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WO2025230768A1 - Compositions et procédés associés à des thérapies contre un coronavirus - Google Patents

Compositions et procédés associés à des thérapies contre un coronavirus

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Publication number
WO2025230768A1
WO2025230768A1 PCT/US2025/025730 US2025025730W WO2025230768A1 WO 2025230768 A1 WO2025230768 A1 WO 2025230768A1 US 2025025730 W US2025025730 W US 2025025730W WO 2025230768 A1 WO2025230768 A1 WO 2025230768A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
domain
fusion protein
protein
coronavirus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/025730
Other languages
English (en)
Inventor
Ashley UTZ
Adrian HUGENMATTER
Matthew ARMBRUST
Peter S. Kim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leland Stanford Junior University
CZ Biohub SF LLC
Original Assignee
Leland Stanford Junior University
CZ Biohub SF LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Leland Stanford Junior University, CZ Biohub SF LLC filed Critical Leland Stanford Junior University
Publication of WO2025230768A1 publication Critical patent/WO2025230768A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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

  • Coronaviruses are a large family of viruses that cause human illness ranging from the common cold to more severe diseases, such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). Coronaviruses are zoonotic, meaning they can be transmitted between animals and humans. COVID-19 is a respiratory disease caused by SARS- CoV-2, a betacoronavirus. It led to a global pandemic with over 750 million cases and nearly seven million deaths worldwide.
  • MERS Middle East Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • SARS-CoV-1and MERS coronavirus (MERS-CoV, a betacoronavirus) originated in bats, and it is likely that SARS-CoV-2 did as well.
  • MERS-CoV SARS-CoV-1and MERS coronavirus
  • Viral mutation and zoonotic transfer are anticipated to lead to future pandemics and large-scale outbreaks of disease caused by novel coronaviruses.
  • active pharmaceutical agents that are effective for treating COVID-19 or other coronavirus infections in patients.
  • fusion proteins comprising at least one neutralizing domain capable of specifically binding to a receptor binding domain (RBD) of a coronavirus spike protein, and at least one antibody domain capable of specifically binding an epitope in a conserved region of the coronavirus spike protein.
  • RBD receptor binding domain
  • At least one antibody domain comprises: a light chain CDR1 comprising SEQ ID NO:5; a light chain CDR2 comprising SEQ ID NO:6; a light chain CDR3 comprising SEQ ID NO:7; a heavy chain CDR1 comprising SEQ ID NO:8; a heavy chain CDR2 comprising SEQ ID NO:9; and a heavy chain CDR3 comprising SEQ ID NO:10.
  • at least one antibody domain comprises: a light chain CDR1 comprising SEQ ID NO:11; a light chain CDR2 comprising SEQ ID NO:12; a light chain CDR3 comprising SEQ ID NO:13.
  • At least one antibody domain comprises: a light chain variable region comprising a sequence having at least 90% sequence identity to SEQ ID NO:1 and a heavy chain variable region comprising a sequence having at least 90% sequence identity to SEQ ID NO:2.
  • at least one antibody domain comprises: a light chain variable region comprising a sequence having at least 90% sequence identity to SEQ ID NO:3 and a heavy chain variable region comprising a sequence having at least 90% sequence identity to SEQ ID NO:4.
  • the above fusion proteins further comprise a dimerization domain or a trimerization domain.
  • fusion proteins comprising at least one neutralizing domain capable of specifically binding to an RBD of a coronavirus spike protein, at least one antibody domain capable of specifically binding an epitope in a conserved region of the coronavirus spike protein, and a trimerization domain.
  • fusion proteins comprising at least one neutralizing domain capable of specifically binding to an RBD of a coronavirus spike protein, at least one antibody domain capable of specifically binding an epitope in a conserved region of the coronavirus spike protein, and a dimerization domain.
  • N-terminus of the antibody domain is linked to C-terminus of the at least one neutralizing domain
  • C-terminus of the antibody domain is linked to N-terminus of the dimerization domain or the trimerization domain.
  • N-terminus of the dimerization domain or the trimerization domain is linked to C-terminus of the antibody domain, and C-terminus of the 2 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 dimerization domain or the trimerization domain is linked to N-terminus of the at least one neutralizing domain.
  • the antibody domain comprises: a light chain CDR1 comprising SEQ ID NO:5; a light chain CDR2 comprising SEQ ID NO:6; a light chain CDR3 comprising SEQ ID NO:7.
  • the antibody domain comprises: a light chain CDR1 comprising SEQ ID NO:11; a light chain CDR2 comprising SEQ ID NO:12; a light chain CDR3 comprising SEQ ID NO:13.
  • the antibody domain comprises: a light chain variable region comprising a sequence having at least 90% sequence identity to SEQ ID NO:1 and a heavy chain variable region comprising a sequence having at least 90% sequence identity to SEQ ID NO:2.
  • the antibody domain comprises: a light chain variable region comprising a sequence having at least 90% sequence identity to SEQ ID NO:3 and a heavy chain variable region comprising a sequence having at least 90% sequence identity to SEQ ID NO:4.
  • the dimerization domain is an Fc domain.
  • the Fc domain has at least 90% sequence identity to SEQ ID NO:62.
  • the trimerization domain is a cartilage matrix protein (CMP) trimerization domain.
  • CMP trimerization domain has at least 90% sequence identity to SEQ ID NO:63.
  • the neutralizing domain comprises a coronavirus receptor polypeptide.
  • the coronavirus receptor polypeptide comprises an ACE2 receptor ectodomain polypeptide or a DPP4 receptor ectodomain polypeptide.
  • the coronavirus receptor polypeptide comprises the ACE2 receptor ectodomain polypeptide.
  • the ACE2 receptor ectodomain polypeptide has at least 90% sequence identity to SEQ ID NO:17 or SEQ ID NO:131. In some embodiments, the ACE2 receptor ectodomain polypeptide lacks peptidase activity. In some embodiments, the ACE2 receptor ectodomain polypeptide that lacks peptidase activity comprises one or more substitutions, in reference to SEQ ID NO:51, at R273, H378, E402, H374, or H345. In some embodiments, the one or more substitutions are R273A, H378A, E402A, H374N, or H345L.
  • the 3 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 ACE2 receptor ectodomain polypeptide that lacks peptidase activity has at least 90% sequence identity to SEQ ID NO:18.
  • the coronavirus receptor polypeptide comprises the DPP4 receptor ectodomain polypeptide.
  • the DPP4 receptor ectodomain polypeptide has at least 90% sequence identity to SEQ ID NO:19 or SEQ ID NO:20.
  • the DPP4 receptor ectodomain polypeptide comprises two or more DPP4 receptor ectodomain polypeptide sequences.
  • the DPP4 receptor ectodomain polypeptide comprising two or more DPP4 receptor ectodomain polypeptide sequences has at least 90% sequence identity to SEQ ID NO:120.
  • the DPP4 receptor ectodomain polypeptide lacks peptidase activity.
  • the DPP4 receptor ectodomain polypeptide that lacks peptidase activity comprises one or more substitutions, in reference to SEQ ID NO:52, at S630, Y547, D708, H740, or H750.
  • the one or more substitutions are S630A, Y547F, D708A, H740L, or H750E.
  • the DPP4 receptor ectodomain polypeptide that lacks peptidase activity has at least 90% sequence identity to SEQ ID NO:20.
  • the antibody domain is capable of specifically binding an epitope in a conserved region of the coronavirus spike protein.
  • the conserved region is located at a trimeric interface of the coronavirus spike protein.
  • the conserved region comprises 90% or greater conservation across betacoronaviruses.
  • the betacoronaviruses are SARS-CoV-1, SARS-CoV-2, HCoV-NL63, SL-CoV-WIV1, or a merbecovirus.
  • the merbecovirus is MERS-CoV or MjHKU4r-CoV-1.
  • the antibody domain comprises CH1 constant region. [0008] Some embodiments of the fusion proteins according to the present invention and described in the present disclosure further comprise a peptide linker between the neutralizing domain and the antibody domain (linker A).
  • Some embodiments of the fusion proteins according to the present invention and described in the present disclosure further comprise at least one of: a peptide linker between the at least one neutralizing domain and the dimerization domain or the trimerization domain (linker B), or a peptide linker between the at least one antibody domain and the dimerization domain or the trimerization domain (linker C).
  • at least one of linker A, linker B, or linker C comprises at least 5 amino acids.
  • At least one 4 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 of linker A, linker B, or linker C comprises an amino acid sequence with at least 90% sequence identity to one of SEQ ID NOs 72-78, 81-94, and 108-114.
  • Some embodiments of the fusion proteins according to the present invention and described in the present disclosure comprise an amino acid sequence with at least 90% sequence identity to one of SEQ ID NOs 21-44, 96, or 97.
  • the fusion protein comprises an amino acid sequence having at least 90% sequence identity to one of SEQ ID NOs SEQ ID NOs 21-44, 96, 97, 116-119, and 123-130.
  • recombinant nucleic acids encoding the fusion proteins according to the embodiments of the present invention and described in the present disclosure, such as the fusion proteins described above.
  • a recombinant nucleic acid is RNA.
  • a recombinant nucleic acid is DNA.
  • nucleic acid construct comprising one or more nucleic acids according to the present disclosure.
  • a nucleic acid construct is an RNA construct comprising the recombinant nucleic acid of claim 37 and one or more of at least one untranslated region, 3’ poly (A) tail, or 5’ cap.
  • a nucleic acid construct is a DNA construct comprising a promoter operably linked to recombinant DNA according to the present disclosure.
  • delivery vehicles comprising one or more recombinant nucleic acids and/or nucleic acid constructs according to the present disclosure.
  • the delivery vehicle is a lipid nanoparticle (LNP).
  • the LNP comprises one or more recombinant RNAs and/or RNA constructs according to the present disclosure.
  • the delivery vehicle is a lipid nanoparticle (LNP).
  • the delivery vehicle is a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • a nanoparticle is an inorganic nanoparticle, such as, but not limited to, a gold nanoparticle.
  • a vector comprises one or more recombinant DNA and/or DNA constructs.
  • a vector is a viral vector.
  • Such methods comprise a step of introducing into a cell one or more of the following: one or more nucleic acids according to the present disclosure, some examples of which are described above; one or more nucleic acid constructs according to the present disclosure, some examples of which are described 5 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 above; one or more delivery vehicles according to the present disclosure, some examples of which are described above; or one or more vectors according to the present disclosure, some examples of which are described above.
  • the step of introducing comprises stably integrating a nucleic acid encoding the fusion protein into genome of the cell.
  • such methods further comprise a step of incubating the cell under conditions allowing for expression of the fusion protein.
  • such methods further comprise a step isolating the fusion protein.
  • the cell is a eukaryotic cell.
  • host cells comprising one or more nucleic acids according to the present disclosure, some examples of which are described above; one or more nucleic acid constructs according to the present disclosure, some examples of which are described above; one or more delivery vehicles according to the present disclosure, some examples of which are described above; or one of the vectors according to the present disclosure, some examples of which are described above.
  • a host cell is a eukaryotic cell.
  • methods of producing the fusion protein comprising culturing a host cell under conditions sufficient for production of the fusion protein by the host cell.
  • kits comprising such pharmaceutical preparations.
  • such kits comprise a device for administering the pharmaceutical preparation.
  • a coronavirus such as a betacoronavirus
  • methods comprise a step of administering to the subject a therapeutically effective amount of a pharmaceutical preparation according to the present disclosure, some 6 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 examples of which are described above.
  • the subject has a confirmed coronavirus infection, such as a betacoronavirus infection.
  • Such methods comprise a step of administering to the subject a therapeutically effective amount of a pharmaceutical preparation according to the present disclosure, some examples of which are described above.
  • the pharmaceutical preparation is administered in an amount effective for treating or preventing a coronavirus infection, such as a betacoronavirus infection or an alphacoronavirus infection in the subject.
  • the coronavirus is SARS-CoV-1, SARS-CoV-2, SL-CoV-WIV1, or a merbecovirus.
  • the merbecovirus is MERS-CoV or MjHKU4r-CoV-1.
  • an alphacoronavirus is HCoV-NL63.
  • the subject is human.
  • the pharmaceutical preparation is administered to the subject in an amount effective for treating or preventing the coronavirus infection in the subject.
  • FIG. 1C are schematic illustrations of structures of trimeric fusion proteins according to the present disclosure. Black ovals - antibody domain; circles - neutralizing domain; grey ovals - trimerization domain.
  • FIG. 2 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure. Bold – signal; Cursive – ACE2 domain; Regular – variable heavy chain; Regular with gray highlight – variable light chain; Underlined – linker.
  • FIG. 3 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 4 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 4 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 4 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure
  • FIG. 5 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 6 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 7 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • Bold – signal ; Bold underlined – FLAG tag; Bold with gray highlight – inactivating ACE2 mutation; Cursive – iACE2 domain; Cursive with gray highlight – cartilage matrix protein trimerization domain (tri); Regular – variable heavy chain; Regular with gray highlight – constant heavy chain; Underlined – linker.
  • FIG. 7 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • Bold – signal Bold underlined – FLAG tag; Bold with gray highlight – inactivating ACE2 mutation; Cursive – iACE2 domain; Cursive with gray highlight – cartilage matrix protein trimerization domain (tri); Regular – variable heavy chain; Regular with gray highlight – constant heavy chain; Underlined – linker.
  • FIG. 7 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present
  • FIG. 8 is a schematic illustration of amino acid sequences of Fab-based antibody domains used in exemplary fusion proteins according to the present disclosure.
  • Bold – signal ; Regular – variable light chain; Regular with gray highlight – constant light chain.
  • FIG. 9 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • Bold – signal ; Cursive – DPP4 domain; Regular – variable heavy chain; Regular with gray highlight – variable light chain; Underlined – linker.
  • FIG. 10 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 11 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 12 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 13 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 8 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 [0028] FIG.
  • FIG. 14 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 15A, FIG. 15B, and FIG. 15C are schematic illustrations of structures of dimeric fusion proteins according to the present disclosure. Black ovals - antibody domain; circles - neutralizing domain; grey ovals - dimerization domain.
  • FIG. 16 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 17 is a dot plot illustrating betacoronavirus neutralization potency of exemplary fusion proteins according to the present disclosure. The symbols indicating different pseudotyped lentiviruses are indicated in the legend on the right.
  • FIG. 18 is a heat map illustrating betacoronavirus neutralization potency of exemplary fusion proteins according to the present disclosure.
  • FIG. 19 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 20 is a dot plot illustrating betacoronavirus neutralization potency of exemplary fusion proteins according to the present disclosure. The symbols indicating different pseudotyped lentiviruses are indicated in the legend on the right.
  • FIG. 21 is a heat map illustrating betacoronavirus neutralization potency of exemplary fusion proteins according to the present disclosure.
  • FIG. 22 is a dot plot illustrating HCoV-NL63 neutralization potency of exemplary fusion proteins according to the present disclosure. The symbols indicating different fusion proteins are indicated in the legend on the right.
  • FIG. 23A and FIG. 23B are heat maps illustrating HCoV-NL63 neutralization potency of exemplary fusion proteins according to the present disclosure. 9 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 [0038]
  • FIG. 24A, FIG. 24B, and FIG. 24C are schematic illustrations of structures of dimeric fusion proteins according to the present disclosure.
  • FIG. 25A, FIG. 25B, and FIG. 25C are schematic illustrations of structure of trimeric fusion proteins according to the present disclosure. Black ovals - antibody domain; rhombuses - iDPP4-iDPP4 neutralizing domain; grey ovals - trimerization domain.
  • FIG. 26 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 27 is a schematic illustration of an amino acid sequence of an exemplary fusion protein according to the present disclosure.
  • FIG. 28 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 29 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 30 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 31 is a schematic illustration of amino acid sequences of exemplary fusion proteins according to the present disclosure.
  • FIG. 32 is a dot plot illustrating merbecovirus neutralization potency of exemplary fusion proteins according to the present disclosure. The symbols indicating pseudotyped merbecoviruses are indicated in the legend on the right. [0047] FIG.
  • FIG. 33 is a dot plot illustrating merbecovirus neutralization potency of exemplary fusion proteins according to the present disclosure.
  • the symbols indicating pseudotyped merbecoviruses are indicated in the legend on the right. The circle indicates that, in contrast to fusion proteins according to present disclosure, clone 834 anti-MERS antibody does not neutralize pangolin merbecovirus MjHKU4r-CoV-1.
  • FIG. 34 is a heat map illustrating merbecovirus neutralization potency of exemplary fusion proteins according to the present disclosure.
  • FIG. 35 is a dot plot illustrating merbecovirus neutralization potency of exemplary fusion proteins according to the present disclosure.
  • FIG. 36 is a heat map illustrating merbecovirus neutralization potency of exemplary fusion proteins according to the present disclosure.
  • FIG. 37 is a line plot illustrating % body weight change in mice challenged with live Omicron variant XBB.1.5 (squares) and control group mice (“Vehicle” (PBS) – triangles). Body weights are plotted as the mean for each group, with Standard Error of the Mean (SEM) shown as vertical bars. The dotted line shows the body weight at the start of the study.
  • FIG. 37 is a line plot illustrating % body weight change in mice challenged with live Omicron variant XBB.1.5 (squares) and control group mice (“Vehicle” (PBS) – triangles). Body weights are plotted as the mean for each group, with Standard Error of the Mean (SEM) shown as vertical bars. The dotted line shows the body weight at the start of the study.
  • SEM Standard Error of the Mean
  • the mAbs neutralize the virus by blocking binding between the RBD and angiotensin converting enzyme 2 (ACE2), which serves as a receptor for SARS-CoV-2 virus and is located on the cell membrane of many host cells, particularly epithelial cells in the respiratory tract (Chen et al. 2023, Scialo 2020).
  • ACE2 angiotensin converting enzyme 2
  • known mAb therapies gradually become ineffective with the emergence of new SARS-CoV-2 variants. This requires the development and approval of new mAb therapies, which is time-consuming and 11 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 expensive.
  • Fusion proteins containing ACE2 domain linked to a fragment of non-neutralizing anti- SARS-CoV-2 spike protein antibody are described in International Patent Publication No. WO202271863 and Weidenbacher et al. 2022.
  • the antibody domain binds an epitope on the S2 subunit of SARS- CoV-2 spike protein, which is highly conserved across coronaviruses, and the ACE2 domain serves as the neutralizing domain.
  • the antibody domain acts as an anchor to increase the local effective concentration of ACE2 domain, enhancing its neutralizing potency.
  • the fusion proteins target a conserved epitope on SARS-CoV-2 spike protein and use the virus’ own host receptor ACE2, which is also highly conserved, as the neutralizing component. Accordingly, SARS-CoV-2 variants are less likely to escape the binding of fusion proteins than neutralizing mAbs targeting RBD of SARS-CoV-2 Spike proteins.
  • the inventors conceived, and the present disclosure describes improved fusion proteins containing an antibody domain. Some embodiments of the improved fusion proteins contain an antibody domain that binds a highly conserved epitope of betacoronavirus spike proteins. The resulting fusion proteins bind a wide range of betacoronaviruses and offer greater breadth of protection than previously described fusion proteins.
  • Some embodiments of the improved fusion proteins use enzymatically inactive ACE2 domain, which prevents potential adverse side effects of administering fusion proteins containing enzymatically active ACE2 domain. Such adverse side effects stem from the functions of ACE2 in regulating blood pressure and vascular tone.
  • Some embodiments of the improved fusion proteins are constructed to form dimers and trimers, which leads to their improved efficacy.
  • Some embodiments of the improved fusion proteins are anti- merbecovirus, such as anti-MERS, fusion proteins and include dipeptidyl peptidase IV (DPP4) or catalytically inactive DPP4 as a neutralizing domain.
  • DPP4 dipeptidyl peptidase IV
  • the inventors also discovered that, surprisingly, altering the domain order of the fusion proteins according to the present disclosure improves both neutralization potency and neutralization 12 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 breadth of the fusion proteins, as well as their expression (for example, purity and/or yield). Accordingly, the inventors conceived, and the present disclosure describes improved designs of fusion proteins with various domain orders. Also conceived by the inventors and described in the present disclosure are nucleic acids encoding improved fusion proteins, as well as constructs, expression cassettes, and vectors containing such nucleic acids, and host cells capable of expressing the improved fusion proteins.
  • improved fusion proteins incorporating enzymatically inactive ACE2 domain and described in the present disclosure possess several advantages over known anti-coronavirus therapeutics. For example, such improved fusion proteins according to the present disclosure are more efficacious in vitro than known anti-coronavirus mAbs. Such improved fusion proteins according to the present disclosure are also efficacious in vivo.
  • the improved fusion proteins according to the present disclosure also possess a broad neutralizing range that includes not only all tested SARS-CoV-2 variants, SARS-CoV-1, and bat coronavirus SL-CoV-WIV1, but also alphacoronaviruses, such as HCoV-NL63.
