified DNA fragment may be used for formulation in a composition for administration.
  • FIG.3 also shows the amino acid sequence of an exemplary Fc-coronavirus antigen fusion protein described herein (SEQ ID NO:4), which may be produced in a host cell from the Fc-coronavirus antigen fusion protein expression vector.
  • the present disclosure provides therapeutic compositions comprising any of the Fc- coronavirus antigen fusion proteins, nucleic acids encoding the Fc-coronavirus antigen fusion proteins, or vectors comprising nucleic acids encoding the Fc-coronavirus antigen fusion proteins, described herein, in an admixture with a pharmaceutically-acceptable excipient.
  • the present disclosure provides therapeutic compositions comprising any of the Fc-coronavirus antigen fusion proteins, nucleic acids encoding the Fc- coronavirus antigen fusion proteins, or vectors comprising nucleic acids encoding the Fc- coronavirus antigen fusion proteins, described herein, in an admixture with a pharmaceutically- acceptable nanoparticle formulation, such as a poly( ⁇ -amino esters) (PBAE) nanoparticle formulation suitable for delivery to a subject of the Fc-coronavirus antigen fusion proteins, nucleic acids and/or vectors described herein.
  • PBAE poly( ⁇ -amino esters)
  • the present disclosure provides a host cell (or a population of a host cell) harboring the expression vector comprising at least one promoter operably linked to a nucleic acid which encodes the Fc-coronavirus antigen fusion proteins described herein.
  • the host cell or a population of the host cell
  • the host cell or a population of the host cell
  • the present disclosure provides a method for preparing a Fc-coronavirus antigen fusion protein described herein, comprising culturing a population of the host cell which harbors the expression vector comprising at least one promoter operably linked to a nucleic acid which encodes a Fc-coronavirus antigen fusion protein described herein, under conditions suitable for expressing the Fc-coronavirus antigen fusion protein. In one embodiment, the conditions ar suitable for forming a Fc-coronavirus antigen fusion protein dimer. [00156] In one embodiment, the method further comprises the step of: recovering from the host cells the expressed Fc-coronavirus antigen fusion protein.
  • FIG.4A is an exemplary schematic showing an overview of preparation and administration of a nanoparticle formulation of an example Fc-coronavirus antigen fusion protein (“Fc-Spike S1 antigen”).
  • Fc-Spike S1 antigen an example Fc-coronavirus antigen fusion protein
  • FIG.4A the Fc region 401 is shown, and a SARS-CoV-2 spike S1 protein domain fragment 402 is shown.
  • the nanoparticle formulation is administered to a subject by intramuscular injection.
  • the Fc region of the Fc-Spike S1 antigen fusion protein binds to the Fc receptor of dendritic cells, resulting in antigen presentation of the spike S1 protein by the dendritic cells.
  • the dendritic cells may internalize the Fc-Spike S1 antigen fusion protein following binding to the dendritic cell’s Fc receptor. Processing of the Fc-Spike S1 antigen fusion protein in the cytoplasm of the dendritic cell may then result in presentation of the spike S1 protein via MHC I/II complexes on the dendritic cells.
  • FIG.4B is an exemplary schematic showing an overview of preparation and administration of a nanoparticle formulation of an example Fc-coronavirus antigen fusion protein expression vector configured to express an Fc-Spike S1 fusion protein (“Fc-Spike S1 DNA construct”).
  • Fc-Spike S1 DNA construct an Fc-Spike S1 fusion protein
  • the Fc region DNA 403 is shown, and a SARS-CoV-2 spike S1 protein domain fragment DNA 404 is shown.
  • the nanoparticle formulation is administered to a subject by intramuscular injection.
  • the Fc-Spike S1 antigen fusion protein (401 and 402) may then be expressed in the subject’s muscle cells transduced with the Fc-Spike S1 DNA construct.
  • the muscle cells may then secrete the Fc-Spike S1 antigen fusion protein.
  • the Fc region of the Fc-Spike S1 antigen fusion protein binds to the Fc receptor of dendritic cells, resulting in antigen presentation of the spike S1 protein by the dendritic cells.
  • the dendritic cells may internalize the Fc-Spike S1 antigen fusion protein following binding to the dendritic cell’s Fc receptor.
  • the Fc-Spike S1 DNA construct may be taken up by dendritic cells, which then express the Fc-Spike S1 antigen fusion protein.
  • FIG.4C is an exemplary schematic showing an overview of preparation and administration of a nanoparticle formulation of an example mRNA encoding an Fc-coronavirus antigen fusion protein configured to be translated into an Fc-Spike S1 fusion protein (“Fc-Spike S1 mRNA construct”).
  • Fc-Spike S1 mRNA construct an Fc-Spike S1 fusion protein
  • the nanoparticle formulation is administered to a subject by intramuscular injection.
  • the Fc-Spike S1 antigen fusion protein (401 and 402) is then expressed in the subject’s muscle cells transduced with the Fc-Spike S1 mRNA construct.
  • the muscle cells secrete the Fc-Spike S1 antigen fusion protein.
  • the Fc region of the Fc-Spike S1 antigen fusion protein binds to the Fc receptor of dendritic cells, resulting in antigen presentation of the spike S1 protein by the dendritic cells.
  • the dendritic cells may internalize the Fc-Spike S1 antigen fusion protein following binding to the dendritic cell’s Fc receptor.
  • the Fc-Spike S1 mRNA construct may be taken up by dendritic cells, which then express the Fc-Spike S1 antigen fusion protein. Processing of the Fc-Spike S1 antigen fusion protein in the cytoplasm of the dendritic cell may then result in presentation of the spike S1 protein via MHC I/II complexes on the dendritic cells.
  • the Fc-coronavirus antigen fusion proteins of the present disclosure may comprise coronavirus antigen epitopes that bind to both MHC class I (for the cytotoxic CD8 Tc cell response) and MHC class II (for the helper CD4 Th cell response).
  • the Fc-coronavirus antigen fusion proteins of the present disclosure may include motifs (e.g., GC rich regions) to allow immunogenicity and uptake directly by dendritic cells.
  • the Fc-coronavirus antigen fusion proteins of the present disclosure include an Fc region or fragment thereof which is capable of binding to macrophages, dendritic cells, and other antigen presenting cells (APCs) via the cells’ Fc receptors.
  • APCs antigen presenting cells
  • the Fc-coronavirus antigen fusion proteins may allow dendritic cells and other APCs to initiate both direct and cross-presentation of coronavirus protein epitopes to T cells.
  • a nucleic acid or vector encoding the Fc-coronavirus antigen fusion proteins may be taken up and processed directly by dendritic cells, whereas the Fc- coronavirus antigen fusion proteins (which may be produced from the DNA or mRNA in cells (e.g. muscle cells at the site of the injection, or administered to the subject as a fusion protein) binds to the Fc receptor on dendritic cells leading to cross-presentation.
  • the T cells will have an increased avidity, for example up to 100-fold increased potency as compared to direct or cross-presentation alone.
  • Immunization of a subject with a Fc-coronavirus antigen fusion protein, nucleic acids encoding the Fc-coronavirus antigen fusion proteins, and/or expression vectors comprising the nucleic acids encoding the Fc-coronavirus antigen fusion proteins described herein, may generate both cytotoxic CD8 T cell responses and helper T CD4 responses.
  • the Fc-coronavirus antigen fusion proteins of the present disclosure may be provided using a suitable expression system known to one skilled in the art.
  • coronavirus antigen proteins such as S, E, or M proteins or fragments thereof may be amplified from SARS-CoV-2 genomic DNA or cDNA, wherein the PCR primers used for amplification contain restriction endonuclease sites compatible with restriction endonuclease insertion sites for insertion in the DNA sequence, thereby creating a DNA sequence encoding for a Fc-coronavirus antigen fusion protein having an Fc region linked with a coronavirus antigen protein.
  • U.S. Patent 8,742,088 provides further guidance on generation of ImmunoBody ® constructs that may be adapted to provide the Fc-coronavirus antigen fusion proteins of the present disclosure.
  • the nucleic acids of the present disclosure may be used to stimulate an immune response against the coronavirus antigen protein (e.g., an S1 protein of a coronavirus such as SARS-CoV-2) in a patient such as a mammal, including human. Humoral (antibody) and/or helper and/or cytotoxic T cell responses may be stimulated.
  • the nucleic acids of the disclosure may be administered as a combination therapy, i.e., a nucleic acid encoding the light chain and nucleic acid encoding the heavy chain.
  • the nucleic acid may be administered intravenously, intradermally, intramuscularly, orally or by other routes.
  • nucleic acid will be dependent upon the properties of the agent employed, e.g. its binding activity and in vivo plasma half-life, the concentration of the polypeptide in the formulation, the administration route, the site and rate of dosage, the clinical tolerance of the patient involved, the pathological condition afflicting the patient and the like, as is well within the skill of the physician. For example, doses of 25-100 ⁇ g of nucleic acid per patient per administration may be preferred, although dosages may range from about 10 ⁇ g to 1 mg per dose.
  • the present disclosure provides a host cell containing a nucleic acid as disclosed herein.
  • the nucleic acid may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences that promote recombination with the genome in accordance with standard techniques.
  • the nucleic acid may be on an extra- chromosomal vector within the cell, or otherwise identifiably heterologous or foreign to the cell.
  • the Fc-coronavirus antigen fusion proteins, nucleic acids encoding the Fc-coronavirus antigen fusion proteins, and expression vectors comprising the nucleic acids encoding the Fc- coronavirus antigen fusion proteins described herein, and compositions and pharmaceutical formulations thereof described herein have the properties and functions of a vaccine.
  • the Fc- coronavirus antigen fusion protein, nucleic acid encoding the Fc-coronavirus antigen fusion protein, and/or an expression vector comprising a nucleic acid encoding a Fc-coronavirus antigen fusion protein described herein, and/or a composition or pharmaceutical formulation thereof described herein may be administered as a vaccine to a subject.
  • the present disclosure provides a vaccine comprising a Fc-coronavirus antigen fusion protein, a nucleic acid encoding the Fc-coronavirus antigen fusion protein, and/or an expression vector comprising a nucleic acid encoding a Fc- coronavirus antigen fusion protein described herein, and/or a composition or pharmaceutical formulation thereof described herein.
  • the present disclosure provides a method of administering to a subject a vaccine for preventing or treating a coronavirus infection in a subject, wherein the vaccine comprises a Fc-coronavirus antigen fusion protein, a nucleic acid encoding the Fc-coronavirus antigen fusion protein, and/or an expression vector comprising a nucleic acid encoding a Fc-coronavirus antigen fusion protein described herein, and/or a composition or pharmaceutical formulation thereof described herein.
  • FIG.5 is an exemplary schematic showing a post-immunization response to SARS- CoV-2 in a subject administered with a Fc-coronavirus antigen fusion protein, a nucleic acid encoding the Fc-coronavirus antigen fusion protein, and/or an expression vector comprising a nucleic acid encoding a Fc-coronavirus antigen fusion protein described herein, or a pharmaceutical formulation thereof described herein.
  • MHC Class I/II antigen presentation e.g., presentation of a SARS-CoV-2 spike S1 protein fragment of the Fc- coronavirus antigen fusion protein
  • dendritic cells in the subject generates both cytotoxic CD8 Tc cell responses and helper Th CD4 responses.
  • CD4-positive T-cells activate B-cells to produce neutralizing antibodies against the SARS-CoV-2 spike S1 protein
  • CD8-positive cytotoxic T-cells kill cells infected with SARS-CoV-2.
  • the Fc-coronavirus antigen fusion protein has a serum or plasma in vivo half-life that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours, or more. [00177] In one embodiment, the Fc-coronavirus antigen fusion protein has a serum or plasma in vivo half-life that is at least 10-24 hours, or at least 1, 2, 3, 4, 5 or 6 days or longer. [00178] In one embodiment, the Fc-coronavirus antigen fusion protein comprises an immunoglobulin Fc region comprising a human immunoglobulin Fc region.
  • the Fc-coronavirus antigen fusion protein comprises an immunoglobulin Fc region having effector function, or having reduced effector function.
  • the Fc-coronavirus antigen fusion protein comprises an immunoglobulin Fc region wherein the immunoglobulin Fc region comprises: at least one CH2 domain from an IgG1 immunoglobulin; at least one CH3 domain from an IgG1 immunoglobulin; a CH2 and CH3 domain from an IgG1 immunoglobulin; at least one CH2 domain from an IgG4 immunoglobulin; at least one CH3 domain from an IgG4 immunoglobulin; a CH2 and CH3 domain from an IgG4 immunoglobulin; a CH2 domain from an IgG1 immunoglobulin and a CH3 domain from an IgG4 immunoglobulin; or a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin.
  • the Fc-coronavirus antigen fusion protein comprises an immunoglobulin Fc region wherein the immunoglobulin Fc region binds a type I Fc receptor, including human: Fc ⁇ RI (CD64), Fc ⁇ RIIA (CD32a), Fc ⁇ RIIB (CD32b), Fc ⁇ RIIC (CD32c), Fc ⁇ RIIIA (CD16a) and/or Fc ⁇ RIIIB (CD16b).