  • the improved fusion proteins incorporating enzymatically inactive DPP4 domain and described in the present disclosure displayed neutralizing potency against merbecoviruses in vitro. For example, such improved fusion proteins displayed broad neutralizing potency not only against a range of MERS-CoV strains, but also against a distantly related merbecovirus circulating in pangolins (MjHKU4r-CoV-1).
  • the inventors discovered that a DPP4 domain incorporating two inactivated DPP4 sequences surprisingly resulted in improved expression and increased purity of fusion proteins incorporating such domain.
  • the therapies based on the improved fusion proteins according to the present disclosure are likely to inhibit alphacoronaviruses and betacoronaviruses that use ACE2 as a receptor, making them a suitable candidate for stockpiling in anticipation of the next alphacoronavirus or betacoronavirus jumping from animals to humans.
  • the therapies based on the improved fusion proteins according to the present disclosure are also likely to inhibit betacoronaviruses that use DPP4 as a receptor.
  • the improved fusion proteins according to the present disclosure may be rapidly deployed as an already developed drug.
  • the improved fusion proteins according to the present disclosure are expected to have few, if any, interactions with other medications a patient may be taking.
  • any currently available therapeutics such as Paxlovid, cause multiple 13 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 drug interactions, limiting their use in high-risk patients who often have complex medication regimens.
  • the improved fusion proteins described in the present disclosure can be used as broad and high-potency therapeutics against coronavirus infections.
  • binding refers to a binding reaction in which, under designated conditions, a specific binding molecule or a composition containing it binds to its binding partner or partners and does not bind in a significant amount to 15 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 anything else.
  • Binding to anything else other than the binding partner is typically referred to as “nonspecific binding” or “background.”
  • the absence of binding in a significant amount is considered, for example, to be binding less than 1.5 times background (i.e., the level of non- specific binding or slightly above non-specific binding levels).
  • Some non-limiting examples of specific binding are antibody-antigen or antibody-epitope binding, binding of oligo- or polynucleotides to other oligo- or polynucleotides, binding of oligo- or polynucleotides to proteins or polypeptides (and vice versa), binding or proteins to polypeptides other proteins or polypeptides, receptor-ligand binding, and carbohydrate-lectin binding.
  • specific binding molecules can be or can include a protein, a polypeptide, an antibody, an oligo- or polynucleotide, a receptor, or a ligand.
  • Specific binding molecules can be natural or engineered.
  • naturally occurring antibodies contain binding sites, typically referred to as “antigen-binding sites,” that are capable of specifically binding to their epitopes.
  • an antibody domain of an engineered fusion protein according to the present disclosure is capable of specifically binding an epitope in a conserved region of the coronavirus spike protein.
  • a neutralizing domain of the engineered fusion protein according to the present disclosure is capable of specifically binding to a receptor binding domain (RBD) of a coronavirus spike protein.
  • RBD receptor binding domain
  • protein protein
  • peptide and “polypeptide” are used interchangeably to refer to a polymer of amino acid residues.
  • the terms apply to naturally occurring amino acid polymers and non-natural amino acid polymers, as well as to amino acid polymers in which one (or more) amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid.
  • the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • a “domain” of a protein or a polypeptide refers to a region of the protein or polypeptide defined by structural and/or functional properties.
  • Exemplary function properties include enzymatic activity and/or the ability to bind to or be bound by another protein or non-protein entity.
  • coronavirus spike protein contains S1 and S2 domains.
  • a “binding site” of a protein or a polypeptide refers to a location of a protein or a polypeptide molecule at which another molecule (which can be referred to as a “target,” a “ligand,” or a “binding partner”) can bind with specificity.
  • binding sites are formed in tertiary structure, with some residues actually participating in binding the ligand and other residues acting as a framework to provide correct conformation and orientation.
  • an antigen-binding site of an antibody is capable of specifically binding an epitope, which is a target of the antibody’s antigen-binding site.
  • oligomer and related terms, when used in reference to polypeptides or proteins, refer to complexes formed by two or more polypeptide or protein monomers, which can also be referred to as “subunits” or “chains.”
  • a dimer is an oligomer formed by two polypeptide subunits
  • a trimer is an oligomer formed by three polypeptide subunits.
  • an “isolated” or “purified” polypeptide or protein, or biologically active portion a polypeptide or a protein is substantially or essentially free from components that normally accompany or interact with the polypeptide or protein as found in its naturally occurring environment.
  • an isolated or purified polypeptide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% (total protein) of contaminating protein.
  • optimally culture medium represents less than about 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% (by concentration) of chemical precursors or non-protein-of-interest chemicals.
  • fusion protein refers to polypeptide molecules, including artificial or engineered polypeptide molecules, that include two or more amino acid sequences previously found in separate polypeptide molecule, that are joined or linked in a fusion protein amino acid sequence to form a single polypeptide.
  • a fusion protein can be an engineered recombinant protein containing amino acid sequence from at least two unrelated proteins that have been joined together, via a peptide bond, to make a single protein.
  • proteins are considered unrelated, if their amino acid sequences are not normally found joined together via a peptide bond in their natural environment, for example, inside a cell.
  • the present disclosure describes fusion proteins that include an amino acid sequence of a coronavirus receptor polypeptide and an amino acid sequence of an antibody, which are unrelated proteins.
  • the amino acid sequences of a fusion protein are encoded by corresponding nucleic acid sequences that are joined “in frame,” so that they are transcribed and translated to produce a single polypeptide.
  • amino acid sequences of a fusion protein can be contiguous or separated by one or more spacer, linker, or hinge sequences. Fusion proteins can include additional amino acid sequences, such as, for example, signal sequences, tag sequences, and/or linker sequences.
  • amino acid refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally occurring ⁇ -amino acids and their stereoisomers, as well as unnatural (non-naturally occurring) amino acids and their stereoisomers.
  • Step 292S-PC “Stereoisomers” of a given amino acid refer to isomers having the same molecular formula and intramolecular bonds but different three-dimensional arrangements of bonds and atoms (e.g., an L- amino acid and the corresponding D-amino acid).
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Naturally occurring ⁇ -amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and their combinations.
  • Stereoisomers of a naturally occurring ⁇ - amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D- asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D- Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and their combinations.
  • D-Ala D-alan
  • Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in either the L- or D-configuration that function in a manner that is similar to the naturally occurring amino acids.
  • amino acid analogs can be unnatural amino acids that have the same basic chemical structure as naturally occurring amino acids (i.e., a carbon that is bonded to a hydrogen, a carboxyl group, an amino group) but have modified side-chain groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Unnatural amino acids that is, those that are not naturally found in proteins
  • Beta and gamma amino acids are known.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC- IUB Biochemical Nomenclature Commission.
  • the expression “chemically modified amino acid” refers to an amino acid whose side chain has been chemically modified.
  • a side chain can be modified to comprise a signaling moiety, such as a fluorophore or a radiolabel.
  • a side chain can also be modified to comprise a new functional group, such as a thiol, carboxylic acid, or amino group.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and their polymers.
  • Nucleic acid sequences as discussed in the present disclosure, encompass all forms of nucleic acids, including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. When an RNA sequence is described, its corresponding DNA sequence is also described, wherein uridine is represented as thymidine.
  • nucleic acid and the related terms and expressions encompass nucleic acids containing known analogues of natural nucleotides that have similar properties as the reference nucleic acid, and are metabolized in a manner similar to naturally occurring nucleotides.
  • a nucleic acid sequence can include combinations of deoxyribonucleic acids and ribonucleic acids.
  • deoxyribonucleic acids and ribonucleic acids include both naturally 19 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 occurring molecules and synthetic analogues.
  • a particular nucleic acid sequence also implicitly encompasses degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • Degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.
  • nucleic acid or amino acid sequences refer to a sequence that has at least 60% sequence identity to a reference sequence. Examples include at least: 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity, as compared to a reference sequence using the programs for comparison of nucleic acid or amino acid sequences, such as BLAST using standard parameters.
  • sequence comparison For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default (standard) program parameters can be used, or alternative parameters can be designated.
  • the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window” includes reference to a segment of any one of the number of contiguous positions (from 20 to 600, usually about 50 to about 200, more commonly about 100 to about 150), in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • sequence identity between sequence A and sequence B aligned using the software above or manually (to maximize alignment), can be determined by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, by the sum of the residue matches between sequence A and sequence B, times one hundred.
  • Algorithms that are suitable for determining percent sequence identity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. 1990 20 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 and Altschul et al. 1977, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site.
  • NCBI National Center for Biotechnology Information
  • the algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold.
  • These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff 1989).
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (Karlin and Altschul 1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10 -5 , and most preferably less than about 10 -20 .
  • the terms “individual”, “subject”, and “patient” can be used interchangeably in the present disclosure to refer to a non-human animal or a human.
  • subjects include, but are not limited to: humans and other primates, including non-human primates, such as chimpanzees and other apes and monkey species; farm animals, such as cattle, sheep, pigs, seals, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs; birds, including domestic, wild and game birds, such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • An infected subject is a subject that is known to have been infected by an infectious organism, such as coronavirus, for example, a betacoronavirus, such as, but not limited to, SARS-CoV-2.
  • an infectious organism such as coronavirus, for example, a betacoronavirus, such as, but not limited to, SARS-CoV-2.
  • Administration encompasses direct administration, such as administration to a subject by a medical professional or self-administration, or indirect administration, which may be the act of prescribing a composition described in the present disclosure.
  • Virus is used in both the plural and singular senses. “Virion” refers to a single virus.
  • coronavirus virion refers to a coronavirus particle.
  • Coronaviruses are a group of enveloped, single-stranded RNA viruses that cause diseases in mammals and birds. Currently, coronaviruses include four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus.
  • Alphacoronaviruses and betacoronaviruses infect mammals, while gammacoronaviruses and deltacoronaviruses primarily infect birds.
  • alphacoronaviruses are: Alphacoronavirus 1 (TREEV, Feline coronavirus, Canine coronavirus), Human coronavirus 229E (HCoV-229E), Human coronavirus HCoV-NL63 (NL63, New Haven coronavirus), Miniopterus bat coronavirus 1, Miniopterus bat coronavirus HKU8 (Bat-CoV HKU8), Porcine epidemic diarrhea virus, Rhinolophus bat coronavirus HKU2 (Bat-CoV HKU2), Scotophilus bat coronavirus 512.
  • TREEV Feline coronavirus, Canine coronavirus
  • Human coronavirus 229E HoV-229E
  • Human coronavirus HCoV-NL63 NL63, New Haven coronavirus
  • Betacoronaviruses include embecoviruses, hibecoviruses, merbecoviruses (such as MERS-CoV, MjHKU4r-CoV-1, which currently circulates in pangolins, bat coronavirus HKU4 (Bat-CoV HKU4), and bat coronavirus HKU25 (BatCoV HKU25), nobecoviruses, and sarbecoviruses (such as SARS-CoV-1 and SARS--CoV-2).
  • merbecoviruses such as MERS-CoV, MjHKU4r-CoV-1, which currently circulates in pangolins
  • bat coronavirus HKU4 Bat-CoV HKU4
  • bat coronavirus HKU25 bat coronavirus HKU25
  • nobecoviruses such as SARS-CoV-1 and SARS--CoV-2).
  • Coronavirus hosts include bats, pigs, dogs, cats, mice, rats, cows, rabbits, 22 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 chickens, and turkeys.
  • coronaviruses cause mild to severe respiratory tract infections. Coronaviruses vary significantly in risk factors. Some can kill more than 30% of infected subjects. Coronaviruses circulating in animals are recognized as having significant pandemic potential due to their ability to jump from non-human to human animal hosts and acquiring the traits allowing for high transmissibility in human population.
  • human coronaviruses are: Human coronavirus 229E (HCoV-229E); Human coronavirus OC43 (HCoV-OC43); Severe acute respiratory syndrome coronavirus (SARS-CoV); Human coronavirus HCoV-NL63 (NL63, New Haven coronavirus); Human coronavirus HKU1 (HCoV-HKU1), which originated from infected mice, was first discovered in January 2005 in two patients in Hong Kong; Middle East respiratory syndrome-related coronavirus (MERS-CoV), also known as novel coronavirus 2012 and HCoV-EMC; and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as 2019-nCoV or “novel coronavirus 2019.” In human, SARS-CoV-2 causes coronavirus disease termed COVID-19, which can cause severe symptoms and death.
  • SARS-CoV-2 causes coronavirus disease termed COVID-19, which can cause severe symptoms and death.
  • Spike protein is a coronavirus surface protein that is able to mediate receptor binding and membrane fusion between a coronavirus virion and its host cell. Characteristic spikes on the surface of coronavirus virions are formed by ectodomains of homotrimers of spike protein. In comparison to trimeric glycoproteins found on other human-pathogenic enveloped RNA viruses, coronavirus spike protein is considerably larger, and totals nearly 450 kDa per trimer.
  • Ectodomains of coronavirus spike proteins contain an N-terminal domain named S1, which is responsible for binding of receptors on the host cell surface, and a C-terminal S2 domain responsible for fusion.
  • S1 domain of SARS-CoV-2 spike protein is able to bind to angiotensin- converting enzyme 2 (ACE2) of host cells.
  • ACE2 angiotensin- converting enzyme 2
  • the region of SARS-CoV-2 spike protein S1 domain that recognizes ACE2 is a 25 kDa domain called the receptor binding domain (RBD) (Walls et al. 2020).
  • RBD receptor binding domain
  • S1 domain of merbecoviruses and alphacoronaviruses contain the RBD that binds to the receptor on host cells.
  • the host receptor is dipeptidyl peptidase-4 (DPP4).
  • DPP4 dipeptidyl peptidase-4
  • Human alphacoronavirus HCoV-NL63 uses ACE2 as a receptor
  • human alphacoronavirus HCoV-229E uses human aminopeptidase N (hAPN).
  • antibody refers to an immunoglobulin or its fragment that binds to a particular spatial and polar organization of another molecule.
  • Immunoglobulins include various classes and isotypes, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgG4, IgM, etc.
  • An antibody can be monoclonal or recombinant, and can be prepared by laboratory techniques, such as by preparing continuous hybrid cell lines and collecting the secreted protein, or by cloning and expressing nucleotide sequences or their mutagenized versions coding at least for the amino acid sequences required for binding.
  • Antibodies as referenced herein may have sequences derived from non-human antibodies, human sequence, chimeric sequences, and wholly synthetic sequences.
  • the term “antibody” encompasses natural, artificially modified, and artificially generated antibody forms, such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies and their fragments.
  • the term “antibody” also includes composite forms including but not limited to fusion proteins containing an immunoglobulin moiety.
  • Antibody also refers to non-quaternary antibody structures (such as camelids and camelid derivatives) and antigen-binding fragments of antibodies, minibodies, bispecific antibodies, nanobodies (also referred to as V H H fragments), and diabodies.
  • Antibodies and antigen-binding portions thereof include domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.
  • SMIPs small modular immunopharmaceuticals
  • Antibody fragments may include Fab, Fv, F(ab’)2, Fab’, scFv, dsFv, ds-scFv, Fd, dAb, Fc, and the like.
  • a natural antibody digested by papain yields three fragments: two Fab fragments and one Fc fragment.
  • the Fc fragment is dimeric and contains two CH2 and two CH3 heavy chain domains. CH3 domains interact to form a homodimer.
  • Fc domains in antibodies may also be optimized to alter antibody characteristics of interest (e.g., bioavailabilty, serum half-life).
  • aggregates, polymers and conjugates of immunoglobulins or their fragments can be used where appropriate.
  • antibody encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class.
  • a natural immunoglobulin G (IgG) antibody molecule is a tetramer that contains two identical light (L) chains and two identical heavy (H) chains.
  • L light
  • H heavy
  • each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each 24 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • 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.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the term antibody also encompasses an antibody fragment, for example, an antigen-binding fragment.
  • Antigen-binding fragments comprise at least one antigen-binding domain, which, in turn, comprises at least one antigen-binding site.
  • an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer.
  • Antibodies and antigen-binding fragments can be described by the antigen to which they specifically bind.
  • the terms “antigen-binding portion” and “antigen-binding fragment” are used interchangeably in the present disclosure and refer to one or more fragments of an antibody that retains the ability to specifically bind to an antigen. The fragments are not necessarily generated from contiguous antibody sequences but can be engineered proteins containing antibody sequences needed to form an antigen-binding site.
  • antigen-binding fragments include, but are not limited to, a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains), F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), a single chain Fv (scFv) fragment (a fusion protein of the VH and VL regions), a disulfide-linked Fv (dsFv), complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), nanobodies, and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to target antigen.
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL, and CH1 domains
  • F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • scFv single chain Fv
  • variable domains Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions (“CDRs”). The more highly conserved portions of the variable domains are called the framework (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.
  • the CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. See Kabat et al.
  • CDR sequences on the heavy chain may be designated as CDRH1, 2, 3, while CDR sequences on the light chain (VL) may be designated as CDRL1, 2, 3.
  • VH heavy chain
  • VL light chain
  • CDRL1 CDRL1
  • an antibody variable region comprises four conserved “framework” regions interspersed with three hypervariable “complementarity determining regions.”
  • CDR complementarity determining region
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.
  • a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • the part of a variable region not contained in the CDRs is called the framework.
  • the “framework regions” of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
  • Framework sequences can be obtained from public DNA databases or published references 26 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 that include germline antibody gene sequences.
  • germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBASE2” germline variable gene sequence database for human and mouse sequences.
  • the amino acid sequences of the CDRs and framework regions can be determined using various well-known methods, some of which are described elsewhere in the present disclosure.
  • a term “monoclonal antibody” refers to antibodies produced by a single clone of cells or a single cell line and consisting of or consisting essentially of antibody molecules that are identical in their primary amino acid sequence.
  • a monoclonal antibody preparation comprises a population of antibodies that are identical and bind to the same epitope of an antigen, except for mutations that arise during monoclonal antibody production.
  • the term “monoclonal antibody” includes synthetic antibodies and antigen- binding fragments thereof.
  • Fc region” or “Fc sequence” is the “tail” region of an antibody sequence.
  • Fc region contains at least the amino acid sequences of the CH2 and CH3 domains of an antibody (and, in some antibody isotypes, can also contain CH4 domain sequence).
  • Fc fragment contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • An Fc domain introduced into a fusion protein may promote dimerization. In some instances, An Fc domain introduced into a fusion protein may improve its stability in vitro or upon administration in vivo.
  • a “Fab fragment” is comprised of one light chain, and the CH1 and variable regions of one heavy chain and can specifically recognize a target epitope, such as an epitope of a spike protein.
  • a Fab domain introduced into a fusion protein according to the present disclosure results in binding of the fusion protein to the target.
  • Such a fusion protein may be referred to as “Fab fusion protein,” “Fab-based fusion protein,” and by other related expressions.
  • a “single-chain variable fragment” or “scFv fragment” is a fusion protein comprising the variable regions of a heavy chain and a light chain from an antibody. The heavy chain and light chain portions may be connected by a linker peptide.
  • An scFv fragment may retain the binding specificity of the antibody from which it is derived.
  • a scFv domain introduced into a fusion protein according to the present disclosure results in binding of the fusion protein to the target.
  • Such a fusion protein may be referred to as “an scFv fusion protein,” “scFv-based fusion protein,” and by other related expressions.
  • a “neutralizing polypeptide” is a polypeptide that, when present at physiologically and/or pharmaceutically acceptable concentrations, is capable of keeping an infectious agent, such as a virus, from infecting a cell by neutralizing or inhibiting one or more parts of the life cycle of the infectious agent.
  • a common type of neutralizing polypeptide is a neutralizing antibody or a fragment of a neutralizing antibody, however other polypeptides that can bind specifically to an infectious agent can also be neutralizing (e.g., polypeptides based on the receptor bound by a virus).
  • neutralizing antibodies typically specifically bind to the receptor binding domain (RBD) of the spike protein and typically act to disrupt or prevent interaction of the virus spike with its receptor, such that virus entry into the target cell is prevented or reduced. As such, neutralizing antibodies can act to prevent or reduce the incidence of coronavirus infection. Because of the neutralizing ability of neutralizing antibodies, coronaviruses face evolutionary pressure to decrease or eliminate binding of neutralizing antibodies through mutation. Coronaviruses that have epitopes that can be bound by neutralizing antibodies will not propagate as effectively in hosts expressing the neutralizing antibodies relative to coronaviruses that have mutations in the epitopes that reduce or prevent binding of neutralizing antibodies.
  • RBD receptor binding domain
  • non-neutralizing antibody refers to an antibody that, when present at physiologically and/or pharmaceutically acceptable concentrations, has little to no ability of keeping an infectious agent, such as a virus, from infecting a cell by neutralizing or inhibiting one or more parts of the life cycle of the infectious agent.
  • the fusion proteins include at least the following domains: (a) at least one antibody domain capable of specifically binding an epitope in a conserved region of the coronavirus spike protein; and (b) at least one neutralizing domain that is capable of specifically binding to a receptor binding domain (RBD) of a coronavirus spike protein.
  • Fusion proteins according to the present disclosure may contain additional domains and/or sequences. In some embodiments, fusion proteins according to the present disclosure may contain additional domain or domains.
  • Non-limiting examples of such additional domain are multimerization domains, such as, but not limited to, dimerization, trimerization, or tetramerization 28 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 domains.
  • fusion proteins include linkers, which can be polypeptide linkers, between different domains. Fusion proteins may also include various sequences useful in their purification, expression, etc. Some examples of such additional amino acid sequences are signal sequences, purification tags, etc.
  • Exemplary amino acid sequences of the fusion proteins according to the present disclosure and components of such amino acid sequences are illustrated in FIG. 2 – FIG. 14, FIG. 16, FIG. 19, and FIG. 26 – FIG. 31.
  • a fusion protein according to the present disclosure comprises or consists of an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:96, SEQ ID NO:
  • Fusion proteins according to the present disclosure include at least one antibody domain capable of specifically binding an epitope in a conserved region of a coronavirus spike protein.
  • the antibody domain is capable of binding a spike protein epitope that is conserved across a range of coronaviruses.
  • the antibody domain may be capable of binding a spike protein epitope that is conserved across alphacoronaviruses and betacoronaviruses.
  • the antibody domain may be capable of binding a spike protein epitope that is conserved across alphacoronaviruses and/or betacoronaviruses.
  • Betacoronoviruses which are also know as “group 2 coronaviruses,” ⁇ -CoVs, or Beta-CoVs, is a genus of coronaviruses.
  • HCoV-229E and HCoV-NL63 are two examples of human alphacoronaviruses.
  • Other examples of alphacoronaviruses are feline coronavirus and canine coronavirus SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-OC43, and HCoV-HKU1 are all betacoronaviruses.
  • betacoronaviruses are bovine coronavirus, China Rattus coronavirus HKU24, murine coronavirus (M-CoV), Myodes coronavirus 2JL14, bat coronaviruses SL-CoV-WIV1 and RaTG13, Hedgehog coronavirus 1, Pipistrellus bat coronavirus HKU5, Tylonycteris bat coronavirus HKU4, Eidolon bat 29 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 coronavirus C704, Rousettus bat coronavirus GCCDC1, Rousettus bat coronavirus HKU9, and bat Hp-betacoronavirus Zhejiang2013.
  • M-CoV murine coronavirus
  • Myodes coronavirus 2JL14 Myodes coronavirus 2JL14
  • an epitope to which the antibody domain of a fusion disclosure is capable of specifically binding is located in a conserved region of a coronavirus spike protein that is located in the S2 subunit of the coronavirus spike protein, for example, in the trimeric interface, stem helix, or fusion peptide (FP) regions of the S2 subunit of the coronavirus spike protein .
  • the antibody domain comprises amino acid sequences derived from one or more non-neutralizing anti-coronavirus spike protein antibody.
  • the antibody domain is derived from a non-neutralizing anti-coronavirus spike protein antibody that can specifically bind to a coronavirus spike protein at a conserved epitope while having little to no ability of inhibiting viral infection.
  • the level of inhibition of viral infection induced by such a non- neutralizing antibody is 20% or less (e.g., 19%, 18%, 17%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%) as compared to the level of inhibition of viral infection observed in the absence of the non-neutralizing antibody.
  • Some embodiments of the fusion proteins according to the present disclosure include antibody domain amino acid sequences derived from anti-coronavirus spike protein mAbs CoV2-11 or CoV2-26, particularly from the amino acid sequences of the heavy and light chain sequences of these mAbs (SEQ ID NOs 65, 67, 69, and 71).
  • Exemplary amino acid sequences derived from mAbs CoV2-11 or CoV2-26 are shown in FIG. 2 – FIG. 14, FIG. 16, FIG. 19, FIG. 26 – FIG. 31, and Tables 2-4 of the present disclosure.
  • the antibody domain can specifically bind to a coronavirus spike protein at a conserved epitope, while not interfering with the interaction between the spike protein RBD and the coronavirus receptor protein (e.g., ACE2 or DPP4).
  • the coronavirus receptor protein e.g., ACE2 or DPP4
  • non-neutralizing coronavirus antibodies are able to bind to different coronaviruses (e.g., SARS- CoV-1, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-HKU1, HCoV-229E, HCoV-NL63, etc.) and variants thereof, allowing fusion proteins according to the present disclosure to specifically bind to and facilitate neutralization of various coronaviruses.
  • coronaviruses e.g., SARS- CoV-1, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-HKU1, HCoV-229E, HCoV-NL63, etc.
  • the term “conserved” in relation to a viral polypeptide sequence indicates that the sequence is identical or highly similar across viral species or strains.
  • conservation of a sequence may indicate that the sequence has been maintained by natural selection and that the sequence has some functional importance.
  • amino acid sequences can be conserved to maintain the structure and/or function of a polypeptide or domain.
  • conserveed polypeptide sequences undergo fewer amino acid replacements or are more likely to substitute amino acids with similar biochemical properties (known as conservative substitutions, as discussed below).
  • amino acids that are important for folding, structural stability, or that form a binding site may be more highly conserved than other amino acids.
  • Various methods for identifying conserved sequences are known in the art, and generally involve bioinformatic approaches based on sequence alignment. Approaches include homology searches (e.g., BLAST, HMMER, OrthologR, and Infernal, with acceptable conservative substitution identified using substitution matrices such as PAM and BLOSUM), multiple sequence alignments (e.g., CLUSTAL format), whole genome alignments, and scoring systems (e.g., Genomic Evolutionary Rate Profiling (GERP), Local Identity and Shared Taxa (LIST), Aminode, PhyloP and PhyloHHM).
  • homology searches e.g., BLAST, HMMER, OrthologR, and Infernal, with acceptable conservative substitution identified using substitution matrices such as PAM and BLOSUM
  • multiple sequence alignments e.g., CLUSTAL format
  • whole genome alignments e.g.
  • conserveed regions may also be estimated by measuring sequence identity for a region of a protein (e.g., coronavirus spike protein) across a group of related coronaviruses.
  • related coronaviruses are betacoronaviruses.
  • related coronaviruses are coronaviruses in which at least one protein in each coronavirus proteome comprises substantial amino acid sequence identity to a selected reference protein amino acid sequence.
  • related coronaviruses are coronaviruses in which at least one protein in each coronavirus proteome comprises a higher level of sequence identity to a selected reference protein amino acid sequence as compared to an unrelated coronavirus.
  • the reference spike protein is SARS-CoV-2 spike protein (for example, having the amino acid sequence set forth in SEQ ID NO:45), and related coronaviruses are those comprising spike proteins with amino acid sequences having 30% or greater (e.g., 35% or greater, 40% or greater, 45% or greater, 50% or greater, 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, or 90% or greater) amino acid sequence identity to SARS-CoV-2 spike protein.
  • SARS-CoV-2 spike protein for example, having the amino acid sequence set forth in SEQ ID NO:45
  • related coronaviruses are those comprising spike proteins with amino acid sequences having 30% or greater (e.g., 35% or greater, 40% or greater, 45% or greater, 50% or greater, 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, or 90% or greater) amino acid sequence identity to SARS-CoV
  • the reference spike protein is SARS- CoV-1 spike protein (e.g., having the amino acid sequence set forth in SEQ ID NO:46), MERS- CoV spike protein (e.g., having the amino acid sequence set forth in SEQ ID NO:47), HCoV-OC43 spike protein (e.g., having the amino acid sequence set forth in SEQ ID NO:48), HCoV-HKU1 spike protein (e.g., having the amino acid sequence set forth in SEQ ID NO:49), HCoV-229E spike protein (e.g., having the amino acid sequence set forth in SEQ ID NO:50), SL-CoV-WIV1 spike protein (e.g., having the amino acid sequence set forth in SEQ ID NO:135), MjHKU4r-CoV-1 (e.g., having the amino acid sequence set forth in SEQ ID NO:134), or spike proteins of related coronaviruses with amino acid sequences having 30% or greater (e.g., 35% or greater, 40% or
  • the conserved region (e.g., in a coronavirus spike protein) comprises 75% or greater (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 37 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 or 100%) sequence identity to a reference spike protein (for example, SARS-CoV-2 spike protein (SEQ ID NO:45), SARS-CoV-1 spike protein (SEQ ID NO:46), MERS-CoV spike protein (SEQ ID NO:47), HCoV-OC43 spike protein (SEQ ID NO:48), HCoV-HKU1 spike protein (SEQ ID NO:49), HCoV-229E spike protein (SARS-Co
  • the conserved region has substantial sequence identity across betacoronaviruses. In some embodiments, the conserved region has substantial sequence identity across all known human coronaviruses.
  • the conserved region of a coronavirus spike protein may reside at a trimeric interface. Exemplary conserved regions within a coronavirus spike protein include amino acid residues 740-746, 815- 837, 855-866, 894-905, 910-931, 965-1034, 1039-1054, 1076-1082, and 1198-1206 of SEQ ID NO:45. Additional information regarding sequence conservation among coronavirus spike proteins can be foundin Jungreis et al. 2021, Gupta et al. 2021, and Kumavath et al. 2021.
  • An antibody domain of the the fusion proteins according to the present disclosure includes amino acid sequences that make up a binding site capable of specifically binding an epitope in a conserved region of a coronavirus spike protein. This binding site can be referred to as “antigen- binding site.”
  • the antibody binding domain comprises an antibody or an antigen-binding fragment thereof comprising at least one antigen-binding site capable of specifically binding an epitope in a conserved region of a coronavirus spike protein.
  • the antigen-binding fragment included in the antibody domain comprises at least one variable heavy chain sequence and at least one variable light chain sequence.
  • an antibody domain comprises a light chain sequence, or a portion or portions thereof, and/or a heavy chain sequence, or a portion or portions thereof, derived from any of the antibodies described in Example 2 of the present disclosure.
  • an 38 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC;
  • S23-308 antibody domain comprises a variable region of a light chain sequence, or a portion or portions thereof, and/or a variable region of a heavy chain sequence, or a portion thereof, derived from mAb CoV2-11 or mAb CoV2-26.
  • an antigen-binding site of the antibody domain of the fusion proteins according to the present disclosure comprises heavy chain CDRs and/or light chain CDRs that are disclosed in Table 2 of the present disclosure.
  • an antigen binding site comprises the following CDRs: a light chain CDR1 comprising or consisting of SEQ ID NO:5; a light chain CDR2 comprising or consisting of SEQ ID NO:6; a light chain CDR3 comprising or consisting of SEQ ID NO:7; a heavy chain CDR1 comprising or consisting of SEQ ID NO:8; a heavy chain CDR2 comprising or consisting of SEQ ID NO:9; and a heavy chain CDR3 comprising or consisting of SEQ ID NO:10.
  • an antigen binding site comprises the following CDRs: a light chain CDR1 comprising or consisting of SEQ ID NO:11; a light chain CDR2 comprising or consisting of SEQ ID NO:12; a light chain CDR3 comprising or consisting of SEQ ID NO:13; a heavy chain CDR1 comprising or consisting of SEQ ID NO:14; a heavy chain CDR2 comprising or consisting of SEQ ID NO:15; and a heavy chain CDR3 comprising or consisting of SEQ ID NO:16.
  • Table 2 Exemplary CDR amino acid sequences.
  • an antibody domain comprises a light chain variable region sequence and/or a heavy chain variable region sequence that is disclosed in Table 3 of the present disclosure.
  • an antibody domain comprises: a heavy chain variable region comprising an amino acid sequence that has at least 75% sequence identity (for example, at least 39 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:2; and/or a light chain variable region comprising an amino acid sequence that has at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:1.
  • a heavy chain variable region comprising an amino acid sequence that has at least 75% sequence identity (for example, at least 39 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB
  • the antibody comprises a heavy chain variable region consisting of SEQ ID NO:2, and/or a light chain variable region consisting of SEQ ID NO:1.
  • an antibody domain comprises: a heavy chain variable region comprising an amino acid sequence that has at least 75% sequence identity (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO:2 and comprises a CDR1 (for example, SEQ ID NO:8), a CDR2 (for example, SEQ ID NO:9), and a CDR3 (for example, SEQ ID NO:10) that is identical to the CDRs of that heavy chain variable region sequence, and a light chain variable region comprising an amino acid sequence that has at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
  • an antibody domain comprises: a heavy chain variable region comprising an amino acid sequence that has at least 75% sequence identity (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:4; and/or a light chain variable region comprising an amino acid sequence that has at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:3.
  • a heavy chain variable region comprising an amino acid sequence that has at least 75% sequence identity (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:3.
  • the antibody comprises a heavy chain variable region consisting of SEQ ID NO:4, and/or a light chain variable region consisting of SEQ ID NO:3.
  • an antibody domain comprises: a heavy chain variable region comprising an amino acid sequence that has at least 75% sequence identity (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO:4 and comprises a CDR1 (for example, SEQ ID NO:14), a CDR2 (for example, SEQ ID NO:15), and a CDR3 (for example, SEQ ID NO:16) that is identical to the CDRs of that heavy chain variable region sequence, and a light chain variable region comprising an amino acid sequence that has at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
  • variable heavy and/or variable light sequence includes the disclosure of the associated (inherent) CDRs. Accordingly, the disclosure of each heavy chain variable region sequence is a disclosure of the heavy chain CDR sequences (CDR1, CDR2, and CDR3), and the disclosure of each light chain variable region sequence is a disclosure of the of the heavy chain CDR sequences (CDR1, CDR2, and CDR3).
  • CDR1, CDR2, and CDR3 An exemplary comparison of CDR numbering is shown in Table 4 below. Table 4. CDR numbering.
  • an antibody domain of a fusion protein comprises a constant region of a light chain sequence, or a portion or portions thereof, and/or a heavy chain sequence, or a portion or portions thereof.
  • an antigen-binding site of antibody domain may be engineered as a Fab fragment and include a CH1 region of a heavy chain of an antibody.
  • Constant chain antibody sequences are known to be the same or highly similar among immunoglobulins of the same isotype (see, for example, Janeway et al.
  • an antibody domain of a fusion protein according to the present disclosure comprises a constant region of a light chain sequence, or a portion or portions thereof, and/or a heavy chain sequence, or a portion or portions thereof derived from any of the antibodies described in Example 2 of the present disclosure.
  • an antibody domain comprises a constant region of a light chain sequence, or a portion or portions thereof, and/or a constant region of a heavy chain sequence, or a portion thereof, derived from mAb CoV2-11 or mAb CoV2-26.
  • an antibody domain comprises an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:64, SEQ ID NO:66; SEQ ID NO:68, or SEQ ID NO:70.
  • nucleic acid sequences [0117] It is contemplated that an antibody domain or antigen-binding sites of the fusion proteins according to the present disclosure may be encoded by any nucleic acid sequence that encodes the appropriate amino acid sequence.
  • An example is a nucleic acid sequence encoding amino acid sequence comprising SEQ ID NOs 5, 6, 7, 8, 9, and 10 (light chain CDRs 1, 2 and 3; and heavy chain CDRs 1 and 2, 3, respectively).
  • nucleic acid sequence encoding an amino acid sequence comprising a sequence that has at least 75% sequence identity (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1.
  • nucleic acid sequence encoding amino acid sequence comprising a sequence comprising an amino acid sequence that has at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2.
  • nucleic acid sequence encoding amino acid sequence comprising SEQ ID NOs 11, 12, 13, 14, 15, and 16 (light chain CDRs 1, 2 and 3; and heavy chain CDRs 1 and 2, 3, respectively).
  • nucleic acid sequence encoding an amino acid sequence comprising a sequence that has at least 43 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 75% sequence identity (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:3.
  • nucleic acid sequence encoding amino acid sequence comprising a sequence comprising an amino acid sequence that has at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4.
  • Nucleic acids include DNA and RNA. The design and production of nucleic acids encoding a desired amino acid sequence is well known. The codons selected for encoding each amino acid may be modified to optimize expression of the nucleic acid in the host cell of interest.
  • Fusion proteins according to the present disclosure include at least one neutralizing domain capable of specifically binding to a receptor binding domain (RBD) of a coronavirus spike protein.
  • RBD receptor binding domain
  • the neutralizing domain comprises a coronavirus receptor polypeptide.
  • the coronavirus receptor polypeptide comprises an ACE2 receptor ectodomain polypeptide. In some embodiments, the coronavirus receptor polypeptide comprises a receptor ectodomain polypeptide. In some embodiments, the coronavirus receptor polypeptide comprises DPP4 receptor ectodomain polypeptide. In some embodiments, the coronavirus receptor polypeptide comprises a human aminopeptidase N polypeptide. Some coronaviruses bind to various glycan structures on the cell surface, such as nonsialylated N-glycans, heparan sulfate, or modified sialic acid, for example, 9-O- acetylated sialic acid.
  • a neutralizing domain comprises one or more glycan molecules that serve as coronavirus receptors.
  • a coronavirus receptor polypeptide used in a neutralizing domain of the fusion proteins according to the present disclosure are modified to become catalytically inactive, that is, to lose their physiologically relevant catalytic activity.
  • Such catalytically inactive coronavirus receptor polypeptides can be referred to as “inactivated.”
  • ACE2 is a zinc metalloenzyme and carboxypeptidase located as an ectoenzyme on the surface of endothelial and other cells (Turner 2015).
  • ACE2 acts as its physiological counterbalance providing homeostatic regulation of circulating angiotensin II (Ang II) levels.
  • Ang II angiotensin II
  • modified ACE2 without peptidase 44 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 activity, as described in the present disclosure, avoids such side effects.
  • DPP4 is expressed on cells throughout the body, and is a key regulator of incretin hormone, which plays an important role in the maintenance of glucose homeostasis (Deacon 2019). Accordingly, exogenous administration of catalytically active ACE2 or DPP4 can lead to undesirable adverse side effects.
  • fusion proteins according to the present disclosure include catalytically inactive variants of ACE2 or DPP4 polypeptides in their neutralizing domains.
  • a coronavirus receptor polypeptide used in a neutralizing domain of the fusion proteins according to the present disclosure comprises two or more receptor ectodomain polypeptides sequences.
  • Some exemplary embodiments according to the present disclosure include two or more, such as two, DPP4 receptor ectodomain polypeptide sequences. [0121] Some examples of coronavirus receptor polypeptide sequences are listed in Table 5.
  • inactivating mutations shown in Table 5 for ACE2 ectodomain polypeptide can be included in various combinations in embodiments of inactivated ACE2 ectodomain polypeptide.
  • inactivating mutations shown in Table 5 for DPP4 ectodomain polypeptide can be included in various combinations in embodiments of inactivated DPP4 ectodomain polypeptide.
  • Table 5 Coronavirus receptor polypeptide sequences.
  • a coronavirus receptor polypeptide included in a neutralizing domain of fusion proteins according to the present disclosure is human ectodomain ACE2 receptor polypeptide.
  • Full-length human ACE2 is 805 amino acids in length (SEQ ID NO:51), of which amino acids 1-17 is a signal peptide that is cleaved from the mature protein. See NCBI Reference Sequence NP_001358344.1; see also UniProtKB Reference Q9BYF1.
  • the coronavirus receptor polypeptide included in a neutralizing domain of fusion proteins according to the present disclosure comprises an ACE2 receptor ectodomain polypeptide that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to one or more of the amino acid sequences of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:131, SEQ ID NO:98.
  • the ACE2 receptor ectodomain polypeptide comprises one or more mutations relative to wild-type ACE2 human sequence.
  • the one or more mutations result in an ACE2 receptor domain polypeptide with reduced or no peptidase activity.
  • One such mutation is H345L substitution in SEQ ID NO:51, shown in SEQ ID NO:18.
  • One more such mutation is H374N substitution in SEQ ID NO:51, shown 49 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 in SEQ ID NO:131.
  • the one or more mutations increase binding affinity of the ACE2 receptor ectodomain polypeptide for the RBD of a coronavirus spike protein.
  • the ACE2 receptor ectodomain polypeptide comprises amino acid substitutions at one or more of the following residues (the positions listed are relative to SEQ ID NO:51): the arginine at position 273 (R273), the histidine at position 378 (H378), the glutamate at position 402 (E402), the histidine at position 374 (H374), and the histidine at position 345 (H345).
  • the ACE2 receptor ectodomain polypeptide comprises one or more of the following amino acid substitutions relative to SEQ ID NO:51: R273A (i.e., the arginine residue at position 273 (relative to SEQ ID NO:51) is substituted for an alanine residue), H378A, E402A, H374N, and H345L.