  • the Fc-coronavirus antigen fusion protein comprises an immunoglobulin Fc region which mediates antibody-dependent cell-mediated cytotoxicity (ADCC) activity.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the Fc-coronavirus antigen fusion protein comprises an immunoglobulin Fc region which mediates antibody-dependent cellular phagocytosis (ADCP) activity.
  • the Fc-coronavirus antigen fusion protein comprises an immunoglobulin Fc region wherein the immunoglobulin Fc region comprises an interchain steric complementarity comprising a knob or hole structure (Ridgeway 1996 Protein Engineering 9(7):617-621).
  • the Fc-coronavirus antigen fusion protein comprises an immunoglobulin Fc region wherein the immunoglobulin Fc region comprises a hinge region or lacks a hinge region.
  • the hinge region is joined to the N-terminus of the immunoglobulin Fc region.
  • the Fc-coronavirus antigen fusion protein comprises an affinity purification tag (e.g., His-tag) at the N-terminus or C-terminus.
  • the Fc-coronavirus antigen fusion protein comprises an immunoglobulin Fc domain comprising a mutation that creates a protrusion (e.g., knob) on one chain and a socket (e.g., hole) on the other chain so that the protrusion and socket associate with each other.
  • the protrusion and socket promote association between the polypeptide chains (e.g., heavy chains) to promote dimerization.
  • one of the polypeptide chains is mutated by substituting a small amino acid with a larger one to create a protrusion (e.g., in the first or second half Fc region).
  • another polypeptide chain is mutated by substituting a larger amino acid with a smaller one to create a socket.
  • Fc domain knob-in-hole mutations comprise a substitute mutation at any one Fc location or any combination of two or more Fc locations selected from a group consisting of T366, L368, T394, F405, Y407 and K409 (numbering is based on Kabat system).
  • Fc domain knob-in-hole mutations comprise any one or any combination of two or more of the following mutations: T366Y, T366W, T366S, L368A, T394S, T394W, F405A, F405W, Y407A, Y407V, Y407T (numbering based on Kabat system).
  • Neutralizing antibodies generated by an immunized subject may specifically bind to the ectodomain of a membrane protein on any coronavirus, where the membrane protein may include an S (spike) protein, such as having an S1 and/or S2 subunits, an M (membrane) protein, or an E (envelope) protein.
  • Coronavirus includes, but is not limited to, SARS-CoV, MERS-CoV and SARS-CoV-2.
  • SARS-CoV and SARS-CoV-2 the S1 subunit carries a receptor binding motif located in a receptor binding domain (RBD) which binds a target receptor angiotensin converting enzyme 2 (ACE2) protein on a target cell.
  • RBD receptor binding domain
  • ACE2 target receptor angiotensin converting enzyme 2
  • MERS-CoV the S1 subunit binds dipeptidyl peptidase-4 (DPP4) on a target cell.
  • DPP4 dipeptidyl peptidase-4
  • the neutralizing antibodies that bind a coronavirus spike protein can bind an epitope on the S1 subunit that binds a target receptor ACE2 or DPP4 on a target cell.
  • the neutralizing antibody can bind a receptor binding motif located in a receptor binding domain (RBD) of a coronavirus S1 subunit and block binding between a coronavirus S1 subunit and its target receptor ACE2 or DPP4.
  • RBD receptor binding domain
  • the neutralizing antibody can block attachment of coronavirus to the target cell and prevent viral entry into the target cell.
  • the coronavirus S1 subunit comprises a cell surface coronavirus S1 subunit antigen or a soluble coronavirus S1 subunit antigen. In one embodiment, the coronavirus S1 subunit antigen comprises receptor binding motif. In one embodiment, the coronavirus S1 subunit antigen comprises receptor domain (RBD). In one embodiment, the coronavirus S1 subunit antigen comprises a human or non-human coronavirus S1 subunit antigen.
  • the present disclosure provides methods for treating a subject having a coronavirus infection, the method comprising: administering to the subject an effective amount of Fc- coronavirus antigen fusion proteins, nucleic acids encoding the Fc-coronavirus antigen fusion proteins, and/or expression vectors comprising the nucleic acids encoding the Fc-coronavirus antigen fusion proteins described herein, and/or a composition of pharmaceutical formulations thereof described herein.
  • the Fc-coronavirus antigen fusion proteins, nucleic acids encoding the Fc-coronavirus antigen fusion proteins, and/or expression vectors comprising the nucleic acids encoding the Fc-coronavirus antigen fusion proteins described herein, and/or a composition of pharmaceutical formulations thereof described herein can be administered to the subject in combination with at least one anti-viral agent and/or at least one viral entry inhibitor.
  • One skilled in the art can routinely select an appropriate anti-viral agent or viral entry inhibitor to be administered with the Fc-coronavirus antigen fusion proteins, nucleic acids encoding the Fc- coronavirus antigen fusion proteins, and/or expression vectors comprising the nucleic acids encoding the Fc-coronavirus antigen fusion proteins described herein, and/or a composition of pharmaceutical formulations thereof described herein.
  • the anti-viral agent and/or the viral entry inhibitor can be administered prior to, during, or after, administration of the Fc-coronavirus antigen fusion proteins, nucleic acids encoding the Fc-coronavirus antigen fusion proteins, and/or expression vectors comprising the nucleic acids encoding the Fc-coronavirus antigen fusion proteins described herein, and/or a composition of pharmaceutical formulations thereof described herein.
  • a subject can be provided with one, two, three, four, or more doses of a vaccine composition as provided herein that includes an S1-Fc fusion protein or a nucleic acid molecule encoding an S1-Fc fusion protein.
  • the doses may be separated in time by weeks or months, for example, or may be given yearly or in alternate years.
  • Doses subsequent to a first dose may include one or more S1-Fc fusion proteins having a different S1 protein sequence, for example, may have an S1 protein moiety of a different coronavirus variant than was the source of the S1 protein moiety of an earlier vaccine dose.
  • a single dose may include more than one S1-Fc fusion protein (or more than one nucleic acid sequence or molecule encoding an S1-Fc fusion protein), where the different S1-Fc fusion proteins include different S1 protein sequences.
  • the S1 protein sequence in various embodiments is a SARS-CoV-2 S1 sequence.
  • the subject may be a subject at risk of becoming infected with coronavirus and may be a human subject.
  • the subject treated with a composition as provided herein may be infected with a coronavirus, such as SARS-CoV-2.
  • the present disclosure provides therapeutic compositions comprising any of the Fc- coronavirus antigen fusion proteins, nucleic acids encoding the Fc-coronavirus antigen fusion proteins, and/or expression vectors comprising the Fc-coronavirus antigen fusion proteins described herein in an admixture with a pharmaceutically-acceptable adjuvant.
  • adjuvant refers to a compound or mixture that enhances the immune response to an antigen.