  • R273A i.e., the arginine residue at position 273 (relative to SEQ ID NO:51) is substituted for an alanine residue
  • H378A i.e., the arginine residue at position 273 (relative to SEQ ID NO:51) is substituted for an alanine residue
  • H378A i.e., the arginine residue at position 273 (relative to SEQ ID NO:51) is substituted for an alanine residue
  • H378A i.e., the arginine residue at position 273 (rel
  • an ACE2 receptor ectodomain polypeptide included in a neutralizing domain of a fusion protein according to the present disclosure can include substitutions at the following residues (all in reference to SEQ ID NO:51), in any combination: H345; R273; H374; H378; and E402.
  • An ACE2 receptor ectodomain polypeptide included in a neutralizing domain of a fusion protein according to the present disclosure can include one or more of the following substitutions (all in reference to SEQ ID NO:51), in any combination: H345L; R273A; H374N; H378A; H378N; and E402A.
  • a coronavirus receptor polypeptide included in a neutralizing domain of fusion proteins according to the present disclosure is human ectodomain DPP4 receptor polypeptide.
  • Full-length human DPP4 is 766 amino acids in length (SEQ ID NO:52) and comprises an ectodomain at amino acids 29-766.
  • the DPP4 ectodomain polypeptide can comprise amino acids 29-766 of SEQ ID NO:52 or variants thereof that are longer and/or shorter at one or both ends, such as, for example, a polypeptide comprising SEQ ID NO:19.
  • the coronavirus receptor polypeptide included in a neutralizing domain comprises a DPP4 receptor ectodomain polypeptide that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to one or more of the amino acid sequences of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, or SEQ ID NO:120.
  • a DPP4 receptor ectodomain polypeptide includes two or more, such as two, DPP4 receptor ectodomain polypeptide sequences.
  • the DPP4 receptor ectodomain polypeptide comprises one or more mutations relative to wild-type DPP4 human sequence.
  • the one or more mutations result in a DPP4 receptor domain polypeptide with reduced or no peptidase activity.
  • the DPP4 receptor ectodomain polypeptide comprises one or more of the mutations described in Li et al. 2020, Song et al. 2014, or Wang et al. 2013.
  • the DPP4 receptor ectodomain polypeptide comprises amino acid substitutions at one or more of the following residues (the positions listed are relative to SEQ ID NO:52): the serine at position 630 (S630), the tryptophane at position 547 (Y547), the aspartate at position 708 (D708), the histidine at position 740 (H740), and the histidine at position 750 (H750).
  • the DPP4 receptor ectodomain polypeptide comprises one or more of the following amino acid substitutions: S630A (i.e., the serine residue at position 630 (relative to SEQ ID NO:52) is substituted for an alanine residue), Y547F, D708A, H740L, and H750E.
  • S630A i.e., the serine residue at position 630 (relative to SEQ ID NO:52
  • Y547F i.e., the serine residue at position 630 (relative to SEQ ID NO:52) is substituted for an alanine residue
  • Y547F i.e., the serine residue at position 630 (relative to SEQ ID NO:52) is substituted for an alanine residue
  • Y547F i.e., the serine residue at position 630 (relative to SEQ ID NO:52) is substituted for an alanine residue
  • Y547F i
  • a DPP4 receptor domain polypeptide included in a neutralizing domain of a fusion protein according to the present disclosure can include substitutions at the following residues (all in reference to SEQ ID NO:52), in any combination: S630; Y547; D708; H740; and H750.
  • An DPP4 receptor ectodomain polypeptide included in a neutralizing domain of a fusion protein according to the present disclosure can include one or more of the following substitutions (all in reference to SEQ ID NO:52), in any combination: S630A; Y547F, D708A, H740L; and H750E. iii.
  • the domain comprises an antigen-binding site of a coronavirus neutralizing antibody.
  • the neutralizing antibody binds to the RBD of a coronavirus spike protein.
  • neutralizing domain comprising a neutralizing antibody may be engineered in any suitable configuration (e.g., as an intact immunoglobulin, an scFv, an antigen-binding fragment, or as part of a CrossMAb), as discussed further below for antibodies that specifically bind an epitope in a conserved region of a coronavirus spike protein.
  • a neutralizing domain based on a neutralizing antibody may, in some embodiments, be based on bispecific antibodies, which may be also be generated using any method known in the art for generating bispecific antibodies (see, for example, Brinkmann and Kontermann 2017).
  • neutralizing antibody sotrovimab binds to a conserved site on the RBD (see International Patent Publication No. WO2021252878).
  • An antigen-binding site based on a neutralizaing antibody that is able to bind to the RBD and promote viral neutralization may be useful in the fusion proteins according to the present disclosure.
  • Exemplary neutralizing antibodies (along with clinical names where applicable) are listed in Table A below (neutralizing antibodies indicated in bold have been shown to neutralize the Omicron SARS-CoV-2 variant effectively), and larger lists are available in the Coronavirus Antibody Database (Raybould et al. 2021).
  • fusion proteins according to the present disclosure comprise any of the neutralizing antibodies discussed herein.
  • Heavy chain (HC) and light chain (LC) sequences of particular neutralizing antibodies are shown in Table 7.
  • the neutralizing antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to any one of SEQ ID NOs 53, 55, 56, 58, or 60.
  • the neutralizing antibody comprises a light chain variable region comprising an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs 54, 57, 59, or 61. In some embodiments, the neutralizing antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 90% identity to any one of any one of SEQ ID NOs 53, 55, 56, 58, or 60, and a light chain variable region comprising an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs 54, 57, 59, or 61.
  • the neutralizing antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:53 and a light chain variable region comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:55. In some embodiments, the neutralizing antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:54 and a light chain variable region comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:55.
  • the neutralizing antibody comprises a heavy chain variable 52 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 region comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:56 and a light chain variable region comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:57.
  • the neutralizing antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:58 and a light chain variable region comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:59.
  • the neutralizing antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:60 and a light chain variable region comprising an amino acid sequence that has at least 90% identity to SEQ ID NO:61.
  • Table 6 Exemplary coronavirus neutralizing antibodies. 53 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 Table 7. Exemplary coronavirus neutralizing antibody sequences (HC – heavy chain; LC – light chain).
  • Other domains, sequences, and sequence modifications i. Multimerization domains [0129] Fusion proteins according to the present disclosure may contain additional domain or domains.
  • an additional domain is a multimerization domain, which may be a dimerization, trimerization, or tetramerization domain.
  • Various dimerization, trimerization, and tetramerization domains are known and can be used.
  • Amino acid sequences of exemplary multimerization domains are shown in Table 8.
  • One example of a suitable dimerization domain can be derived from antibody Fc region of various antibody isotypes.
  • a dimerization domain included in a fusion protein according to the present disclosure can be derived from Fc region of IgGs.
  • a dimerization domain comprises or consists of an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:62.
  • a suitable trimerization domain can 54 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 be derived from a cartilage matrix protein (CMP) trimerization domain.
  • CMP cartilage matrix protein
  • a trimerization domain comprises or consists of an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:63.
  • a suitable trimerization domain is GCN4-pII trimerization domain.
  • a trimerization domain comprises or consists of an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:104.
  • a suitable trimerization domain is GCN4-pIL trimerization domain.
  • a trimerization domain comprises or consists of an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:105.
  • a suitable tetramerization domain is a vasodilator-stimulated phosphoprotein (VASP) tetramerization domain.
  • VASP vasodilator-stimulated phosphoprotein
  • a tetramerization domain comprises or consists of an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:106.
  • a suitable tetramerization domain is a tetramerization domain derived from right-handed coiled-coil (RHCC) protein.
  • a trimerization domain comprises or consists of an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:107.
  • a suitable tetramerization domain is GCN4- pLI tetramerization domain.
  • a trimerization domain comprises or consists of an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:115.
  • Linkers [0130] Fusion proteins according to the present disclosure may include at least one linker.
  • Linkers also referred to as spacers, are flexible molecules or a flexible stretch of molecules that joins or connects two portions, such as domains, of a fusion protein according to the present disclosure.
  • a linker may increase the range of orientations that may be adopted by the domains of the fusion protein or modified protein.
  • a linker may be optimized to produce desired effects in the fusion protein or modified protein. Aspects of linker design and considerations are described, for example, in Chen et al. 2013 and Klein et al. 2014.
  • fusion proteins according to the present disclosure include at least one (such as one, two, three, four, five, etc.) peptide linker.
  • fusion proteins according to the present disclosure include at least one (such as one, two, three, four, five, etc.) non-peptide linker. In some embodiments, fusion proteins according to the present disclosure include at least one peptide linker and at least one non- peptide linker. Fusion proteins according to the present disclosure may also include a plurality of different linkers, including at least one peptide linker, at least one non-peptide linker, or at least one peptide linker and at least one non-peptide linker.
  • the length of a linker may affect the ability of the fusion protein to bind to and/or neutralize a coronavirus virion, for example, by facilitating the binding of one or 56 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 both the neutralizing domain and the antibody domain to their respective viral protein binding sites.
  • a longer linker may be desirable when the RBD of a coronavirus spike protein (i.e., to which the neutralizing domain of the fusion protein binds) and the conserved epitope of the coronavirus spike protein (i.e., to which the antibody domain of the fusion protein binds) are far away from each other.
  • a shorter linker may be desirable when the RBD and the conserved epitope are close to each other.
  • the length of the linker may also be selected based on the binding orientation of the antibody domain of the fusion protein.
  • the antibody domain binds the epitope in the conserved region of the coronavirus spike protein in such a way that the neutralizing domain is brought into close proximity of the coronavirus spike protein RBD, a shorter linker may be desirable.
  • Various factors may influence the binding orientation of the antibody that specifically binds an epitope in a conserved region of a coronavirus spike protein (and, thus, the fusion protein or modified protein), including, but not limited to, the order of the domains in the fusion protein or modified protein and the antibody format used.
  • the linkers used in the fusion proteins according to the present disclosure are peptide linkers that are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long.
  • the linkers of the fusion proteins according to the present disclosure are about 40 angstroms ( ⁇ ) to about 450 ⁇ in length (e.g., about 40 ⁇ to about 300 ⁇ , about 150 ⁇ to about 400 ⁇ , about 150 ⁇ to about 350 ⁇ , about 200 ⁇ to about 300 ⁇ , about 200 ⁇ to about 400 ⁇ , about 150 ⁇ to about 45 ⁇ , about 100 ⁇ , about 150 ⁇ , about 200 ⁇ , about 250 ⁇ , about 300 ⁇ , about 350 ⁇ , about 400 ⁇ , or about 450 ⁇ ).
  • a peptide linker may be, for example, 5 to 100 more amino acids in length (e.g., 5 aa, 10 aa, 15 aa, 20 aa, 25 aa, 30 aa, 35 aa, 40, 45 aa, 50 aa, 55 aa, 60 aa, 65 aa, 70 aa, 75 aa, 80 aa, 85 aa, 90 aa, 95 aa, or 100 aa).
  • Non-peptide linkers may similarly be selected based on the size of the repeating molecule(s) which they are made.
  • a polyethylene glycol (PEG) monomer is also approximately 4 ⁇ .
  • a polyethylene glycol (PEG) linker may be, for example, 10 to 100 PEG linkages in length (e.g., 10 PEG linkages, 15 PEG linkages, 20 PEG linkages, 25 PEG linkages, 30 PEG linkages, 35 PEG linkages, 40 PEG linkages, 45 PEG linkages, 50 PEG linkages, 55 PEG linkages, 60 PEG linkages, 65 PEG linkages, 70 PEG linkages, 75 PEG linkages, 80 PEG linkages, 85 PEG linkages, 90 PEG linkages, 95 PEG linkages, or 100 PEG linkages).
  • linker sequence may have various conformations in secondary structure, such as helical, ⁇ -strand, coil/bend, and turns.
  • a linker sequence may have an extended conformation and function as an independent domain that does not interact with the adjacent protein domains.
  • Linker sequences may be flexible or rigid.
  • Flexible linkers provide a certain degree of movement or interaction between the polypeptide domains and are generally rich in small or polar amino acids such as Gly and Ser (e.g., at least 90%, at least 95%, at least 98%, at least 99%, or all of the amino acid residues of the linker are either Gly or Ser).
  • a rigid linker can be used to keep a fixed distance between the domains and to help maintain their independent functions.
  • Linker attachment can be through an amide linkage (e.g., a peptide bond) or other functionalities as discussed further below.
  • a peptide linker according to the present disclosure comprises an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs 72-83 and 108-114, which are shown in Table 9.
  • the linker comprises one or more repeats of GGGGS (SEQ ID NO:84) and/or one or more repeats of GSSGSS (SEQ ID NO:85).
  • Additional exemplary peptide linkers include, but are not limited to, peptide linkers comprising SGSETPGTSESATPE (SEQ ID NO:86), SGSETPGTSESATPES (SEQ ID NO:87), (GGGGS) 3 (SEQ ID NO:88), (GGGGS) 5 (SEQ ID NO:89), (GGGGS) 10 (SEQ ID NO:90), GGGGGGGG (SEQ ID NO:91), GSAGSAAGSGEF (SEQ ID NO:92), A(EAAAK)3A (SEQ ID NO:93), or A(EAAAK) 10 A (SEQ ID NO:94).
  • Additional non-limiting exemplary linkers that can be used include those disclosed in Chen et al. 2014 and Rosemalen et al.
  • the peptide linker comprises a protease recognition site, e.g., a Tobacco Etch Virus (TEV) protease cut site ENLYFQG (SEQ ID NO:95).
  • TSV Tobacco Etch Virus
  • Such protease recognition sites may be useful for testing binding of the fusion proteins compared to the individual domains, as in the Examples herein.
  • a non-peptide linker can comprise one or more of a number of known chemical linkers.
  • Exemplary chemical linkers can include one or more units of beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminobexanoic acid (Ahx), PEG multimers, and trioxatricdeacan-succinamic acid (Ttds).
  • the non- peptide linker comprises one or more units of polyethylene glycol (PEG), which is commonly used as a linker for conjugation of polypeptide domains due to its water solubility, lack of toxicity, low immunogenicity, and well-defined chain lengths.
  • PEG polyethylene glycol
  • the number of PEG linkage units may be selected based on the desired length of the linker.
  • Modified proteins comprising a linker can be produced in a variety of ways.
  • a neutralizing polypeptide and an antibody that specifically binds an epitope in a conserved region of a coronavirus spike protein may be produced separately (e.g., in vitro or by expression in and 59 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 purification from host cells) and chemically linked in vitro.
  • a neutralizing polypeptide, an antibody that specifically binds an epitope in a conserved region of a coronavirus spike protein, and a linker can each be produced separately and chemically linked in vitro.
  • a partial modified protein comprising a neutralizing polypeptide with or without a linker.
  • a partial modified protein comprising an antibody that specifically binds an epitope in a conserved region of a coronavirus spike protein as described above with or without a linker.
  • Various chemical linkers may be used to cross link two amino acid residues. ii.
  • Fusion proteins according to the present disclosure can include sequences useful for protein isolation.
  • the fusion proteins according to the present disclosure can comprise an affinity tag, for example an AviTagTM, a Myc tag, a polyhistidine tag (such as 8XHis tag), an albumin-binding protein, an alkaline phosphatase, an AU1 epitope, an AU5 epitope, a biotin-carboxy carrier protein (BCCP), or a FLAG epitope, to name a few.
  • an affinity tag for example an AviTagTM, a Myc tag, a polyhistidine tag (such as 8XHis tag), an albumin-binding protein, an alkaline phosphatase, an AU1 epitope, an AU5 epitope, a biotin-carboxy carrier protein (BCCP), or a FLAG epitope, to name a few.
  • the affinity tags are useful for protein isolation. See, for example, Kimple et al. 2013.
  • fusion proteins according to the present disclosure comprise a signal sequence useful for protein isolation, for example a mutated Interleukin-2 signal peptide sequence, which promotes secretion and facilitates protein isolation. See, for example, Low et al. 2013.
  • a fusion protein or modified protein comprises a protease recognition site, for example, TEV protease cut site, which may be useful for, among other things, removal of a signal peptide or affinity purification tag following isolation of the fusion protein or modified protein.
  • the amino acid sequences of fusion proteins according to the present disclosure can include modifications that improve binding, stability, or other properties.
  • one or more cysteine substitutions, or substitutions with noncanonical amino acids containing long side-chain thiols may be introduced into the polypeptides that can form disulfide bonds between two polypeptides that have interacted to form a dimer.
  • the substitutions improve polypeptide stability.
  • amino acids found to not contribute to the binding specificity and/or affinity of a fusion protein or modified protein can be deleted without a loss in the respective activity. Insertions, deletions, substitutions, or other selected mutations of particular regions or specific amino acids residues can be made, provided the activity of the fragment is not significantly altered or impaired compared to the non-mutated fusion protein can be made. Modifications of amino acid sequences can be made by known methods.
  • Nucleic acid sequences encoding fusion proteins according to the present disclosure or their components can be codon-optimized to alter, for example, maximize expression, in a host cell or organism.
  • the amino acids used in the amino acid sequences of the fusion proteins according to the present disclosure can be any of the 20 naturally occurring amino acids, D-stereoisomers of the naturally occurring amino acids, unnatural amino acids, and/or chemically modified amino acids.
  • Amino acid sequences of the fusion proteins according to the present disclosure and their components can also include conservative amino acid substitutions.
  • Amino acid sequences of the fusion proteins according to the present disclosure can be further modified. The modifications can be covalent or non-covalent modifications.
  • modifications can be introduced into the fusion protein or modified protein by, e.g., reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • Suitable sites for modification can be chosen using any of a variety of criteria including, e.g., structural analysis or amino acid sequence analysis of the fusion proteins according to the present disclosure.
  • the fusion proteins according to the present disclosure may be labeled by a variety of means for use in diagnostic and/or pharmaceutical applications. [0140]
  • the fusion proteins according to the present disclosure can be conjugated to a heterologous moiety.
  • the heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin.
  • a therapeutic agent e.g., a toxin or a drug
  • a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin.
  • Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)), or chloramphen
  • Suitable radioactive labels include, e.g., 32 P, 33 P, 14 C, 125 I, 131 I, 35 S, and 3 H.
  • Suitable fluorescent labels include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLightTM 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor ® 700, Cy5, allophycocyanin, and Cy7.
  • Luminescent labels include, e.g., any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates.
  • suitable europium chelates include the europium chelate of diethylene triamine pentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).
  • Enzymatic labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase. Another labeling technique which may result in greater sensitivity is coupling the fusion proteins according to the present disclosure to low molecular weight haptens.
  • haptens can then be specifically altered by means of a second reaction.
  • haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific antihapten antibodies.
  • Two proteins e.g., an antibody and a heterologous moiety
  • cross linkers are those that link two amino acid residues via a linkage that includes a “hindered” disulfide bond.
  • a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase.
  • One suitable reagent 4-succinimidyloxycarbonyl- -methyl- (2-pyridyldithio) toluene (SMPT)
  • SMPT 4-succinimidyloxycarbonyl- -methyl- (2-pyridyldithio) toluene
  • Heterobifunctional reagents that cross-link by a different coupling moiety on each protein can also be used.
  • cross-linkers include, without limitation, reagents which link two amino groups (e.g., N-5-azido-2- nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate).
  • reagents which link two amino groups e.g., N-5-azido-2- nitrobenzoyloxysuccinimide
  • two sulfhydryl groups e.g
  • a radioactive label can be directly conjugated to the amino acid backbone of the fusion protein or modified protein.
  • the radioactive label can be included as part of a larger molecule (e.g., 125 I in meta-[ 125 I]iodophenyl-N-hydroxysuccinimide ([ 125 I]mIPNHS), which binds to free amino groups to form meta-iodophenyl (mIP) derivatives of relevant proteins or chelates (e.g., to DOTA or DTPA), which is in turn bound to the protein backbone.
  • a larger molecule e.g., 125 I in meta-[ 125 I]iodophenyl-N-hydroxysuccinimide ([ 125 I]mIPNHS)
  • Such methods involve incubating the proteins with the radioactive label/chelate under conditions (e.g., pH, salt concentration, and/or temperature) that facilitate binding of the radioactive label/chelate to the protein.
  • conditions e.g., pH, salt concentration, and/or temperature
  • Methods for conjugating a fluorescent label (sometimes referred to as a fluorophore) to a protein are known in the art of protein chemistry.
  • fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores.
  • the fluorophores can be conjugated to a heterobifunctional cross-linker moiety such as sulfo-SMCC. Suitable conjugation methods involve incubating an antibody protein or fragment thereof with the fluorophore under conditions that facilitate binding of the fluorophore to the protein.
  • fusion proteins according to the present disclosure can be modified with a moiety that improves the stabilization and/or retention of the fusion protein or modified protein in circulation, e.g., in blood, serum, or other tissues.
  • the fusion protein or modified protein can be PEGylated or HESylated (coupled to biodegradable hydroxyethyl starch (HES)).
  • the stabilization moiety can improve the stability, or retention of, the fusion protein or modified protein (or fragment) by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.
  • fusion proteins according to the present disclosure or their components can be glycosylated.
  • a fusion protein or modified protein can be subjected to enzymatic or chemical treatment, or produced from a cell, such that the fusion protein or modified protein has reduced or absent glycosylation.
  • Fusion protein domain configurations [0145] The domains of the fusion proteins according to the present disclosure may be present in a variety of configurations.
  • Such configurations may be selected for various reasons, for example, but not limited to, to optimize binding of and/or neutralization by the fusion protein for one or more coronavirus spike protein targets.
  • Exemplary, non-limiting embodiments are provided below. Descriptions are given, along with specific examples. Domains listed in order are intended to show fusion proteins according to the present disclosure from the amino-terminus (N-terminus) to the carboxy-terminus (C-terminus). However, it will be appreciated that additional configurations are possible (for example, either the N-terminus or C-terminus of a given domain may be joined to the next domain).
  • the fusion proteins comprise a single neutralizing domain (ND) and a single antibody domain (AD).
  • a fusion protein comprises a single neutralizing that binds to a RBD of a coronavirus spike protein and a single antibody domain that specifically binds an epitope in a conserved region of a coronavirus spike protein.
  • the two domains 63 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 can be ordered in either orientation in a fusion protein molecule, for example, ND-linker-AD or AD-linker-ND).
  • the neutralizing domain can include an ACE2-based polypeptide (such as one single DPP4-based polypeptide or two or more DPP4-based polypeptides).
  • the neutralizing domain can include a DPP4-based polypeptide (such as one DPP4-based polypeptide or two or more DPP4-based polypeptides).
  • the neutralizing domain and the antibody domain of a fusion protein may be linked in any orientation.
  • the N-terminus or C-terminus of the neutralizing domain may be linked to the N-terminus or C-terminus of the antibody domain.
  • the neutralizing domain and/or the antibody domain may be linked internally to another domain of the fusion protein.
  • the neutralizing domain may be flanked by two antibody domains or two portions of the antibody.
  • the fusion proteins according to the present disclosure include more than one (two, three, four, five, six, etc.) neutralizing domains and more than one (two, three, four, five, six, etc.) antibody domain.
  • the fusion proteins according to the present disclosure include at least one neutralizing domain.
  • the fusion proteins include more than one neutralizing domain (for example, two or more, three or more, four or more, five or more etc. neutralizing domains).
  • each of the neutralizing polypeptides in a fusion protein or modified protein bind to the same coronavirus spike protein.
  • two or more neutralizing domains bind to spike proteins of different coronaviruses.
  • a single fusion protein may include at least one (for example, one, two, three etc.) ACE2-based neutralizing domain, at least one (for example, one, two, three etc.) DPP-based neutralizing domain, and at least one (for example, one, two, three etc.) antibody-based neutralizing domain.
  • a single fusion protein may include an ACE2-based neutralizing domain and a DPP-based neutralizing domain, a DPP-based neutralizing domain, and at least one (for example, one, two, three etc.) antibody-based neutralizing domain, or an ACE2-based neutralizing domain and at least one antibody-based neutralizing domain (for example, one, two, three etc.).
  • Such configurations may allow a given fusion protein or modified protein to bind to and/or neutralize different coronaviruses (e.g., SARS-CoV-2 and MERS-CoV).
  • a single fusion protein can include two or more ACE2-based neutralizing domains (ACE2 ND) along with an antibody- based neutralizing domain (Ab ND), in various configurations, such as, ACE2 ND-linker-Ab ND-linker-ACE2 ND or ACE2 ND-linker-ACE2 ND-linker-Ab ND.
  • ACE2 ND ACE2-based neutralizing domains
  • Ab ND antibody- based neutralizing domain
  • a single fusion protein can include two or more DPP4-based neutralizing domains 64 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 (DPP4 ND) along with an antibody-based neutralizing domain in various configurations, for example, DPP4 ND-linker-Ab ND-linker-DPP4 ND-linker-DPP4 ND-linker-AD).
  • One or more of the neutralizing domains may be linked to the other domains of the fusion protein in any order and orientation.
  • fusion proteins include at least one antibody domain. In some embodiments, fusion proteins include more than one antibody domain (for example, two or more, three or more, four or more, five or more, etc. antibody domains).
  • such multiple antibody domains can specifically bind to the same coronavirus spike protein epitope or to different coronavirus spike protein epitopes.
  • the antibody domains are connected via linkers, which are discussed elsewhere in the present disclosure.
  • a fusion protein according to the present disclosure may comprise two antibody domains, a first antibody domain specific for a first coronavirus spike protein and a second antibody domain specific for a second coronavirus protein (e.g., first ND-linker-first AD-linker-second AD-linker-second ND).
  • a fusion protein according to the present disclosure may comprise two antibody domains that each specifically bind to a conserved epitope on a coronavirus spike protein (i.e., two antibody domains derived from the same antibody or two antibody domains derived from different antibodies that specifically bind to the same coronavirus spike protein; e.g., ND-linker-first AD-linker-second AD- linker).
  • One or more of the antibody domains may be linked to the other domains of the fusion protein or modified protein in any order and orientation.
  • an amino-terminus (N-terminus) or carboxy-terminus (C-terminus) of an antibody domain may be linked to an N-terminus or C-terminus of another antibody domain or to the N-terminus or C-terminus of a neutralizing domain.
  • a fusion protein or modified protein may have an N-terminus or C-terminus of a first antibody domain linked to the N-terminus of a first neutralizing domain and an N-terminus or C-terminus of a second antibody domain, which is then linked to the C-terminus of the second neutralizing domain.
  • the one or more antibody domains may be linked internally to another domain of the fusion protein or modified protein.
  • the fusion proteins according to the present disclosure include at least one (one or more) multimerization domain, such as a dimerization domain, a trimerization domain, or a tetramerization domain.
  • Some embodiments of the fusion proteins according to the present disclosure include a dimerization domain derived from antibody Fc region of various antibody isotypes. Such a dimerization domain may be referred to as “Fc” in the present disclosure.
  • a dimerization domain included in a fusion protein according to the present disclosure can be derived from Fc region of IgGs.
  • Some embodiments of the fusion proteins according to the present disclosure include a trimerization domain derived from a cartilage matrix protein (CMP) trimerization domain. Such a trimerization domain may be referred to as “tri” in the present disclosure.
  • Some embodiments of the fusion proteins according to the present disclosure include a trimerization domain derived from GCN4-pII trimerization domain.
  • Some embodiments of the fusion proteins according to the present disclosure include a tetramerization domain derived from vasodilator-stimulated phosphoprotein (VASP) tetramerization domain.
  • VASP vasodilator-stimulated phosphoprotein
  • fusion proteins are expressed and form a multimer in a host cell which can be isolated.
  • the fusion protein monomers forming the multimer are the same fusion protein.
  • the multimer includes fusion proteins that are different fusion proteins according to the present disclosure.
  • the fusion proteins according to the present disclosure comprise multimerization domains to facilitate dimerization.
  • the fusion proteins are expressed, isolated, and then linked chemically to form dimers. Methods of producing fusion proteins according to the present disclosure are discussed in more detail elsewhere in the present disclosure.
  • a multimerization domain such as a dimerization domain, a trimerization domain, or a tetramerization domain
  • a multimerization domain may be linked to the other domains of the fusion protein or modified protein in any order and orientation.
  • an amino-terminus (N-terminus) or carboxy- terminus (C-terminus) of a multimerization domain may be linked to an N-terminus or C-terminus of an antibody domain or to the N-terminus or C-terminus of a neutralizing domain.
  • a multimerization domain may be flanked by two portions of a neutralizing domain or an antibody domain.
  • N-terminus of neutralizing domain is linked to C-terminus of an antibody domain
  • C-terminus of a neutralizing domain is linked to N-terminus of a multimerization domain 66 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 (AD – ND – MD orientation).
  • N-terminus of neutralizing iACE2 domain is linked to C-terminus of CoV2-26 Fab domain, and C-terminus of neutralizing iACE2 domain is linked to N-terminus of Fc domain (CoV2-26-Fab – iACE2 – Fc orientation illustrated in FIG. 15A).
  • N-terminus of neutralizing iACE2 domain is linked to C-terminus of CoV2-26 Fab domain, and C-terminus of neutralizing iACE2 domain is linked to N-terminus of CMP trimerization domain (CoV2-26-Fab – iACE2 – tri orientation illustrated in FIG. 1A).
  • N-terminus of neutralizing iDPP4 or iDPP4-iDPP4 domain is linked to C-terminus of CoV2-26 Fab domain
  • C-terminus of neutralizing iDPP4 or iDPP4- iDPP4 domain is linked to N-terminus of Fc domain (CoV2-26-Fab – iDPP4 – Fc or CoV2-26-Fab – iDPP4-iDPP4 – Fc orientation illustrated in FIG. 124A).
  • N-terminus of neutralizing iDPP4 or iDPP4-iDPP4 domain is linked to C-terminus of CoV2-26 Fab domain
  • C-terminus of neutralizing iDPP4 or iDPP4-iDPP4 domain is linked to N-terminus of CMP trimerization domain (CoV2-26-Fab – iDPP4 – tri or CoV2-26-Fab – iDPP4-iDPP4 – tri orientation illustrated in FIG. 25A).
  • N-terminus of an antibody domain was linked to C-terminus of a neutralizing domain
  • C-terminus of an antibody domain was linked to N-terminus of a multimerization domain (ND – AD – MD orientation).
  • N-terminus of CoV2-26 Fab domain was linked to C-terminus of iACE2 domain
  • C-terminus of CoV2-26 Fab domain was linked to N-terminus of Fc domain (iACE2 – CoV2-26-Fab – Fc orientation illustrated in FIG. 15B).
  • N-terminus of CoV2-26 Fab domain was linked to C-terminus of iACE2 domain
  • C-terminus of CoV2-26 Fab domain was linked to N-terminus of CMP trimerization domain (iACE2 – CoV2-26-Fab – tri orientation illustrated in FIG. 1B).
  • N-terminus of CoV2-26 Fab domain was linked to C-terminus of iDPP4 or iDPP4- iDPP4 domain
  • C-terminus of CoV2-26 Fab domain was linked to N-terminus of Fc domain (iDPP4 – CoV2-26-Fab – Fc or iDPP4-iDPP4 – CoV2-26-Fab – Fc orientation illustrated in FIG. 24B).
  • N-terminus of CoV2-26 Fab domain was linked to C-terminus of iDPP4 or iDPP4-iDPP4 domain
  • C-terminus of CoV2-26 Fab domain was linked to N-terminus of CMP trimerization domain (iDPP4 – CoV2-26-Fab – tri or iDPP4-iDPP4 – CoV2-26-Fab – tri orientation illustrated in FIG. 25B).
  • N-terminus of a multimerization domain was linked to C-terminus of an antibody domain, and C-terminus of a multimerization domain was linked to N-terminus neutralizing 67 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 domain (AD – MD – ND orientation).
  • N-terminus of Fc domain was linked to C-terminus of CoV2-26 Fab domain, and C-terminus of Fc domain was linked to N-terminus of iACE2 domain (CoV2-26-Fab – Fc – iACE2 orientation illustrated in FIG. 15C).
  • N-terminus of CMP trimerization domain was linked to C-terminus of CoV2-26 Fab domain, and C-terminus of CMP trimerization domain was linked to N-terminus of iACE2 domain (CoV2-26-Fab – tri – iACE2 orientation illustrated in FIG. 1C).
  • N-terminus of Fc domain was linked to C-terminus of CoV2-26 Fab domain
  • C-terminus of Fc domain was linked to N-terminus of iDPP4 or iDPP4-iDPP4 domain (CoV2-26-Fab – Fc – iDPP4 CoV2-26-Fab – Fc – iDPP4-iDPP4 orientation illustrated in FIG. 24C).
  • N-terminus of CMP trimerization domain was linked to C-terminus of CoV2-26 Fab domain
  • C-terminus of CMP trimerization domain was linked to N-terminus of iDPP4 or iDPP4-iDPP4 domain (CoV2-26-Fab – tri – iDPP4 CoV2-26-Fab – tri – iDPP4-iDPP4 orientation illustrated in FIG. 24C).
  • an antibody domain need not be a CoV2-26-Fab-based antibody domain and may be any antibody domain described elsewhere in the present disclosure.
  • a neutralizing domain need not be an ACE2-based or a DPP4-based neutralizing domain and may be any neutralizing domain described elsewhere in the present disclosure.
  • a multimerization domain need not be an Fc domain or a CMP trimerization domain and may be any multimerization domain described elsewhere in the present disclosure.
  • a nucleic acid sequence according to the present disclosure encodes one or more polypeptides comprising an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:64, SEQ ID NO:66; SEQ ID NO:68, or SEQ ID NO:70.
  • a nucleic acid sequence according to the present disclosure encodes one or more polypeptides of an antibody domain of fusion proteins according to the present disclosure.
  • a nucleic acid sequence according to the present disclosure encodes a polypeptide comprising an 68 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 antigen-binding site of the antibody domain of the fusion proteins according to the present disclosure.
  • a nucleic acid encodes a polypeptide comprising one or more of the following amino acid sequences: a light chain CDR1 comprising or consisting of SEQ ID NO:5; a light chain CDR2 comprising or consisting of SEQ ID NO:6; a light chain CDR3 comprising or consisting of SEQ ID NO:7; a heavy chain CDR1 comprising or consisting of SEQ ID NO:8; a heavy chain CDR2 comprising or consisting of SEQ ID NO:9; and a heavy chain CDR3 comprising or consisting of SEQ ID NO:10.
  • an antigen binding site comprises the following CDRs: a light chain CDR1 comprising or consisting of SEQ ID NO:11; a light chain CDR2 comprising or consisting of SEQ ID NO:12; a light chain CDR3 comprising or consisting of SEQ ID NO:13; a heavy chain CDR1 comprising or consisting of SEQ ID NO:14; a heavy chain CDR2 comprising or consisting of SEQ ID NO:15; and a heavy chain CDR3 comprising or consisting of SEQ ID NO:16.
  • a nucleic acid sequence according to the present disclosure encodes a polypeptide comprising one or more of the following sequences: a heavy chain variable region comprising an amino acid sequence that has at least 75% sequence identity (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:2; and/or a light chain variable region comprising an amino acid sequence that has at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:1.
  • a nucleic acid sequence according to the present disclosure encodes a polypeptide comprising a heavy chain variable region consisting of SEQ ID NO:2, and/or a light chain variable region consisting of SEQ ID NO:1.
  • a nucleic acid sequence according to the present disclosure encodes a polypeptide comprising one or more of the following sequences: a heavy chain variable region comprising an amino acid sequence that has at least 75% sequence identity (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO:2 and comprises a CDR1 (for example, SEQ ID NO:8), a CDR2 (for example, SEQ ID NO:9), and a CDR3 (for example, SEQ ID NO:10) that is identical to the CDRs of that heavy chain variable region sequence, and a light chain variable region comprising an amino acid sequence that has at least 70% sequence identity (for
  • a nucleic acid sequence according to the present disclosure encodes a polypeptide comprising one or more of the following sequences: a heavy chain variable region comprising an amino acid sequence that has at least 75% sequence identity (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:4; and/or a light chain variable region comprising an amino acid sequence that has at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:3.
  • a heavy chain variable region comprising an amino acid sequence that has at least 75% sequence identity (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:3.
  • a nucleic acid sequence according to the present disclosure encodes a polypeptide comprising the following sequences: a heavy chain variable region consisting of SEQ ID NO:4, and/or a light chain variable region consisting of SEQ ID NO:3.
  • a nucleic acid sequence according to the present disclosure encodes a polypeptide comprising one or more of the following sequences: a heavy chain variable region comprising an amino acid sequence that has at least 75% sequence identity (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO:4 and comprises a CDR1 (for example, SEQ ID NO:14), a CDR2 (for example, SEQ ID NO:15), and a CDR3 (for example, SEQ ID NO:16) that is identical to the CDRs of that heavy chain variable region sequence, and a light chain variable region comprising an amino acid sequence that has at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO:3 and comprises a
  • a nucleic acid sequence according to the present disclosure encodes a polypeptide comprising one or more amino acid sequence that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:64, SEQ ID NO:66; SEQ ID NO:68, or SEQ ID NO:70.
  • a nucleic acid sequence according to the present disclosure encodes a polypeptide comprising one or more amino acid sequences of the neutralizing domains of the fusion proteins according to the present disclosure.
  • a nucleic acid sequence according to the present disclosure encodes one or more polypeptides comprising one or more amino acid sequences at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or 70 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 SEQ ID NO:20.
  • a nucleic acid sequence according to the present disclosure encodes one or more polypeptides comprising one or more amino acid sequences In some embodiments, a nucleic acid sequence according to the present disclosure encodes one or more of the amino acid sequences of SEQ ID NOs 53-61. [0161] In some embodiments, a nucleic acid sequence according to the present disclosure encodes a polypeptide comprising a fusion protein according to the embodiments of the present disclosure.
  • a nucleic acid sequence according to the present disclosure encodes one or more polypeptides comprising one or more amino acid sequences at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:40,
  • recombinant nucleic acids according to the embodiments of the present disclosure can be DNA or RNA.
  • nucleic acid constructs including nucleic acids according to the embodiments of the present disclosure are also provided.
  • An exemplary DNA construct comprises a promoter operably linked to recombinant DNA according to the present disclosure.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • Numerous promoters can be used in the constructs described herein.
  • a promoter is a region or a sequence located upstream and/or downstream from the start of transcription that is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • the promoter can be a eukaryotic or a prokaryotic promoter. In some embodiments the promoter is an inducible promoter. In some embodiments, the promoter is a constitutive promoter. Also provided is an RNA construct comprising recombinant RNA described according to the present disclosure.
  • An exemplary RNA construct comprises recombinant RNA and one or more additional sequences, such as one or more untranslated regions (UTRs), 3’ poly (A) tail (poly A), or 5’ cap. As discussed in, for example, Lin et al. 2023, UTRs can regulate locations of mRNA and translation efficiency.
  • 5’ cap also known as m7GpppN, can bind to the multi- subunit initiation factor eIF4F to promote mRNA binding with ribosomes and translation of 71 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 mRNA. It can also protect mRNA from degradation by exonucleases. Poly A influences stability and translation efficiency of mRNA. [0163] The recombinant nucleic acids provided herein can be included in expression cassettes for expression in a host cell or an organism of interest.
  • the cassette includes 5′ and 3′ regulatory sequences operably linked to a recombinant nucleic acid provided herein that allows for expression of the modified polypeptide.
  • the cassette may additionally contain at least one additional gene or genetic element to be co-transformed into the organism. Where additional genes or elements are included, the components are operably linked. Alternatively, the additional gene(s) or element(s) can be provided on multiple expression cassettes.
  • Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the polynucleotides to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette will include in the 5′ to 3′ direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a polynucleotide disclosed herein, and a transcriptional and translational termination region (i.e., termination region) functional in the cell or organism of interest.
  • a transcriptional and translational initiation region i.e., a promoter
  • a polynucleotide disclosed herein i.e., termination region
  • a transcriptional and translational termination region i.e., termination region
  • heterologous in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. Additional regulatory signals include, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See Sambrook et al. Molecular Cloning: A Laboratory Manual, 4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2012) (“Sambrook”); Davis et al., eds. Advanced Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  • the expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Marker genes include genes conferring antibiotic resistance, such as those conferring hygromycin resistance, ampicillin resistance, gentamicin resistance, neomycin resistance, to name a few. Additional selectable markers are known and any can be used.
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other 72 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • a vector comprising a nucleic acid or expression cassette set forth herein. The vector is contemplated to have the necessary functional elements that direct and regulate transcription of the inserted nucleic acid.
  • These functional elements include, but are not limited to, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers that can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region that may serve to facilitate the expression of the inserted gene or hybrid gene. See generally, Sambrook.
  • the vector for example, can be a plasmid.
  • E. coli expression vectors known to one of ordinary skill in the art, which are useful for the expression of a nucleic acid.
  • microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Senatia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilis
  • Enterobacteriaceae such as Salmonella, Senatia, and various Pseudomonas species.
  • prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • yeast expression can be used.
  • nucleic acid encoding a polypeptide of the present invention, wherein the nucleic acid can be expressed by a yeast cell. More specifically, the nucleic acid can be expressed by Pichia pastoris or S. cerevisiae.
  • Mammalian cells also permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein.
  • Vectors useful for the expression of active proteins in mammalian cells are known in the art and can contain genes conferring hygromycin resistance, geneticin or G418 resistance, or other genes or phenotypes suitable for use as selectable markers, or methotrexate resistance for gene amplification.