  • An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al., Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p.384). Often, a primary challenge with an antigen alone, in the absence of an adjuvant, will fail to elicit a humoral or cellular immune response.
  • Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • the adjuvant is pharmaceutically acceptable.
  • the term “molecular adjuvant” is defined as a protein, lipid, nucleic acid, carbohydrate, or chemical compound for which dendritic cells (DCs), macrophages, B cells, T cells, and/or NK cells have a known receptor whose occupancy leads to a defined sequence of intracellular signal transduction and a change in the phenotype resulting in an improvement in the quantity or quality of the ensuing immune response.
  • DCs dendritic cells
  • macrophages B cells, T cells, and/or NK cells have a known receptor whose occupancy leads to a defined sequence of intracellular signal transduction and a change in the phenotype resulting in an improvement in the quantity or quality of the ensuing immune response.
  • the cells as described above are collectively referred to as “immune cells.”
  • the term “antigen-presenting cell” or “APC” refers to those highly specialized cells that can process antigens and display their peptide fragments on the cell surface together with molecules required for lymphocyte activation.
  • the main antigen-presenting cells for T-cells are DC, macrophages, and B-cells, whereas the main antigen-presenting cells for B-cells are follicular dendritic cells.
  • dendritic cell or “DC” is defined as those APCs that are found in T-cell areas of lymphoid tissues. (Banchereau et al., Nature 392:245-251, 1998).
  • DCs are a sparsely distributed, migratory group of bone-marrow-derived leukocytes that are specialized for the uptake, transport, processing and presentation of antigens to T-cells.
  • Non-lymphoid tissues also contain DCs, but these do not stimulate T-cell responses until they are activated and migrate to lymphoid tissues.
  • dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes, dendrites, visible in vitro); their ability to take up, process and present antigens with high efficiency; and their ability to activate naive T- cell responses.
  • the term “immunogenic” refers to the ability of an antigen to elicit an immune response, either humoral or cell mediated.
  • An “immunogenically effective amount” as used herein refers to the amount of antigen sufficient to elicit an immune response, either a cellular (T cell) or humoral (B cell or antibody) response, as measured by standard assays known to one skilled in the art.
  • the effectiveness of an antigen as an immunogen can be measured either by proliferation assays, by cytolytic assays, such as chromium release assays to measure the ability of a T cell to lyse its specific target cell, or by measuring the levels of B cell activity by measuring the levels of circulating antibodies specific for the antigen in serum, or by measuring the number of antibody spot-forming cells in the spleen.
  • the level of protection of the immune response may be measured by challenging the immunized host with a replicating virus or cell containing the antigen that has been injected.
  • the antigen to which an immune response is desired is a virus or a tumor cell
  • the level of protection induced by the “immunogenically effective amount” of the antigen is measured by detecting the level of survival after virus or tumor cell challenge of the animals.
  • protection can also be measured as the reduction in viral replication or tumor growth following challenge of the animals.
  • the term “vaccine” includes an immunostimulatory treatment designed to elicit an immune response against an antigen, whether administered prophylactically or for the treatment of an already existing condition.
  • a vaccine may elicit acquired immunity to a particular infectious disease in a subject administered with the vaccine.
  • a vaccine typically contains an agent that resembles a disease-causing microorganism or virus or a portion thereof and is often made from weakened or killed forms of a microorganism or virus, its toxins, or one or more of its surface proteins.
  • the agent may stimulate a subject’s immune system to recognize the agent as a threat, destroy and/or neutralize it, and to further recognize and destroy and/or neutralize any of the microorganisms or viruses having one or more components resembling the agent that it may encounter.
  • Vaccines can function as prophylactic (e.g., to prevent or ameliorate the effects of a future infection in a subject by a microorganism or virus), or therapeutic (e.g., to treat a subject already infected by a microorganism or virus).
  • the administration of a vaccine to a subject is herein referred to as vaccination or immunization.
  • a “genetic vaccine” relates to the use of genetic material (e.g., nucleic acid sequences) encoding a protein of interest which is used as an immunizing agent.
  • This term includes, but is not limited to, nucleic acids transported into host cells within viruses or viral vectors (e.g., modified forms of adenoviruses, poxviruses, rhabdoviruses, alphaviruses, herpesviruses, influenza viruses, retroviruses, or lentiviruses) or bacteria (e.g., modified forms of Salmonella species, Listeria species, or mycobacterial species).
  • the term also includes, but is not limited to, nucleic acids administered directly, such as plasmid DNA, which is referred to as a “DNA vaccine.”
  • DNA encoding a protein of interest can also be administered in a non-plasmid form as a linear, double-stranded molecule as a minimalist expression construct.
  • RNA messenger RNA
  • a nucleic acid encoding a protein of interest is administered as a DNA vaccine comprising a plasmid or circular or a linear DNA molecule.
  • non-viral gene delivery The general class of gene or nucleic acid delivery that does not rely on microbial delivery, usually with viruses, has been called ‘non-viral gene delivery’.
  • An alternative designation is ‘synthetic vectors’ or ‘artificial viruses’ for gene delivery. These typically involve polymers which form complexes, nanoparticles (defined as less than 1 micron in diameter), or even microparticles (defined as 1 micron in diameter or greater) with DNA plasmids and other nucleic acids.
  • polyethylenimine can be used as a delivery agent.
  • PEI polyethylenimine
  • PEI is one of the most well established polymers for DNA delivery. PEI is positively charged which allows it to complex with negatively charged DNA. In its mannosylated form, it directs plasmid DNA into resting macrophages and dendritic cells which endocytose it using their mannose receptors (sold as Man jetPEI by QBioGene, Inc.).
  • PEI effectively buffers the normally acidic pH in endosomal vesicles, thereby serving as a “proton sponge” that prevents acid damage to the DNA cargo.
  • cationic lipids can be used as delivery agents for nucleic acids.
  • Cationic lipids and related compounds have been used to enhance the effectiveness of vaccines and the expression of genes encoded by nucleic acids in cells.
  • DNA or RNA can also be encapsulated into microspheres comprised of an aminoalkyl glucosaminide 4-phosphate (AGP).
  • AGP aminoalkyl glucosaminide 4-phosphate
  • lipid-DNA complexes have direct inflammatory activity that is immunostimulatory and augments the antitumor effect of the plasmid DNA.
  • DNA was formulated with DOTMA:DOPE (Avanti Polar Lipids, Alabaster, AL) prior to injection (see for example, Li et al. (2013) Int J Cardiol.168:3659-3664).
  • DOTMA:DOPE Advanti Polar Lipids, Alabaster, AL
  • Cationic polymers such as poly-L-lysine, poly-L-glutamate, or block co-polymers may also be delivery agents for nucleic acids.
  • poly-L-arginine was found to synergize with oligodeoxynucleotides containing CpG-motifs (CpG-ODN) for enhanced and prolonged immune responses and prevented the CpG-ODN-induced systemic release of pro-inflammatory cytokines.
  • Pharmaceutical compositions comprising an antigen, an immunogenic oligodeoxynucleotide containing CpG motifs (CpG-ODN), and a polycationic polymer are known in the art.
  • CpG-ODN refers to a single-stranded oligodeoxynucleotide produced using phosphorothioate linkages and containing an unmethylated cytosine-guanosine motif.
  • dendrimeric polymer delivery agents include Starburst polymers (Dow Chemical).
  • poloxamine delivery agents may be used, including both poloxamer and polxamine compositions.
  • Poly-lactide-co-glycolide (PLGA) is used to make surgical sutures. It can also be formulated to deliver vaccine components. For example, a plasmid DNA encoding an HIV protein was formulated with PLGA with cetyl trimethyl ammonium bromide (CTAB), and the resulting PLG-CTAB-DNA microparticles were found to elicit an improved immune response. PLGA can also be combined with polyethylenimine (PEI) to make microspheres for DNA delivery.
  • PEI polyethylenimine
  • Microparticles formed from PLGA and other materials that incorporate DNA and TLR agonists have been developed by Chiron.
  • the polymer component was selected (1) from the group consisting of a poly(a-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a polyanhydride and a polycyanoacrylate and (2) a detergent, and used to deliver a polynucleotide, a polynucleoside, a polypeptide, an immunomodulator, an antigen, and an adjuvant.
  • Another type of gene delivery polymer is formed from beta-amino esters. Agents in this series, such as C32, U28, and JJ28, were identified using a combinatorial library approach.
  • C32 may be especially useful for tumor immunotherapy as it has been shown to increase plasmid DNA gene expression in tumors 4-fold.
  • the polymer is first dissolved in DMSO (100 mg/ml).
  • DNA 50 ⁇ g
  • DNA is then suspended in 25 ⁇ l of 25 mM sodium acetate buffer (pH 5.0) and mixed with the polymer solution (1,500 ⁇ g or 25 ⁇ g), also diluted in 25 ⁇ l of 25 mM sodium acetate buffer (pH 5.0).
  • 10 ⁇ l of 30% glucose in PBS is added to the 50- ⁇ l polymer/DNA mixture.
  • DNA vaccination one approach for targeting DNA to DCs is to adsorb it onto cationic poly(lactic-co-glycolic acid) (PLGA) particles, which then targets the DNA to phagocytic APCs and enhances CD8+ T cell responses and antibody titers by 100-fold and 1,000-fold respectively.
  • PLGA cationic poly(lactic-co-glycolic acid)
  • PLGA microparticles can produce an extremely low pH microclimate (pH ⁇ 3.5) after only 3 days in an aqueous environment. This level of acidity has been shown to severely reduce the activity of plasmid DNA. PLGA microparticles also remain confined to phagolysosomal vesicles, which limits gene expression in the transfected DCs.
  • PBAE pH-sensitive poly- amino ester
  • PBAE/PLGA mixtures are preferred: 15% PBAE/85% PLGA and 25% PBAE/75% PLGA. Although the 25% PBAE mixture was significantly more stimulatory for DCs in vitro, the 15% and 25% PBAE mixtures were nearly equivalent when used for intradermal DNA vaccination.
  • the final lyophylized microparticle preparation is resuspended for use in PBS at a concentration of 10 ⁇ g/50 ⁇ l, where the 10 ⁇ g refers to the amount of DNA in the particles.
  • Peritumoral injections of “naked” plasmid DNA for IL-12 have been used for antitumor treatment in mice.
  • PAGA poly[alpha-(4-aminobutyl)-1-glycolic acid]
  • WSLP water-soluble lipopolymer
  • IL-12 interleukin-12
  • Polyethylenimine-based vesicle-polymer hybrid gene delivery as another way to deliver plasmid DNA expression vectors, including the use of poly(propylenimine) dendrimers as delivery agents
  • polyethylene glycol (PEG) copolymers were found to improve plasmid DNA delivery, including various kinds of polymers that can be used for the controlled release of plasmid DNA and other nucleic acids.
  • Such molecules include poly(lactic acid) and its derivatives, PEGylated poly(lactic acid), poly(lactic-co-glycolic acid) and its derivatives, poly(ortho esters) and their derivatives, PEGylated poly(ortho esters), poly(caprolactone) and its derivatives, PEGylated poly(caprolactone), polylysine and its derivatives, PEGylated polylysine, poly(ethylene imine) and its derivatives, PEGylated poly(ethylene imine), poly(acrylic acid) and its derivatives, PEGylated poly(acrylic acid), poly(urethane) and its derivatives, PEGylated poly(urethane), and combinations of all of these.
  • Self-assembling particle delivery systems are often composite substances, including self-assembling particles that can be made as polyplexes between nucleic acids and a hybrid polymer composed of mannose-polyethylene glycol (PEG)-PAMAM-G3.0, -G4.0, or -G5.0, where PAMAM refers to a branching dendrimer of poly(amidoamine) and the G indicates the number of branches.
  • PEG mannose-polyethylene glycol
  • PAMAM refers to a branching dendrimer of poly(amidoamine)
  • the G indicates the number of branches.
  • mannose is used to form the outer shell of this nanoparticle because immature DCs and macrophages avidly take up mannosylated substances using their mannose receptors (which are downregulated upon DC maturation) and possibly other mannose-binding receptors such as DC-SIGN.
  • the resulting polyplexes of a luciferase plasmid with Man-PEG-PAMAM-G5.0 or -G6.0 resulted in 4-fold more gene expression than plasmid complexation with commercially available JetPEI (QBioGene, Inc.), whereas the G4.0 polymer was equivalent to JetPEI.
  • the ⁇ G6.0 polymer was mildly toxic to these cells, but the G5.0 polymer was essentially nontoxic at concentrations 100 ⁇ greater than the toxic dose of JetPEI.
  • Polymeric gene delivery systems need not be biologically inert. Indeed, they may be even more effective if they are immunostimulatory in their own right, in which case they may be preferred for vaccination and tumor immunotherapy. For example, polymers many have intrinsic anticancer effects. Polypropylenimine (PPI) dendrimers have been observed to augment the antitumor effects of TNF plasmid DNA.
  • PPI Polypropylenimine
  • PPI dendrimers alone had some antitumor effects, as did linear polyethylenimine (PEI) and polyamidoamine dendrimer, including that PPI dendrimers induce gene expression in transfected cells, a property that could be useful in immunostimulation or antitumor activity.