  • Suitable host cell lines capable of secreting intact human proteins include CHO cells, HEK293 cells, HeLa cells, COS-7 cells, myeloma cell lines, Jurkat cells, derivatives of any of the above (e.g., Expi-HEK cells, Expi-CHO cells), etc.
  • Expression vectors for these cells can include 73 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc.
  • Several possible vector systems are available for the expression of cloned heavy chain and light chain polypeptides from nucleic acids in mammalian cells.
  • One class of vectors relies upon the integration of the desired gene sequences into the host cell genome.
  • Cells that have stably integrated DNA can be selected by simultaneously introducing drug resistance genes, such as E. coli gpt.
  • the selectable marker gene can be either linked to the DNA gene sequences to be expressed or introduced into the same cell by co-transfection.
  • a second class of vectors utilizes DNA elements that confer autonomously replicating capabilities to an extrachromosomal plasmid.
  • These vectors can be derived from animal viruses, such as bovine papillomavirus, CMV, polyoma virus, or SV40 virus. Including among the embodiments of the present disclosure are viral vectors including nucleic acids and/or nucleic acid constructs according to the present disclosure.
  • Non- limiting examples of such viral vectors are adenovirus (Ad) vectors, lentivirus / retrovirus vectors, and Adeno-associated virus (AAV) vectors (including recombinant AAVs or rAAVs).
  • Ad adenovirus
  • AAV Adeno-associated virus
  • HSV Herpes simplex virus
  • Various viral vector platforms are described, for example, in Bulcha et al. 2021.
  • the vectors according to the present disclosure can also include the nucleic acids as described herein under the control of an inducible promoter such as the tetracycline inducible promoter or a glucocorticoid inducible promoter.
  • the nucleic acids of the present invention can also be under the control of a tissue-specific promoter to promote expression of the nucleic acid in specific cells, tissues, or organs.
  • a tissue-specific promoter to promote expression of the nucleic acid in specific cells, tissues, or organs.
  • Any regulatable promoter such as a metallothionein promoter, a heat-shock promoter, and other regulatable promoters, of which many examples are well known in the art are also contemplated.
  • a Cre-loxP inducible system can also be used, as well as a Flp recombinase inducible promoter system, both of which are known in the art.
  • RNA delivery systems are described, for example, in Lin et al. 2023.
  • An example of an RNA delivery system is mRNA-LNP (lipid nanoparticle).
  • LNPs contain four components: a cationic or ionizable lipid, cholesterol, a helper lipid, and a poly(ethylene glycol) (PEG)-lipid.
  • PEG poly(ethylene glycol)
  • non-vector based delivery systems include 74 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 polymers, such as, but not limited to, Polyethyleneimine (PEI) or poly(lactic-co-glycolic acid) (PLGA) or poly(l-lysine) (PLL) or poly(beta-amino ester)s (PBAEs). Polymers may be used in the form of polymeric micelles or dendrimers.
  • Other examples of non-vector based delivery systems inorganic nanoparticles, such as gold nanoparticles (AuNPs).
  • the nucleic acid constructs and vectors provided herein may comprise the nucleic acids described above in a variety of configurations to allow expression and purification of the fusion proteins according to the present disclosure or their domains.
  • the domains of the fusion proteins may be present in a variety of configurations (e.g., one or more neutralizing domains, one or more antibody domains, peptide and/or non-peptide linkers, various orientations, and orders for domain arrangement, etc.).
  • nucleic acid constructs and vectors that are able to express the fusion proteins or their domains in any desired configuration.
  • a nucleic acid construct or vector may comprise nucleic acids encoding an antibody domain sequence (e.g., a heavy chain sequence, a light chain sequence, or an antibody fragment sequence such as an scFv fragment) linked (e.g., via a peptide linker) to a neutralizing domain sequence (e.g., an ACE2 ectodomain polypeptide, a DPP4 ectodomain polypeptide, or a neutralizing antibody).
  • an antibody domain sequence e.g., a heavy chain sequence, a light chain sequence, or an antibody fragment sequence such as an scFv fragment
  • a neutralizing domain sequence e.g., an ACE2 ectodomain polypeptide, a DPP4 ectodomain polypeptide, or a neutralizing antibody.
  • various domains e.g., antibody domains or neutralizing domains
  • domain components e.g., antibody heavy chains and antibody light chains
  • one nucleic acid construct or vector may comprise nucleic acids encoding an antibody heavy chain sequence linked to a neutralizing domain polypeptide
  • another nucleic acid construct or vector may comprise nucleic acids encoding a corresponding antibody light chain sequence.
  • nucleic acid constructs or vectors could be introduced into a host cell, as described below, and the expressed polypeptides allowed to associate to form a dimer (e.g., the antibody heavy chain and light chain may associate) prior to purification.
  • two or more modified protein domains may be produced from separate nucleic acid constructs or vectors.
  • the nucleic acid constructs or vectors may then be introduced into a host cell, as described below, and the expressed polypeptides (e.g., an scFv antibody fragment and a neutralizing domain) may be chemically linked with a non-peptide linker to produce a fusion protein.
  • any of the fusion proteins according to the present disclosure can be purified or isolated from a host cell or population of host cells.
  • a recombinant nucleic acid or a nucleic 75 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 acid construct encoding one or more of the fusion proteins or their domains according to the present disclosure can be introduced into a host cell under conditions that allow expression protein expression.
  • the recombinant nucleic acid is codon-optimized for expression.
  • a recombinant protein can be isolated or purified using purification methods known in the art.
  • a plurality of recombinant nucleic acids each encoding a different fusion protein or a domain of a fusion protein according to the present disclosure can be introduced into a host cell under conditions that allow protein expression.
  • the expressed fusion proteins form multimers.
  • the multimeric fusion protein can be isolated or purified using purification methods known in the art.
  • a fusion protein is isolated as a monomer and allowed to multimerize (dimerize, trimerize, etc.) in vitro.
  • one or more of the domains of a fusion protein can be expressed and/or isolated individually and then assembled with the remaining domains to form the fusion.
  • a host cell comprising a nucleic acid, a nucleic acid construct, or a vector described herein is also provided.
  • the host cell can be an in vitro, ex vivo, or in vivo host cell. Populations of any of the host cells described herein are also provided.
  • a cell culture comprising one or more host cells described herein is also provided. Appropriate host cells for the expression of antibodies or antigen-binding fragments thereof include yeast, bacteria, insect, plant, and mammalian cells.
  • the host cell can be a prokaryotic cell, including, for example, a bacterial cell.
  • the cell can be a eukaryotic cell, for example, a mammalian cell.
  • the cell can be an HEK293T cell, a Chinese hamster ovary (CHO) cell, a COS-7 cell, a HeLA cell, an avian cell, a myeloma cell, a Pichia cell, an insect cell, or a plant cell.
  • CHO Chinese hamster ovary
  • COS-7 COS-7
  • HeLA avian
  • myeloma cell a cell that uses a cell
  • Pichia cell a cell lines
  • insect cell or a plant cell.
  • a number of other suitable host cell lines have been developed and include myeloma cell lines, fibroblast cell lines, and a variety of tumor cell lines such as melanoma cell lines.
  • the vectors containing the nucleic acid segments of interest can be transferred or introduced into the host cell by well-known methods, which vary depending on the type of cellular host. Insect cells also permit the expression of polypeptides.
  • fusion proteins produced in insect cells such as Sf9 with baculovirus vectors undergo post-translational modifications similar to those of wild-type mammalian proteins.
  • the fusion proteins according to the present disclosure and their domains may be produced by recombinant expression in a human or non-human cell.
  • the cell is a synthetic antibody-producing cell, such as non-human cells expressing heavy chains, light chains, or both heavy and light chains; human cells that are not immune cells that express heavy chains, light chains, or both heavy and light chains; and human B cells that produce heavy chains or light 76 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 chains, but not both heavy and light chains.
  • a synthetic antibody-producing cell such as non-human cells expressing heavy chains, light chains, or both heavy and light chains; human cells that are not immune cells that express heavy chains, light chains, or both heavy and light chains; and human B cells that produce heavy chains or light 76 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 chains, but not both heavy and light chains.
  • the fusion proteins according to the present disclosure and their domains may be heterologously expressed, in vitro or in vivo, in cells other than human B cells, such as non-human cells and human cells other than B cells, optionally other than immune cells, and optionally in cells other than cells in a B cell lineage.
  • the fusion proteins according to the present disclosure and their domains can be produced from the cells by culturing a host cell containing the nucleic acid encoding the fusion protein or modified protein, under conditions and for an amount of time sufficient to allow expression of the proteins. Such conditions for protein expression vary with the choice of the expression vector and the host cell. Methods for the culture and production of many cells are available in the art.
  • nucleic acids, DNA constructs, and expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid.
  • introducing in the context of introducing a nucleic acid into a cell refers to the translocation of the nucleic acid sequence from outside a cell to inside the cell.
  • introducing refers to translocation of the nucleic acid from outside the cell to inside the nucleus of the cell.
  • the method of introduction is largely dictated by the targeted cell type, discussed below.
  • Exemplary methods include CaPO 4 precipitation, liposome fusion, cationic liposomes, electroporation, nucleoporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.
  • translocation including but not limited to, electroporation, nanoparticle delivery, viral delivery, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, DEAE dextran, lipofectamine, calcium phosphate or any method now known or identified in the future for introduction of nucleic acids into prokaryotic or eukaryotic cellular hosts.
  • a targeted nuclease system e.g., an RNA-guided nuclease (CRISPR-Cas9), a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a megaTAL (MT) can also be used to introduce a nucleic acid, for example, a nucleic acid encoding a recombinant protein described herein, into a host cell.
  • a fusion protein according to the present disclosure or one or more of its domains can be expressed in, and purified from, transgenic animals (e.g., transgenic mammals).
  • an antibody can be produced in transgenic non-human mammals (e.g., 77 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 rodents) and isolated from milk as described in, e.g., Houdebine 2002, van Kuik-Romeijn et al. 2000 and Pollock et al. 1999. [0178] Following expression, fusion proteins according to the present disclosure pr their domains can be isolated or purified in a variety of ways known in the art depending on what other components are present in the sample.
  • Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography.
  • a fusion protein or modified protein comprising an antibody can be purified using a standard anti-antibody column (e.g., a protein-A or protein-G column).
  • Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes Protein Purification, 3 rd edition, Springer- Verlag, New York City, New York (1994). The degree of purification necessary varies depending on the desired use. In some instances, no purification of the expressed antibody or fragments thereof is necessary.
  • Papain digestion of antibodies can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988). Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab’)2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • the Fab fragments produced in antibody digestion can also contain the constant domains of the light chain and the first constant domain of the heavy chain.
  • Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region.
  • the F(ab’)2 fragment is a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region.
  • Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyl- oxycarbonyl) or Boc (tert-butyloxycarbonoyl) chemistry (Applied Biosystems, Inc.; Foster City, CA).
  • Fmoc (9-fluorenylmethyl- oxycarbonyl) or Boc (tert-butyloxycarbonoyl) chemistry Applied Biosystems, Inc.; Foster City, CA.
  • a fusion protein according to the present disclosure or one or more of its domains, for 78 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 example, can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group that is functionally blocked on the other fragment.
  • peptide condensation reactions these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof.
  • the peptide or polypeptide can by independently synthesized in vivo. Once isolated, these independent peptides or polypeptides may be linked to form a fusion protein via similar peptide condensation reactions.
  • enzymatic ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains.
  • native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction.
  • the first step is the chemoselective reaction of an unprotected synthetic peptide a thioester with another unprotected peptide segment containing an amino terminal Cys residue to give a thioester linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.
  • production of polypeptides involves chemical linkage of unprotected peptide segments where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond.
  • linkers may be used to link two amino acid residues.
  • two amino acid residues may be cross linked via a linkage that includes a “hindered” disulfide bond.
  • a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase.
  • One suitable reagent 4- succinimidyloxycarbonyl- -methyl- (2-pyridyldithio) toluene (SMPT), forms such a linkage between two proteins utilizing a terminal lysine on one of the proteins and a terminal cysteine on the other.
  • SMPT 4- succinimidyloxycarbonyl- -methyl- (2-pyridyldithio) toluene
  • cross-linkers include, without limitation, reagents which link two 79 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis- maleimidobutane), an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p- azidosalicylamido]butylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate).
  • amino groups e.g.,
  • a neutralizing polypeptide or an antibody described herein one of which is attached to a linker having a chemical functional group at the free/unattached end is produced and joined to another neutralizing polypeptide domain or antibody comprising a complementary reactive chemical functional group (e.g., at an end or internally).
  • a complementary reactive chemical functional group e.g., at an end or internally.
  • two domains of the modified protein having a full or partial linker sequence with a chemical functional group at the end are produced and chemically linked in vitro via the free/unattached ends of the full or partial linker.
  • additional non-peptide linkers may be used to join domains of a fusion protein according to the present disclosure.
  • non-peptide linkers comprise functional groups on at least one terminus to allow attachment to a polypeptide domain.
  • PEG linkers can be designed with N-hydroxy-succinimide (NHS) esters at one end or both ends that react specifically and efficiently with lysine and N- terminal amino groups to form amide bonds.
  • linkers can also be designed with sulfhydryl-reactive crosslinkers at one end or both ends that react with reduced sulfhydryls to form stable thioether bonds.
  • Methods for determining the yield or purity of a purified protein or polypeptide include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).
  • a protein stain such as Coomassie Blue or colloidal silver stain.
  • compositions and formulations comprising one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins according to the present disclosure and a pharmaceutically acceptable carrier are also provided.
  • the compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and/or adjuvant to be used with the methods disclosed herein.
  • Such compositions can be used in a subject infected with a coronavirus that would benefit from the activity of any of one or more fusion proteins according to the present disclosure.
  • compositions comprising one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or 80 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 more nucleic acid constructs encoding one or more fusion proteins according to the present disclosure may be referred to as “pharmaceutical compositions,” “therapeutic compositions,” “formulations,” “pharmaceutical formulations,” “therapeutic formulations,” and by other non- limiting terms and expressions. [0186] In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.
  • the formulation materials are selected for subcutaneous, intravenous, intramuscular, intranasal, inhalational, or intraocular administration, or for administration by injection into a specific location, such as into cerebrospinal fluid.
  • a pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • a pharmaceutical composition can contain delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants.
  • suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying amino acids (such
  • the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage.
  • such compositions may influence the physical state, stability, rate of in vivo release and/or rate of in 81 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 vivo clearance of one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins according to the present disclosure.
  • the information regarding pharmaceutical formulation is available.
  • the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • the saline comprises isotonic phosphate-buffered saline.
  • neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • compositions comprise a pH controlling buffer such phosphate-buffered saline or acetate-buffered saline.
  • a composition comprising one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins disclosed herein can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (see Remington) in the form of a lyophilized cake or an aqueous solution.
  • a composition comprising one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins disclosed herein can be formulated as a lyophilizate using appropriate excipients.
  • appropriate excipients may include a cryo-preservative, a bulking agent, a surfactant, or a combination of any thereof.
  • Exemplary excipients include one or more of a polyol, a disaccharide, or a polysaccharide, such as, for example, mannitol, sorbitol, sucrose, trehalose, and dextran 40.
  • cryo- preservative may be sucrose or trehalose.
  • the bulking agent may be glycine or mannitol.
  • the surfactant may be a polysorbate such as, for example, polysorbate-20 or polysorbate-80.
  • the pharmaceutical composition can be selected for parenteral delivery. In certain embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art. [0189] In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the 82 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • the pH may be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.
  • the pH of the pharmaceutical composition may be in the range of 6.6-8.5 such as, for example, 7.0-8.5, 6.6-7.2, 6.8-7.2, 6.8-7.4, 7.2-7.8, 7.0-7.5, 7.5-8.0, 7.2-8.2, 7.6-8.5, or 7.8-8.3.
  • the pH of the pharmaceutical composition may be in the range of 5.5-7.5 such as, for example, 5.5-5.8, 5.5-6.0, 5.7-6.2, 5.8-6.5, 6.0-6.5, 6.2-6.8, 6.5-7.0, 6.8-7.2, or 6.8-7.5.
  • the pH of the pharmaceutical composition may be in the range of 4.0-5.5 such as, for example, 4.0-4.3, 4.0-4.5, 4.2-4.8, 4.5-4.8, 4.5-5.0, 4.8-5.2, or 5.0-5.5.
  • a pharmaceutical composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins in a pharmaceutically acceptable vehicle.
  • a vehicle for parenteral injection is sterile distilled water in which a fusion protein or modified protein is formulated as a sterile, isotonic solution and properly preserved.
  • the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection.
  • an agent such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection.
  • hyaluronic acid can also be used and can have the effect of promoting sustained duration in the circulation.
  • implantable drug delivery devices can be used to introduce the desired molecule.
  • a pharmaceutical composition can be formulated for inhalation.
  • a fusion protein, a nucleic acid encoding one or more fusion proteins, or a nucleic acid construct encoding one or more fusion proteins can be formulated as a dry powder for inhalation.
  • an inhalation solution can be formulated with a propellant for aerosol delivery.
  • solutions can be nebulized.
  • pulmonary administration is further described in International Patent Publication No. WO1994020069, which describes pulmonary delivery of chemically modified proteins.
  • formulations can be administered orally.
  • a fusion protein or modified protein that is administered in this fashion can be formulated with or without carriers customarily used in compounding solid dosage forms, such as tablets and capsules.
  • a capsule can be designed to release the active 83 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized, and pre-systemic degradation is minimized.
  • at least one additional agent can be included to facilitate absorption of a fusion protein or modified protein.
  • a pharmaceutical composition can involve an effective quantity of a fusion protein according to the present disclosure in a mixture with non-toxic excipients suitable for the manufacture of tablets.
  • solutions can be prepared in unit-dose form.
  • suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate
  • binding agents such as starch, gelatin, or acacia
  • lubricating agents such as magnesium stearate, stearic acid, or talc.
  • Additional pharmaceutical compositions can be selected by one skilled in the art, including formulations involving one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins in sustained- or controlled-delivery formulations.
  • sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • Sustained release matrices can include polyesters, hydrogels, polylactides, including, for example, chemically synthesized polymers, starch-based polymers, and polyhydroxyalkanoates (PHAs), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, ethylene vinyl acetate.
  • sustained release compositions can also include liposomes.
  • the pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, sterilization is accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution.
  • the composition for parenteral administration can be stored in lyophilized form or in a solution.
  • parenteral compositions generally are placed into a container 84 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • kits are provided for producing a single-dose administration unit.
  • the kit can contain both a container containing a dried protein and another container containing an aqueous formulation.
  • kits containing single and multi-chambered pre-filled syringes are included.
  • the effective amount of a pharmaceutical composition comprising one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins as described herein to be employed therapeutically depends, for example, upon the therapeutic context and objectives.
  • the appropriate dosage levels for treatment vary depending, in part, upon the molecule delivered, the indication for which a fusion protein is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • the clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins in the formulation used.
  • a clinician administers the composition until a dosage is reached that achieves the desired effect.
  • the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter.
  • appropriate dosages can be ascertained through use of appropriate dose-response data. Dosing considerations and administration are discussed further below. 85 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 VI. Methods of use [0200] Methods of using fusion proteins according to the present disclosure are contemplated and provided.
  • fusion protein or fusion protein encompasses the compositions containing one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins, as discussed above.
  • methods of treating a subject infected with a coronavirus infection comprising administering to the subject a therapeutically effective amount of one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins according to the present disclosure.
  • Fusion proteins, nucleic acids encoding one or more fusion proteins, or nucleic acid constructs encoding one or more fusion proteins according to the present disclosure can also be used as a prophylactic therapy for a coronavirus infection.
  • Compositions including one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins according to the present disclosure can be administered to subjects in order to lessen the likelihood and/or severity of a coronavirus infection or administered to subjects already evidencing active coronavirus infection.
  • compositions including one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins according to the present disclosure according to the present disclosure may be used either in prophylactic and therapeutic administration, as well as in passive immunization with substantially purified polypeptide products or gene therapy by transfer of nucleic acids or nucleic acid constructs according to the present disclosure.
  • the subject has or is determined to have a coronavirus infection or has been exposed to a coronavirus. [0201] It is contemplated that the subject may be asymptomatic or symptomatic.
  • the subject is displaying one or more symptoms indicative of a coronavirus infection, such as, but not limited to, infection with SARS-CoV-2 (or SARS-CoV-2 variant), SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-HKU1, HCoV-229E, HCoV-NL63 etc.
  • symptoms include, but are not limited to, a new loss of taste or smell, myalgia, fatigue, shortness of breath or difficulty breathing, fever, and/or cough.
  • Symptoms may also include pharyngitis, headache, productive cough (i.e.
  • the patient has at least two symptoms selected from the group consisting of a new loss of taste or smell, shortness of breath or difficulty 86 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 breathing, fever, cough, chills, or muscle aches.
  • the patient may have a blood oxygen level reading of 94 or less, e.g., as determined by an oximeter.
  • the subject may have radiographic evidence of pulmonary infiltrates.
  • the subject may have been receiving standard support care, e.g., such as being administered oxygen, fluids, and/or other therapeutic procedures or agents.
  • the subject may not manifest any symptoms that are typically associated with a coronavirus infection.
  • the subject is known or believed to have been exposed to a coronavirus, suspected of having exposure to a coronavirus or believed not to have had exposure to a coronavirus.
  • the subject may have recovered from a prior coronavirus infection.
  • the subject has received a coronavirus vaccine, such as, but not limited to, SARS-CoV-2 vaccine.
  • the coronavirus vaccine can be any of the DNA, RNA, or protein, or inactive virus vaccine that is capable of inducing immune response in a patient to generate anti-coronavirus antibodies.
  • the subject has been free of symptoms suggestive of a coronavirus infection for at least 14 days.
  • the subject may have one or more of other conditions of hypertension, coronary artery disease, diabetes, chronic obstructive pulmonary disease.
  • a coronavirus infection in a subject can be detected by various assays performed on a biological sample from the subject.
  • the biological sample may be from a throat swab, a nasopharyngeal swab, sputum or tracheal aspirate, urine, feces, or blood.
  • nucleic acids are isolated from the biological sample and tested for the presence of viral genomic sequences.
  • PCR is performed to detect coronavirus nucleic acids from the biological sample.
  • a subject may have antibodies that selectively bind to coronavirus proteins, e.g., coronavirus spike protein. Antibodies can be detected in a blood sample from the subject by immunoassay (e.g., lateral flow assay or enzyme-linked immunosorbent assay (ELISA)).
  • immunoassay e.g., lateral flow assay or enzyme-linked immunosorbent assay (ELISA)
  • coronavirus infection can be detected using a proximity-based binding assay for detection of virus and/or anti-virus antibodies, as described for example, in in Lui et al. 2021 and Elledge et al. 2021.
  • “treating” or “treatment” of any disease or disorder refers to preventing or ameliorating a disease or disorder, or one or more of its symptoms, in a subject.
  • ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, for example, a coronavirus infection, such as a betacoronavirus infection, for example, an infection with SARS-CoV-2 (or SARS-CoV-2 variant), SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-OC43, or HCoV-HKU1, etc., or an alphacoronavirus infection with 87 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 HCoV-229E, HCoV-NL63, etc., lessening in the severity or progression, or curing thereof.
  • Treating or treatment also encompass prophylactic treatments that reduce the incidence of a disease or disorder in a subject and/or reduce the incidence or reduce severity of a symptom thereof.
  • treating or treatment includes ameliorating at least one physical parameter or symptom.
  • Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both.
  • Treating or treatment includes delaying, preventing increases in, or decreasing viral load.
  • treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition.
  • a method for treating a coronavirus infection in a subject by administering one or more of a fusion protein, a nucleic acid encoding a fusion protein, or a nucleic acid construct encoding a fusion protein according to the present disclosure is considered to be a treatment if there is a 10% reduction in one or more symptoms of the coronavirus infection in a subject as compared to a control.
  • the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels.
  • formulations comprising a fusion protein or modified protein as described herein are administered to the subject until the subject exhibits amelioration of at least one symptom of a coronavirus infection and/or is demonstrated to have a sustained decrease in viral load, e.g., as measured by immunoassay and/or quantitative amplification method, including PCR or sequencing.
  • the formulation is administered to the subject until viral load is undetectable, i.e. below the level of detection, such that no coronavirus RNA copies can be detected by the assay methodology employed.
  • the subject exhibits undetectable viral load 1-4 weeks, 2-4 weeks, 2-12 weeks, 4-12 weeks, or 12-24 weeks after last administration of the formulation.
  • treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
  • the subject is administered one or more of a fusion protein, a nucleic acid encoding a fusion protein, or a nucleic acid construct encoding a fusion protein according to the present disclosure within 1, 2, 3, 4, or 5 days from the onset of symptoms or within 1, 2, 3, 4, or 5 days from testing positive for a coronavirus infection.
  • the subject is administered one or more compositions including one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins according to the present disclosureas described herein within 88 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 1 or 2 days of hospitalization with one or more symptoms indicative of a coronavirus infection.
  • a composition according to the present disclosure is administered to the subject at least once a day, at least twice a day, or at least three times a day.
  • a composition according to the present disclosure is administered on consecutive days or on non- consecutive days. In some instances, a composition according to the present disclosure is administered to the subject for at least 1 day, at least 2 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, or at least 3 months. In some embodiments, a composition according to the present disclosure is administered to the subject for 2 to 5 or more days after the viral load is undetectable in order avoid “rebound” of virus replication.
  • a composition comprising one or more of a fusion protein, a nucleic acid encoding a fusion protein, or a nucleic acid construct encoding a fusion protein according to the present disclosure can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration.
  • the route of administration can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intramuscular injection (IM), intradermal injection (ID), subcutaneous, transdermal, intracavity, oral, intraocular or intracranial injection, or intrathecal injection (IT).
  • the injection can be in a bolus or a continuous infusion.
  • the route of administration can be intranasal, by inhalation, or by an implant.
  • Techniques for preparing suitable delivery systems are well known. Generally, such systems should utilize components that will not significantly impair the biological properties of the fusion protein according to the present disclosure, such as the capacity to bind the spike RBD (see, for example, Remington), nucleic acids encoding fusion proteins according to the present disclosure, or nucleic acid constructs encoding fusion proteins according to the present disclosure.
  • Those of skill in the art can readily determine the various parameters and conditions for producing delivery system without resorting to undue experimentation.
  • Passive immunization with one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins according to the present disclosure is an option for prevention and/or treatment of a coronavirus infection.
  • Fusion proteins according to the present disclosure can also be used as a prophylactic therapy for a coronavirus infection, such that the fusion proteins are administered to high-risk subjects in a therapeutically effective amount in order to lessen the likelihood and/or severity of a coronavirus infection.
  • compositions containing fusion proteins, nucleic acids encoding fusion proteins according to the present disclosure, or 89 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 nucleic acid constructs encoding fusion proteins according to the present disclosure may be administered prior to the onset of symptoms of an infection.
  • Prophylactic administration may prevent exposure to a coronavirus from progressing to a coronavirus infection or prevent a coronavirus infection from progressing to symptomatic disease.
  • the subject has been exposed to a coronavirus.
  • the subject is at risk of exposure to a coronavirus.
  • the subject may be a healthcare worker who has been exposed to a human patient with a suspected or confirmed coronavirus infection.
  • the subject may be identified through contact tracing efforts as having come into physical contact with a human having a confirmed coronavirus infection.
  • the subject is administered a composition as described herein within 1, 2, 3, 4, or 5 days from exposure or suspected exposure to a coronavirus.
  • the subject is administered a composition as described herein with 1, 2, 3, 4, or 5 days from identification of the subject as having a high risk of coronavirus infection.
  • a composition administered to a subject may comprise an effective amount of one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins.
  • effective amounts can be readily determined by one of ordinary skill in the art as described below. Considerations include the effect of the administered one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins, or the combinatorial effect of the above with one or more additional active agents, if more than one agent is used in or with the pharmaceutical composition.
  • a “therapeutically effective amount” refers to an amount of one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins according to the present disclosure that, when administered to a subject, is effective to achieve an intended purpose, e.g., to reduce viral load or prevent viral load from increasing, to reduce or ameliorate at least one symptom of a coronavirus infection, and/or otherwise reduce the length of time that a patient experiences a symptom of a coronavirus infection, or extend the length of time before a symptom may recur.
  • an intended purpose e.g., to reduce viral load or prevent viral load from increasing, to reduce or ameliorate at least one symptom of a coronavirus infection, and/or otherwise reduce the length of time that a patient experiences a symptom of a coronavirus infection, or extend the length of time before a symptom may recur.
  • a “prophylactically effective amount” of one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins according to the present disclosure is a dosage large enough 90 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 to produce the desired effect in the protection of individuals against a coronavirus infection for a reasonable period of time, such as one to two months or longer following administration.
  • therapeutically effective amount and prophylactically effective amount may each be referred to herein as effective amounts, with the context depending on the subject who is receiving treatment (i.e. having an infection or not).
  • An effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
  • an effective amount is not a dosage so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like.
  • An effective amount may vary with the subject’s age, condition, and sex, the extent of the disease in the subject, frequency of treatment, the nature of concurrent therapy (if any), the method of administration, and the nature and scope of the desired effect(s) (Nies et al., Chapter 3 In: Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. eds., McGraw-Hill, New York, NY (1996)). and can be determined by one of skill in the art.
  • a therapeutically effective amount may vary from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 20 mg/kg, most preferably from about 0.2 mg/kg to about 2 mg/kg, in one or more dose administrations daily, for one or several days.
  • a prophylactically effective amount may vary from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 20 mg/kg, most preferably from about 0.2 mg/kg to about 2 mg/kg, in one or more administrations.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of a fusion protein, a nucleic acid encoding one or more fusion proteins, or a nucleic acid construct encoding one or more fusion proteins lies generally within a range that includes the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of 91 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 administration utilized.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the EC 50 (i.e., the concentration of the construct – e.g., polypeptide – that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, e.g., where local administration is desired, cell culture or animal models can be used to determine a dose required to achieve a therapeutically effective concentration within the local site. Suitable human doses of any of the fusion proteins according to the present disclosure can further be evaluated in, e.g., Phase I dose escalation studies.
  • Toxicity and therapeutic efficacy of a fusion protein, a nucleic acid encoding one or more fusion proteins, or a nucleic acid construct encoding one or more fusion proteins according to the present disclosure can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the disease states described herein). These procedures can be used, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50/ED50.
  • a composition including a fusion protein, a nucleic acid encoding one or more fusion proteins, or a nucleic acid construct encoding one or more fusion proteins that exhibits a high therapeutic index is preferred. While compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such a composition to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects.
  • Wild- type (WT) human recombinant ACE2 hrACE2/APN01 was previously found to be safe in humans for the treatment of hypertension and acute respiratory distress syndrome (see Haschke et al. 2013 and Khan et al. 2017).
  • Fusion proteins, nucleic acid encoding one or more fusion proteins, or nucleic acid constructs encoding one or more fusion proteins according to the present disclosure are expected to be similarly safe for therapeutic use.
  • a fusion protein, a nucleic acid encoding one or more fusion proteins, or a nucleic acid construct encoding one or more fusion proteins according to the present disclosure can be administered to a subject as a monotherapy.
  • a fusion protein, a nucleic acid encoding one or more fusion proteins, or a nucleic acid construct encoding one or more fusion proteins according to the present disclosure can be administered in conjunction with other therapies for viral infection (combination therapy).
  • composition including a 92 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 fusion protein, a nucleic acid encoding one or more fusion proteins, or a nucleic acid construct encoding one or more fusion proteins can be administered to a subject at the same time, prior to, or after, a second therapy.
  • a composition according to the present disclosure and the one or more additional active agents are administered at the same time.
  • a composition can be administered first in time and the one or more additional active agents are administered second in time.
  • the one or more additional active agents are administered first in time and the composition including a fusion protein, a nucleic acid encoding one or more fusion proteins, or a nucleic acid construct encoding one or more fusion proteins is administered second in time.
  • a composition according to the present disclosure and the one or more additional agents can be administered simultaneously by the same or different routes.
  • a composition according to the present disclosure can contain one or more additional therapeutic agents.
  • the other therapies may include administration of, for example, remdesivir, chloroquine, tenofovir, entecavir, and/or protease inhibitors (lopinavir/ritonavir).
  • the other therapies may include administration of annexin-5, anti-PS monoclonal or polyclonal antibodies, bavituximab, and/or bind to viral glucocorticoid response elements (GREs), retinazone and RU486 or derivatives, cell entry inhibitors, uncoating inhibitors, reverse transcriptase inhibitors, integrase inhibitors, transcription inhibitors, antisense translation inhibitors, ribozyme translation inhibitors, prein processing and targeting inhibitors, protease inhibitors, assembly inhibitors, release phase inhibitors, immunosystem modulators and vaccines, including, but not limited to Abacavir, Ziagen, Trizivir, Kivexa/Epzicom, Aciclovir, Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, Balavir, Cidofovir, Combivir, Dolutegravir, Darunavir, Delavird
  • Administration of a composition according to the present disclosure can replace or augment a previously or currently administered therapy.
  • administration of the one or more additional active agents can cease or diminish, e.g., be administered at lower levels or dosages.
  • administration of the previous therapy can be maintained.
  • a previous therapy is maintained until the level of a fusion protein, a nucleic acid encoding one or more fusion proteins, or a nucleic acid construct encoding one or more fusion proteins according to the present disclosure reaches a level sufficient to provide a therapeutic effect.
  • Monitoring a subject e.g., a human patient
  • Monitoring a subject for an improvement of a coronavirus infection refers to evaluating the subject for a change in a disease parameter, e.g., a reduction in one or more symptoms of a coronavirus infection exhibited by the subject.
  • the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration.
  • the subject can be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation can include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered.
  • Systems and kits [0220] Included among the embodiments of the present invention are systems and kits containing one or more of: fusion proteins described to the present disclosure, or their domains, polypeptides including amino acid sequences described in the present disclosure, nucleic acids described in the present disclosure, nucleic acids constructs described in the present disclosure, vectors described in the present disclosure, or compositions including one or more of any of the foregoing. Some embodiments of the systems and kits may contain one or more fusion proteins or nucleic acids encoding the fusion proteins described in the present disclosure.
  • a system or a kit may contain two or more, three or more, four or more, five or more etc. different fusion proteins or fusion protein domains described in the present disclosure.
  • a system or a kit may contain nucleic acids encoding two or more, three or more, four or more, five or more etc. different fusion proteins or fusion protein domains described in the present disclosure. The nucleic acids encoding two or more, three or more, four or more, five or more etc.
  • fusion proteins 94 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 or their domains may be included in the same nucleic acid construct, such as a vector, or in different nucleic acid constructs.
  • a system or a kit can contain one or more, two or more, three or more, four or more, five or more etc. of fusion proteins or nucleic acids encoding fusion proteins.
  • Systems and kits according to the present disclosure may contain one or more components or ingredients in addition to one or more of: fusion proteins according to the present disclosure, or their domains, polypeptides including amino acid sequences described in the present disclosure, nucleic acids described in the present disclosure, nucleic acids constructs described in the present disclosure, vectors described in the present disclosure, or compositions including one or more of any of the foregoing.
  • a system or a kit may include a composition according to the present disclosure and a container for its storage, such as a bag or a vial.
  • a container may have a sterile access port, for example, a bag or vial having a stopper pierceable by a hypodermic injection needle.
  • a system or a kit may include one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins according to the present disclosure in lyophilized or concentrated form and diluent.
  • a diluent may also be a pharmaceutically acceptable carrier or excipient, as described elsewhere in the present disclosure. Examples of diluents that may be included in such systems or kits are saline, buffered saline, water, or sucrose.
  • a system or a kit may include a composition according to the present disclosure and a device for administering the composition.
  • a device for administering the composition may be a syringe for injection or oral administration (for example, the kit may be a syringe pre-filled with a liquid immunogenic composition), a microneedle device, such as a microneedle patch, an inhaler, or a nebulizer.
  • a system or a kit may contain a defined amount of one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins according to the present disclosure capable of eliciting a protective or prophylactic effect against a coronavirus in a subject.
  • a system or a kit may contain multiple doses of a defined amount of one or more fusion proteins, one or more nucleic acids encoding one or more fusion proteins, or one or more nucleic acid constructs encoding one or more fusion proteins capable of eliciting a protective or prophylactic effect against a coronavirus in a subject.
  • a system or a kit may contain multiple vials, syringes or microneedle patches containing a composition according to the present disclosure.
  • Systems and kits according to the present disclosure may comprise a carrier container being compartmentalized to receive in close confinement one or more containers such as vials, tubes, and 95 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 the like, each of the containers comprising one of the separate elements to be used the methods according to the present disclosure.
  • Materials, compositions, components, and ingredients described in the present disclosure can be used in conjunction with or can be used in preparation for the disclosed embodiments. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc.
  • HeLa-ACE2/TMPRSS2 cells were maintained in D10 media – Dulbecco’s Modified Eagle Medium (DMEM, Corning ® , Corning, New York) supplemented with 10% fetal bovine serum (GeminiBio ® , West Sacramento, California, 1% penicillin/streptomycin/L-glutamine (GeminiBio), and 1% 1M HEPES (Gibco).
  • DMEM Modified Eagle Medium
  • GeminiBio fetal bovine serum
  • Expi-293F cells were purchased from Thermo Fisher Scientific and maintained in Freestyle293/Expi-293 media (2:1, v/v, Thermo Fisher Scientific) in polycarbonate shaking flasks (Triforest Labware). StellarTM competent cells were purchased from Takara Bio.
  • Human coronavirus spike protein constructs [0227] Nucleic acid sequences encoding spike proteins from seven human coronaviruses (hCoVs) were cloned into a pADD2 vector between the rBeta-globin intron and ⁇ -globin poly(A). All of the above nucleic acid sequences included sequences encoding a GCN4 trimerization domain, an AviTag TM , and a hexahistidine tag. D.
  • Lentivirus plasmids [0228] The plasmids encoding the full-length spike proteins with native signal peptides were cloned into the background of the HDM-SARS2-spike-delta21 plasmid (Addgene plasmid, 155130). A 21-amino acid C-terminal deletion was included to promote viral expression. SARS- CoV-1 spike protein used had an 18-amino acid C-terminal deletion. The other viral plasmids that were used included pHAGE-Luc2-IRS-ZsGreen (NR-52516), pHDM-Hgpm2 (NR-52517), pRC- CMV-Rev1b (NR-52519), and pHDM-tat1b (NR-52518). E.
  • DNA preparation DNA was isolated using Thermo Fisher Scientific GeneJET plasmid mini-prep kit according to the manufacturer’s recommendations. The sequences of the plasmids were confirmed by whole-plasmid sequencing (Primordium). The plasmid DNA was transformed into Stellar cells and then isolated using the NuceloBond Xtra Maxi Kit following the manufacturer’s recommendations (Macherey-Nagel). The isolated plasmid DNA was filtered through a sterile 0.45- m membrane in a biosafety cabinet and stored at 4 C or -20°C. F. Protein production [0230] Proteins were expressed in Expi293F cells at 37 °C in 8% CO 2 .
  • the cells were diluted to a density of approximately 3–4 ⁇ 10 6 cells per mL.
  • the following protocol was used for transfection of the constructs other than those encoding Fc and Fab of fusion proteins.
  • 120 ⁇ g of plasmid DNA was added to 20 mL of expression media, followed by dropwise addition of 260 ⁇ L of FectoPro transfection reagent (Polyplus) with vigorous mixing.
  • the transfection mixture contained 60 ⁇ g of light chain- encoding plasmid DNA and 60 ⁇ g plasmid DNA encoding a fusion protein.
  • Two plasmids were 97 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 used for fusion proteins with a Fab-based antibody domain.
  • One plasmid encoded heavy chain sequences (illustrated in FIG. 5, FIG. 6, FIG. 7, FIG. 12, FIG. 13, and FIG. 14).
  • Another plasmid encoded light chain sequences illustrated in FIG.
  • HBS HEPES-buffered Saline
  • FPLC fast protein liquid chromatography
  • Filtered cell supernatants were diluted with 1/10 volume of 10 ⁇ HBS.
  • the FPLC system was equilibrated with an equilibration buffer containing the following fluids: A1 – 1 ⁇ HBS; A2 – 100 mM glycine pH 2.8; B1 – 0.5 M NaOH; Buffer line – 1xHBS; and Sample lines – ddH2O.
  • the column was washed with A1, and then the proteins were loaded onto the column eluted with A2 into 50 mL conical tubes containing 2.5 mL of 1M HEPES buffer, pH 7.4. The column was then washed sequentially with A1, B1, and A1.
  • Fc-containing samples were buffer exchanged and concentrated using 30-kDa or 50-kDa cutoff centrifugal concentrators. IgGs used for competition, binding and neutralization experiments were not further purified. Fc-containing proteins were further purified on the FPLC system using Superdex 6Increase (S6) gel filtration column.
  • Ni-NTA resin was added, and the samples were then incubated overnight at 4 °C while stirring. Resin– supernatant mixtures were added to chromatography columns for gravity flow purification.
  • SARS-CoV-2 VOCs SARS-CoV-, CoV-SL-CoV-WIV1 (“WIV1”), MERS-CoV VOCs, and merbecovirus spike pseudotyped lentiviral particles were produced as follows. Viral transfections were done in HEK293F cells using BioT reagent (Bioland Scientific). Cells were diluted to a density of approximately 3–4 ⁇ 10 6 cells per mL.