  • PEI linear polyethylenimine
  • polyamidoamine dendrimer including that PPI dendrimers induce gene expression in transfected cells, a property that could be useful in immunostimulation or antitumor activity.
  • PLGA lactic-co-glycolic acid
  • PBAE poly-amino ester
  • nucleic acids are delivered by electroporation.
  • Electroporation uses electrical pulses to introduce proteins, nucleic acids, lipids, carbohydrates, or mixtures thereof into the host to produce an effect.
  • a typical use of electroporation is to introduce a nucleic acid into the host so that the protein encoded by the nucleic acid is efficiently produced.
  • Ichor (San Diego, CA, USA) manufactures a device (TriGrid) for in vivo delivery of nucleic acids to tissue such as muscle tissue via electroporation.
  • nucleic acids are delivered by particle bombardment.
  • PowderJect Novartis Pharmaceutical Corporation
  • a polynucleotide may be administered/delivered as “naked” DNA, for example as described in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993.
  • the uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
  • a composition can be delivered via a particle bombardment approach, many of which have been described.
  • gas- driven particle acceleration can be achieved with devices such as those manufactured by PowderJect Pharmaceuticals PLC (Oxford, UK) and PowderJect Vaccines Inc. (Madison, Wis.), both now part of the Chiron division of Novartis, some examples of which are described in U.S. Pat. Nos.5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No.0500799.
  • Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Pat. No.5,466,468).
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., vegetable oils
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion
  • isotonic agents for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035- 1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Moreover, for human administration, preparations will of course preferably meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologics standards. [00222] The SARS-CoV-2 virus was first reported in December 2019 and has infected over 250,000 people and caused more than 10,000 deaths worldwide to March 2020.
  • Vaccines are urgently needed for prevention of this contiguous virus.
  • immunization has been achieved using live, weakened forms of the virus, or part or whole of the virus once it has been inactivated by heat or chemicals. These methods have drawbacks.
  • the live form can continue to evolve in the host, for example, potentially recapturing some of its virulence and making the recipient sick, while higher or repeat doses of the inactivated virus are required to achieve the necessary degree of protection.
  • Whole virus vaccine also may produce non-neutralizing antibodies; in some instances, the presence of specific antibodies can be beneficial to the virus. This activity is known as antibody-dependent enhancement (ADE) of virus infection.
  • ADE antibody-dependent enhancement
  • the ADE of virus infection is a phenomenon in which virus-specific antibodies enhance the entry of virus.
  • the present disclosure provides coronavirus antigen proteins such as coronavirus spike S1 protein and viral epitopes thereof that may be associated with neutralization may be used as vaccines with minimum risk for ADE.
  • coronavirus antigen proteins such as coronavirus spike S1 protein and viral epitopes thereof that may be associated with neutralization may be used as vaccines with minimum risk for ADE.
  • Previous studies have demonstrated that targeting immunogens to Fc ⁇ R on antigen- presenting cells (APCs) can selectively elicit uptake by APCs and increase cellular immunity in vitro and in vivo.
  • the Fc can be a human IgG Fc fragment.
  • nucleic acids encoding the Fc-coronavirus antigen fusion proteins can include codon-optimized nucleic acids.
  • the Fc-coronavirus antigen fusion protein may include a linker adapted to link an Fc or fragment thereof to an Fc protein or fragment thereof. Accordingly, the Fc or fragment thereof may be linked directly or indirectly to the coronavirus antigen protein or fragment thereof.
  • a nucleic acids encoding an Fc-coronavirus antigen fusion protein or a vector comprising the nucleic acid may be cloned to a plasmid vector and propagated by cell culture, e.g. bacterial cell culture techniques known in the art, to produce a large quantity of the nucleic acid or vector.
  • the nucleic acid or vector may be formulated in a nanoparticle carrier for delivery to a subject, thereby administering the encoded Fc-coronavirus antigen fusion protein as a DNA vaccine.
  • a nucleic acid encoding a Fc-coronavirus antigen fusion protein may be cloned into an mRNA transcription vector to produce mRNA encoding the Fc- coronavirus antigen fusion protein, e.g. in vitro, and the mRNA may be then formulated into nanoparticles for administration, e.g. intramuscular injection.
  • a Fc-coronavirus antigen fusion protein may be produced in cell culture, e.g. in a suspension of CHO-K1 cells.
  • the Fc-coronavirus antigen fusion protein produced may be purified.
  • purified Fc-coronavirus antigen fusion protein may be formulated e.g. emulsified with an adjuvant.
  • the immunogenicity of a Fc-coronavirus antigen fusion protein, nucleic acids encoding the Fc-coronavirus antigen fusion protein, vectors, compositions and/or formulations thereof may be assessed in preclinical studies, e.g. in mice, e.g.
  • Th1, Th2, Th17, and T-reg cytokine patterns may be evaluated e.g. using an ELISA method to observe whether the Fc-coronavirus antigen fusion protein will induce a more effective Th1 immune response (e.g., IFN- ⁇ , and IL-12) with an absent, low or very low increase in IL-17 and IL-4 and an absent, low or very low increase in TGF- ⁇ .
  • Th1 immune response e.g., IFN- ⁇ , and IL-12
  • the Fc-coronavirus antigen fusion protein may allow selective uptake into APCs, induce cross-presentation of coronavirus antigen proteins or fragments thereof and elicit a robust anti-SARS-CoV-2 response in context of Th1/Th2 and Th17/T-reg balances, which may allow an immune response in a subject providing effective vaccination of a subject, treatment of a coronavirus infection in a subject and minimization or prevention of adverse immune-related effects in the subject.
  • the present disclosure provides administration to a subject of recombinant Fc-coronavirus antigen fusion proteins, nucleic acids (e.g., dsDNA or mRNA) encoding the Fc-coronavirus antigen fusion proteins, and/or vectors comprising the nucleic acids.
  • the Fc-coronavirus antigen fusion proteins can engage with Fc-receptors (FcRs).
  • FcRs Fc-receptors
  • Use of antibody Fc domains as a fusion partner with coronavirus antigen protein or fragment thereof confers stability to the coronavirus antigen protein or fragment thereof and allows for specific targeting of the coronavirus antigen protein or fragment thereof to innate immune effector cells.
  • the coronavirus antigen protein is processed and presented to lymphocytes in the periphery.
  • the Fc-coronavirus antigen fusion proteins described herein can utilize an established antigen detection cellular mechanism to bring non-infectious immunogens into immediate proximity with APCs.
  • the Fc-coronavirus antigen fusion proteins may be engineered to target activating FcRs expressed on the surface of dendritic cells.
  • the Fc-coronavirus antigen fusion proteins may be engineered to minimize engagement of inhibitory FcRs.
  • the Fc-coronavirus antigen fusion proteins described herein may comprise one or more SARS-CoV-2 Spike protein S1 domain fragments, a Fc gamma mutant variant, and optionally inter-domain linkers adapted to link one or more domains, proteins, or fragments thereof.
  • nanoparticle formulations such as those described herein may be used for delivery to subjects of the Fc-coronavirus antigen fusion proteins, nucleic acids and/or vectors described herein as a vaccine candidates, optionally in combination with one or more adjuvants.
  • Fc-coronavirus antigen fusion proteins nucleic acids, vectors, and/or compositions described herein may result in an increased Th1 polarization response and low or absent mixed Th1/Th2 or predominantly Th2 responses.
  • the Fc-coronavirus antigen fusion proteins described herein may enhance APC-specific targeting and enhance Th1 immunization and prevent tolerance induction.
  • the Fc-coronavirus antigen fusion proteins, nucleic acids, vectors, and compositions described herein may be used as vaccines for treating a coronavirus infection
  • Fc-coronavirus antigen fusion proteins having suitable vaccine potency, safety, and scale-up manufacturing attributes can be investigated as preclinical leads, followed by development as clinical therapeutic agents.
  • Existing DNA vaccines in development typically use plasmid DNA. The successful transfection of antigen presenting cells (APC) in vivo was previously demonstrated which resulted in the induction of primary adaptive immune responses.
  • plasmid DNA vaccines Due to the good biocompatibility of plasmid DNA, their cost-efficient production and long shelf life, many researchers have investigated development of DNA vaccine-based immunotherapeutic strategies for treatment of infections and cancer, and also autoimmune diseases and allergies.
  • existing plasmid DNA vaccines have shown poor immunogenicity in human subjects so far since poor transfection efficiency and protein expression, even despite using several optimization steps that improve DNA transfection efficiency including the introduction of DNA-complexing nano- carriers aimed to prevent extracellular DNA degradation, enabling APC targeting, and enhanced endo/lysosomal escape of DNA.
  • the nucleic acids encoding Fc-coronavirus antigen fusion proteins, and vectors comprising the nucleic acids described herein may be prepared as linearized DNA molecules, such as double-stranded linearized DNA molecules.
  • the DNA may be chemical modified DNA.
  • the linearized DNA molecules may be prepared for example as shown in FIG.6.
  • the linearized DNA has the advantages of being stable, easy to manufacture and may circumvent potential inhibitory effects of a plasmid backbone DNA such as antibiotics resistance genes and gene sequences in the plasmid DNA used for propagation in bacteria.
  • the linearized DNA molecule comprising nucleic acids encoding an Fc-coronavirus antigen fusion protein, or an expression vector comprising the nucleic acids, formulated with a nanoparticle carrier described herein, allows increased transfection efficiency.
  • a nuclear localization sequences e.g. a virus-derived nuclear localization sequence, may be included in a nucleic acid sequence comprising an Fc-coronavirus antigen fusion protein described herein, thereby allowing increased nuclear entry of the nucleic acid.
  • the Fc-coronavirus antigen fusion proteins, nucleic acids encoding the Fc-coronavirus antigen fusion proteins, vectors, compositions, and formulations provide improved vaccines, including improved DNA-based vaccines, mRNA-based vaccines, and/or fusion protein-based vaccines useful for treating a coronavirus infection in a subject.
  • EXAMPLES [00243] In the following examples, an S1-Fc fusion protein serves as the antigen for immunization.
  • the S1-Fc fusion protein may be delivered as a protein or a DNA molecule encoding the S1-Fc fusion protein.
  • the fusion protein is designed to include the human immunoglobulin Fc region to engage with Fc ⁇ R + expressing cells such as dendritic cells, macrophages, and B cells.
  • the S1 domain of the SARS-CoV-2 spike protein serves as the immunogenic antigen to mount anti-viral humoral and adaptive immune responses.
  • Example 1 Expression of double-stranded DNA encoding S1-Fc fusion protein in murine muscle cells in vitro and in vivo.
  • a double stranded DNA (dsDNA) expression construct was designed to include a promoter active in mammalian cells operably linked to an S1-Fc encoding sequence which included at its N-terminus a signal peptide for secretion (3,560 bp; FIG.1).
  • the expression construct was produced as a linear dsDNA fragment (FIG.7A), labeled with Cy3, and delivered to cultured murine NIH/3T3 fibroblasts and C2C12 myoblasts by electroporation. Uptake of the labeled S1-Fc construct was analyzed by flow cytometry, which demonstrated that the uptake of the linear dsDNA was very efficient (FIG.7B). Confocal microscopy of transfected cells validated the flow cytometry results (FIG.7C). Subsequently, S1-Fc protein was detected in muscle cell culture supernatant (FIG.7D).
  • the dsDNA construct formulated with a cationic lipid transfection reagent (1:1 DOTAP:DOPE, Avanti Polar Lipids, Alabaster, AL., USA) was introduced into muscle tissue of mice by injection.
  • S1-Fc protein was detected in the biceps femoris muscle 47 days after delivery, which is indicative of continuous S1-Fc synthesis and secretion (FIG.8A).
  • S1-Fc protein was found to be internalized by dendritic cells in the biceps femoris, suggesting an operative targeting (FIG.8B).
  • Example 2 Delivery of recombinant rS1-Fc protein.
  • Recombinant rS1-Fc protein chimera (also referred to herein as rS1-Fc fusion protein) was produced in CHO cells and subjected to analytical size-exclusion chromatography showing a distinct elution of rS1-Fc monomer (84.5%). However, a minor molecular population (13.2%) eluted as self-assembled (rS1-Fc)n aggregate (FIG.9A) which might by due to intrinsic oligomerization activity of the SARS-CoV-2 spike protein. Notably, gel electrophoretic analysis showed reduced rS1-Fc aggregation activity in repeated protein production (FIG.9B).
  • rS1-Fc immunologic identity was validated by immunodetection of S1 and Fc upon Western blotting procedure (FIG.10A).
  • rS1-Fc was conjugated to IRDye® 800RS NHS Ester (Li-cor) using amine-reactive crosslinker chemistry. Briefly, S1-Fc antigen (2.5-3.0 mg/ml) was reacted with 5 equivalents of IRDye® 800RS NHS Ester in 1x DPBS pH 7.4 containing 5% anhydrous DMSO for 3 hours with gentle rotation at room temperature.
  • the reaction mixture was subjected to PD-Minitrap G-25 column (GE Healthcare) to remove unreacted dyes according to the manufacturer’s instructions.
  • the conjugate underwent buffer exchange three times into 1x DPBS pH 7.4 using a 4-ml Amicon Ultra centrifugal filter (30 kDa MWCO, Millipore).
  • the conjugate was characterized using SDS- PAGE, SEC HPLC, and BCA Assay.
  • rS1-Fc Chimeric rS1-Fc was found to be readily internalized by murine RAW 264.7 macrophages in vitro (FIG.10B) and could be found throughout the cell cytoplasm as well as the cell nucleus as demonstrated by confocal laser-scanning microscopy (FIG.10C). Significantly, rS1-Fc was found to bind ACE2 as assessed by ELISA (FIG.10D). The S1 domain of the SARS- CoV-2 spike protein binding to host receptor ACE2 is critical to the initiation of host cell infection.
  • rS1-Fc binding to ACE2 is indicative operative functionality and may also indicate a decoy activity by competition with viral spike protein for ACE2 binding, potentially reducing host cell virus susceptibility.
  • Targeted delivery of rS1-Fc to antigen presenting cells (APCs) is thought to be mediated by Fc::Fc ⁇ R interaction, which requires rS1-Fc homing to secondary lymphoid organs once administered.
  • IRDye800-rS1-Fc was also injected (10 ⁇ l at 1 mg/ml concentration) intramuscularly into the biceps femoris of C57BL/6 mice.
  • splenic APCs including CD11c + dendritic cells, F4/80 + macrophages and CD19 + B cells
  • rS1-Fc fluorescently labeled rS1-Fc at a concentration of 1- mg/ml for 1h.
  • analysis by flow cytometry showed the splenocytes internalized rS1-Fc (FIG.11).
  • mice immunized with linearized dsDNA encoding S1-Fc mounted a significant and robust CD4 + IFN ⁇ + Th1 polarization in vivo in a dose-dependent manner (FIG.13B).
  • S1-antigen specific CD8 + T cells isolated from spleen accumulated upon immunization at increased dose (FIG.13C).
  • high dose immunization favored CD8 + IFN ⁇ + effector T cell in vivo education in a dose-dependent manner (FIG.13D).
  • a mouse monoclonal antibody (SARS-CoV Spike S1 Subunit Antibody) from Sino Biological (Cat#40150-MM02) was used to determine the standard curve between 100 – 0.781 ⁇ g/ml.
  • the standard was prepared using Casein Block Buffer at a 2-fold serial dilution.
  • Mouse serum samples were diluted with Casein Block Buffer at the designated dilution factor during the plate blocking period.
  • the blocked plate was washed once and incubated with the standard or test samples at room temperature for 1.5 hours with shaking at 300- 400 rpm.
  • Y A450 Absorbance with blank subtracted, to a 4-parameter logistic curve generated by the standard serials using non-linear regression in SoftMax Pro GxP.
  • immunization with recombinant rS1-Fc protein facilitated accelerated seroconversion detectable at day 7, with considerable increases in levels of S1-specific serum IgG antibodies over time.
  • Production of S1-specific serum IgG antibodies in vivo experienced a decrease at day 24, which might be due to the anticipated inhibitory FC ⁇ R-B activity, mediating self-regulatory termination of antibody production.
  • FIG.14A to FIG.14C provides a comparison of the antibody response following intramuscular injection of different dosages of dsDNA encoding rS1-Fc and FIG.14D provides the antibody response to intramuscular injection of 100 ⁇ g of the rS1-Fc protein.
  • FIG.17A demonstrates that rS1-Fc migrated to the inguinal lymph node to a greater extent than was observed for rS1, with the difference being less between the Fc and non-Fc recombinant proteins delivered at the lowest dose.
  • FIG.17B demonstrates that the humoral immune response of mice receiving rS1-Fc intramuscular injections measured by ELISA of harvested blood serum was greater than that of mice receiving rS1 except at the lowest protein dose, which elicited a lower antibody response for both proteins.
  • rS1-Fc showed the greatest advantage over rS1 as an immunogen.

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Abstract

La présente divulgation concerne des agents thérapeutiques comprenant des protéines de fusion recombinantes comprenant une région Fc d'anticorps couplée à une protéine d'antigène du coronavirus (ici désignée par " protéine de fusion d'antigène du Fc-coronavirus "), et des acides nucléiques (ADN ou ARNm) codant pour les protéines de fusion d'antigène du Fc-coronavirus, et des vecteurs d'expression, des compositions et des procédés d'utilisation de ceux-ci pour traiter une infection à coronavirus.
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US20210353742A1 (en) * 2020-05-04 2021-11-18 Adimmune Corporation Immunogenic composition and use thereof
WO2022164208A1 (fr) * 2021-01-29 2022-08-04 (주)셀트리온 Protéine de fusion du coronavirus-2 du sars et composition immunogène la comprenant
WO2023130089A1 (fr) * 2021-12-31 2023-07-06 Boost Biopharma, Inc. Polypeptides recombinants contenant au moins un fragment immunogène et une région fc d'anticorps et leurs utilisations
WO2023166054A1 (fr) * 2022-03-02 2023-09-07 ISR Immune System Regulation Holding AB (publ) Composition de vaccin comprenant un antigène et un agoniste de tlr3
CN118184795A (zh) * 2022-12-13 2024-06-14 成都维瑾柏鳌生物医药科技有限公司 抗hiv-1的重组蛋白及其应用
US12409219B2 (en) 2020-06-30 2025-09-09 Boost Biopharma, Inc. Recombinant polypeptides containing at least one immunogenic fragment and uses thereof

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US20210353742A1 (en) * 2020-05-04 2021-11-18 Adimmune Corporation Immunogenic composition and use thereof
US11904013B2 (en) * 2020-05-04 2024-02-20 Adimmune Corporation Immunogenic composition and use thereof
US12409219B2 (en) 2020-06-30 2025-09-09 Boost Biopharma, Inc. Recombinant polypeptides containing at least one immunogenic fragment and uses thereof
WO2022164208A1 (fr) * 2021-01-29 2022-08-04 (주)셀트리온 Protéine de fusion du coronavirus-2 du sars et composition immunogène la comprenant
CN112941091A (zh) * 2021-03-17 2021-06-11 吉林大学 一种猪血凝性脑脊髓炎dna疫苗及其制备方法
WO2023130089A1 (fr) * 2021-12-31 2023-07-06 Boost Biopharma, Inc. Polypeptides recombinants contenant au moins un fragment immunogène et une région fc d'anticorps et leurs utilisations
WO2023166054A1 (fr) * 2022-03-02 2023-09-07 ISR Immune System Regulation Holding AB (publ) Composition de vaccin comprenant un antigène et un agoniste de tlr3
CN118184795A (zh) * 2022-12-13 2024-06-14 成都维瑾柏鳌生物医药科技有限公司 抗hiv-1的重组蛋白及其应用

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