  • transfection mixture For a 50 mL transfection mixture, the following five plasmids were added: 50 ⁇ g of pHAGE-Luc2-IRES-ZsGreen; 17 ⁇ g SARS-CoV-2 variant-encoding plasmid or MERS-CoV variant-encoding plasmid; and 11 ⁇ g of each helper plasmid (pHDM-Hgpm2, pHDM-Tat1b, and pRC-CMV_Rev1b). 5 mL of expression media was then added to the transfection mixture, followed by dropwise addition of 150 ⁇ L of BioT with vigorous mixing. After a 10-minute incubation at room temperature, the transfection mixture was added to 50 mL of cell suspension.
  • D-glucose (4 g/L, Sigma-Aldrich) and valproic acid (3 mM, Acros Organics) were then added to the cells immediately post-transfection to increase recombinant protein production.
  • the cells were harvested 3–5 days after transfection by spinning the cultures at 300 ⁇ g for 5 minutes, followed by filtering through a 0.45- ⁇ m filter. 0.5 mL of 1 mM HEPES was then added to neutralize the pH.
  • the resulting viral stocks were aliquoted, flash-frozen in liquid nitrogen, stored at ⁇ 80 °C, and titrated before further use. [0241] If the viral titers were low, Lenti-X concentrator (Takara Bio) was used following the manufacturer’s protocol.
  • the cultures were spun at 500 ⁇ g for 10 minutes and filtered through a 0.45- ⁇ m filter. Each filtered supernatant (3 volumes) was combined with Lenti-X Concentrator (1 volume) and gently mixed. The mixture was stored at 4°C overnight. The next day, the mixture was centrifuged at 1,500 x g for 45 minutes at 4°C. The supernatant was removed carefully without disturbing the pellet and DMEM was added at 1/10th to 1/20th the original volume. The resulting viral stocks were aliquoted, flash-frozen in liquid nitrogen, stored at ⁇ 80 °C, and titrated before further use.
  • protein solutions were serially diluted (10-fold dilution for fusion proteins, 5-fold dilution for ACE2-Fc) in D10 media.
  • Samples were run in technical duplicates in each experiment.
  • a virus mixture was made containing the virus of interest (for example, SARS-CoV-2 Wild Type), D10 media, and polybrene (1:500).
  • Virus dilutions into media were selected such that a suitable signal (a luminescence of at least >1,000,000 RLU) would be obtained in the virus-only wells. 60 ⁇ L of this virus mixture was added to the dilution plate to make a final volume of 120 ⁇ L in each well.
  • Virus-only wells were made that contained 60 ⁇ L of D10 media and 60 ⁇ L of virus mixture.
  • Cell-only wells were made that contained 120 ⁇ L of D10 media.
  • the inhibitor/virus mixture was left to incubate for 1-2 hours at 37 °C. After incubation, the medium was removed from the cells on the plates made 1 day prior. 100 ⁇ L of inhibitor/virus dilutions were added, and the plates were incubated at 37 °C for 2 days.
  • infectivity readout was performed by removing the media from the wells and adding 80 ⁇ L of a 1:1 dilution of BriteLite in Dulbecco’s Phosphate-Buffered Saline (DPBS; BriteLite Plus, Perkin Elmer). Luminescence values were measured using a microplate reader (BioTek SynergyTM HT or Tecan M200). Each plate was normalized by averaging cell-only (0% infectivity) and virus-only (100% infectivity) wells. Normalized values were fit with a three- parameter non-linear regression inhibitor curve in Prism to obtain 50% inhibitory concentration (IC 50 ) values. The average half-maximal neutralization titers (NT50) of two independent experiments were calculated.
  • Merbecovirus neutralization assays were performed substantially as described above, but HeLa/DPP4 cells were used instead of HeLa/ACE2/TMPRSS2 cells. The cells were plated at either 5,000 cells per well two days before infection or 8,000 cells per well the day before infection. Additionally, on the day of the assay (day 1), purified fusion proteins (stored in HBS with 10% glycerol) were diluted with D10 media to the starting concentration (200 nM for monomeric fusion proteins, 20 nM for dimeric fusion proteins, or 2 nM for trimeric fusion proteins). Concentrations were chosen based on predicted IC50 values to achieve a full infection curve.
  • the blot was incubated with 50 mL of 1XPBST + 10% non-fat dry milk (Bio-Rad) for 1 hour at room temperature, followed by incubation with anti-ACE2 mouse antibody (1:4000 dilution in PBST with 10% non-fat dry milk) for 1 hour at room temperature, and the detection with goat anti-mouse HRP-conjugated (1:4000 dilution in PBST with 10% non-fat dry milk) for 1 hour at room temperature.
  • Luminescent signals were developed using a luminol-based substrate (Pierce ECL, Thermo Fisher Scientific) for imaging on a chemiluminescence imager (GE Amersham Imager 600). N.
  • SARS-CoV-2 variants were obtained from two sources. WA-1/2020 was obtained from World Reference Center for Emerging Viruses and Arboviruses. BA.5 and JN.1 were isolated from de-identified nasopharyngeal (NP) swabs sent to the California Department of Public Health from hospitals in California for surveillance purposes.
  • NP nasopharyngeal
  • NP swab sample from a patient with COVID-19 200 ⁇ L was diluted 1:3 in PBS supplemented with 0.75% BSA (BSA-PBS) and added to confluent Vero E6-TMPRSS2-T2A- ACE2 cells in a T25 flask, allowed to adsorb for 1 hour, inoculum removed, and additional media was added. The flask was incubated at 37 °C with 5% CO2 for 3–4 days, with daily monitoring for cytopathic effects (CPE).
  • CPE cytopathic effects
  • CPE endpoint neutralization assays were performed following the limiting dilution model using sequence-verified viral stocks of WA-1, BA.5 and JN.1 in Vero E6-TMPRSS2-T2A-ACE2. Two-fold serial dilutions of inhibitor were made in BSA-PBS and mixed at a 1:1 ratio with 100 TCID50 of each virus and incubated for 1 hour at 37 °C. Final inhibitor dilutions ranged from 500 nM to 0.06 nM.
  • mice were evaluated by administering a fusion protein in PBS to 6-8 week old male C57BL/6 mice via a 20 mg/kg intravenous (i.v.) bolus tail vein injection.
  • the mice were housed in Innovive IVC systems and provided with chow diet and water ad libitum. The housing room was temperature and humidity controlled with a 12 hour light/dark cycle.
  • the blood samples collected from the mice via lateral tail vein before treatment and at 1, 3, 5, or 24 hours post-treatment. Serum was separated from the collected blood samples by centrifugation at 5000 rpm at 4°C for 10 min.
  • the concentration of fusion proteins in the resulting serum samples was determined by sandwich ELISA as described below.
  • R. Sandwich ELISA [0246] Clear Flat-Bottom Immuno Nonsterile 96-Well plates (Thermo Fisher Scientific) were coated with 100 ⁇ L of AffiniPureTM Goat Anti-Human IgG, F(ab')2 fragment specific antibody (Jackson ImmunoResearch) diluted to 2.4 ⁇ g/mL (1000-fold dilution from 2.4 mg/mL stock). The plates were incubated overnight at 4oC. The coating solution was removed from each plate and replaced with 200 ⁇ L of blocking solution (3% BSA in 0.05% PBS-T, pH 7.4), followed by overnight incubation at 4oC.
  • mice 6-8 week old female AC70 hACE2 mice (Taconic) were anesthetized with isoflurane and challenged intranasally (i.n) with Omicron variant XBB1.5 SARS-CoV-2 virus at 1.96x10 4 PFU/dose on day 0.
  • mice were housed in ABSL3 facility post-challenge and provided with chow diet and water ad libitum. The housing room was temperature and humidity controlled with a 12 hour light/dark cycle. The mice were monitored daily for body weight changes and twice daily for clinical signs (ruffled fur, hunched posture, inactivity), with >20% body weight loss serving as the euthanasia criterion. Oral swabs for viral RNA quantification were collected prior to treatment, on day 1. day 2, day 3, and day 5 post-challenge.
  • Quantitative RT-PCR assay for SARS-CoV-2 RNA [0248] Quantitative RT-PCR assay of the oral swab samples obtained from experimental mice was performed for SARS-CoV-2 genomic RNA (gRNA) targeting a conserved region of Nucleocapsid (N) gene of SARS-CoV-2. The primers and probe used for the RT-PCR assay are shown in Table 10. Table 10.
  • RT-PCR assay primers and probe [0249] Lyophilized primer oligonucleotides were reconstituted to 100 ⁇ M in 10 mM Tris + 0.1 mM EDTA (TE) solution, pH 8.0, and diluted 1:1:50 to working stock in RNAse free water (Sigma), for 2 ⁇ M primer mix. Lyophilized probe was likewise diluted to 2 ⁇ M solution.
  • PCR master mix was prepared by combining 500 ⁇ L RNAse-free water, 1.5 mL primer mix (600 nM final concentration), 350 ⁇ L probe (140nM final concentration), and the following SensiFASTTM Probe Lo-ROX One-Step Kit (Meridian Bioscience) components: 2.5 mL 2X Reaction Buffer, 50 ⁇ L reverse transcriptase, and 100 ⁇ L RiboSafe RNAse inhibitor.
  • Viral RNA was extracted from oral swab samples using a QIAmp MinElute Virus Spin Kit (Qiagen).
  • EXAMPLE 2 EXAMPLE 2.
  • Antibody identification [0252] The antibodies binding SARS-CoV-2 spike protein with high affinity and also cross- reactive with all tested human coronavirus spike proteins (thus targeting a highly conserved epitope) were identified. Eight known non-neutralizing mAbs binding highly conserved S2 subunit on SARS-CoV-2 spike protein were tested. These antibodies included several mAbs that also bound HCoV-OC43 and HCoV-HKU1 human coronaviruses, which are betacoronaviruses that cause cold-like symptoms (Brewer et al. 2022).
  • Binding of eight mAb Fabs (at 200 nM) to SARS- CoV-2 Wuhan spike protein was measured using biolayer interferometry (BLI), and it was determined that Fabs of the seven out of eight mAbs bound SARS-CoV-2 Wuhan spike protein.
  • BLI measurements are summarized in Table 11. The two best candidates for further experiments were identified: (1) mAb CoV2-11, which bound SARS-CoV-2 Wuhan spike protein with sub-nanomolar affinity (0.19 nM); and (2) mAb CoV2-26, which bound SARS-CoV-2 Wuhan spike protein with nanomolar affinity (2.3 nM).
  • CoV2-11 and CoV2-26 Fabs were found to bind with nanomolar or sub-nanomolar affinity to each of the five tested spike proteins, including the spike 106 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 protein of HCoV-229E, a common cold alphacoronavirus (Brewer et al. 2022).
  • the binding data are summarized in Table 12. Table 12. Binding of mAbs CoV2-11 and CoV2-26 to coronavirus spike proteins (measured by BLI).
  • Fusion proteins were created based on CoV2-11 and CoV2-26 by linking their scFvs to catalytically inactive ACE2 according to the procedures described in Weidenbacher et al. 2022. Catalytically inactive ACE2 lacking peptidase activity was used to avoid potential adverse effects in patients due to administration of catalytically active exogenous ACE2 (Patel et al. 2021, Garc ⁇ a- Escobar et al. 2021).
  • H345L ACE2 or iACE2 H345L ACE2
  • iACE2 H345L ACE2
  • Binding of iACE2 to SARS-CoV-2 spike protein and its neutralization properties were tested. No significant differences with catalytically active ACE2 in affinity or neutralization potency were detected.
  • CoV2-11 scFv linked to iACE2 (CoV2-11-scFv – iACE2); (2) CoV2-26 scFv linked to iACE2 (CoV2-26-scFv – iACE2); 108 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 (3) CoV2-11 Fab linked to iACE2 (CoV2-11-Fab – iACE2); and (4) CoV2-26 Fab linked to iACE2 (CoV2-26-Fab – iACE2). SDS-PAGE was used to confirm their purity.
  • the stability of the above fusion proteins was measured by differential scanning fluorimetry. The results are summarized in Table 14. The measured melting temperatures of the fusion proteins were similar to the melting temperature of ACE2 and iACE2. Table 14. Melting temperatures (°C) measured in triplicate by differential scanning fluorimetry.
  • the fusion proteins neutralized all VOCs tested, including all the variants that arose after BQ.1.
  • CoV2-26-scFv – iACE2 fusion protein neutralized all variants with higher consistency than CoV2-11-scFv – iACE2 fusion protein, despite the latter fusion protein having higher binding affinity.
  • CoV2-11 and CoV2-26 IgGs failed to neutralize pseudotyped lentiviruses in the absence of the linked ACE2 component.
  • the yield of the scFv-based fusion proteins was low. Replacing scFv component of the fusion proteins with a Fab component resulted in significant improvements in yield.
  • fusion proteins discussed in the previous Example did not include an Fc domain, they were likely to have a short half-life upon administration, which may lead to decreased in vivo efficacy or require frequent dosing (Tada et al. 2023; Jha et al. 2022). Moreover, it has been shown that Fc-mediated effector functions of the antibodies, such as antibody-dependent cellular cytotoxicity (ADCC), can elicit protective effects in vivo (Pierre et al. 2023). Adding an Fc domain to fusion proteins may enhance their potency in vivo. [0260] Human IgG1 Fc region from the VRC01 vector was incorporated into the fusion protein expression vector.
  • the detected neutralization potency of the trimeric fusion proteins was significantly improved compared to monomeric fusion proteins. Strikingly, the trimeric fusion proteins neutralized all SARS-CoV-2 VOCs with a 10-fold or higher potency than bebtelovimab. Neutralization potency of both scFv-based trimeric fusion proteins and Fab-based trimeric fusion proteins was tested, and no significant differences were detected (Table 20 and Table 21). 113 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 Table 20. Comparative neutralization potency, measured by pseudotyped lentiviral neutralization assay, for CoV2-11 antibody-based fusion proteins.
  • Comparative neutralization potency measured by pseudotyped lentiviral neutralization assay, for CoV2-26 antibody-based fusion proteins. * monomeric; ** dimeric; *** trimeric 115 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 Table 22. Comparative neutralization potency of Fab-based fusion proteins against SARS-CoV-1 pseudovirus. * monomeric; ** dimeric; *** trimeric Table 23. Live virus neutralization testing. * monomeric; ** dimeric; *** trimeric D.
  • Dimeric fusion protein CoV2-26-Fab – iACE2 – Fc had a significantly longer half-life (4.7 hours) than CoV2-26-Fab – iACE2 – tri trimeric fusion protein (0.5 hours).
  • Table 24 Serum concentration over time of CoV2-26-Fab – iACE2 – tri fusion protein in mice.
  • Table 25 Serum concentration over time of CoV2-26-Fab – iACE2 – tri fusion protein in mice.
  • Anti-MERS fusion proteins [0266] As discussed in Example 2, non-neutralizing antibodies CoV2-11 and CoV2-26 bound MERS spike protein with high affinity (Table 11).
  • Anti-MERS fusion proteins were constructed by replacing iACE2 with catalytically active DPP4 or catalytically inactive S630A DPP4 (iDPP4). The resulting fusion proteins were tested and found to bind MERS spike protein with high affinity. Binding affinity measured by BLI was similar between catalytically active (DPP4) and catalytically inactive DPP4 (iDPP4). Neutralization potency of anti-MERS fusion proteins was measured by pseudotyped lentiviral neutralization assay. The data is summarized in Table 26. It was found that both monomeric and dimeric anti-MERS fusion proteins neutralized MERS pseudovirus with higher potency than DPP4 and DPP4-Fc.
  • mice were dosed three times during the study at a dosage of 10 mg/kg, starting at 12 hours post-infection, with subsequent doses every 18 hours. Body weights were recorded once daily, and clinical observations were made twice daily.
  • the mice treated with CoV2-26-Fab – iACE2 – Fc dimeric fusion protein exhibited 80% survival and maintained their body weight, as compared to 20% survival and >20% body weight loss in the control group treated with vehicle (PBS).
  • Body weight change in mice challenged with live Omicron variant XBB.1.5 (treatment group) and control group mice is illustrated in FIG. 37 and summarized in Table 27.
  • mice in the control group exhibited subtle clinical disease, with mild ruffled fur and a hunched back as described in Yinda et 118 US2008313719721 Attorney Docket No.: 110221-1489731-010510WO Client Ref: CZB-292S-PC; S23-308 al. 2021, which progressed to severe clinical disease indicated by ruffled fur in all mice by day 7.
  • mice in the treatment group had only mild ruffled fur and/or a hunched back on days 7 and 8, but returned to normal appearance for the remainder of the study.
  • N-terminus of Fc domain was linked to C-terminus of CoV2-26 Fab domain
  • C-terminus of Fc domain was linked to N-terminus of iACE2 domain (CoV2-26-Fab – Fc – iACE2 orientation; FIG. 15C and FIG. 16).
  • the yield and the purity of both new dimeric fusion protein variants were significantly and unexpectedly higher than those of CoV2-26-Fab – iACE2 – Fc dimeric fusion protein.
  • the neutralization potency of the two new dimeric fusion protein variants was also found to be significantly and unexpectedly higher than that of CoV2-26-Fab – iACE2 – Fc dimeric fusion protein as shown in FIG. 18 and FIG. 19. See also Table 29, A and B. Furthermore, when tested by pseudotyped lentiviral neutralization assay, the two new dimeric fusion protein variants unexpectedly exhibited similar or higher neutralization potency than bebtelovimab. This in contrast to CoV2-26 – iACE2 – Fc dimeric fusion protein, which exhibited similar neutralization potency than bebtelovimab.
  • N-terminus of CMP trimerization domain was linked to C-terminus of CoV2-26 Fab domain
  • N-terminus iACE2 domain was linked to C-terminus of CMP domain (CoV2-26-Fab – tri – iACE2 orientation; FIG. 1C and FIG. 17).
  • the neutralization potency of the two trimeric fusion new variants was also found to be significantly and unexpectedly higher than that of CoV2-26-Fab – iACE2 – tri fusion protein as shown in FIG. 22 and FIG. 23. See also Table 30, A and B. Table 31.
  • each of the new dimeric and trimeric fusion protein variants neutralized HCoV-NL63 with at least ten-fold higher potency, as compared to, respectively, to CoV2-26-Fab – iACE2 – Fc and CoV2-26-Fab – iACE2 – tri (FIG. 24, FIG. 25A, and FIG. 25B; see also Table 31, A and B).
  • EXAMPLE 8 Designing improved DPP4-based fusion proteins [0271] To improve purity and yield of DPP4-based fusion proteins, two iDPP4 molecules were linked together to force iDPP4 domains into a dimeric state.
  • Monomeric DPP4-based fusion protein variants were created by linking CoV2-26 Fab at either the N terminus (CoV2-26-Fab – iDPP4–iDPP4 orientation) or C terminus (iDPP4– iDPP4 – CoV2-26-Fab orientation) of iDPP4–iDPP4 (FIG. 28).
  • the resulting molecules were thermally stable and bound MERS spike with high affinity.
  • Dimeric and trimeric fusion proteins incorporating diDPP4 and CoV2-26 Fab were found to neutralize MjHKU4r-CoV-1, while the neutralizing MERS antibody (Clone 834) did not.
  • the above results are illustrated in FIG. 33, FIG. 34, FIG. 35, and FIG. 36 and summarized in Table 32, A and B, and Table 33, A and B.
  • nine antibodies were also tested as controls against merbecoviruses.
  • the nine antibodies (G2, G4, 4V2, 4C2h, JC57, M336, GD27, REGN antibody #1, and REGN antibody #2) bind several epitopes on MERS-CoV spike proteins (class I, class II, and class III RBD binders, S2 binder, and NTD binder, respectively), including two antibodies that completed phase I clinical trials (Du et al. 2017).
  • the results for the nine antibodies are summarized in Tables 34 and 35.
  • Table 32 The results of pseudotyped lentivirus neutralization assay for dimeric fusion proteins against merbecoviruses.
  • Non-neutralizing SARS-CoV-2 N-terminal domain antibodies protect mice against severe disease using Fc-mediated effector functions.” bioRxiv 2023.07.25.550460 doi:10.1101/2023.07.25.550460 (2023). Pollock et al. “Transgenic milk as a method for the production of recombinant antibodies.” J. Immunol Methods 231(1-2):147-157 (1999). Ramirez-Paz et al. “Thiol-maleimide poly(ethylene glycol) crosslinking of L-asparaginase subunits at recombinant cysteine residues introduced by mutagenesis.” PLoS One 13(7):e0197643 (2016). Raybould et al.

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Abstract

L'invention concerne des protéines de fusion d'au moins un domaine neutralisant capable de se lier spécifiquement à un domaine de liaison au récepteur (RBD) d'une protéine de spicule de coronavirus, et au moins un domaine d'anticorps capable de lier spécifiquement un épitope dans une région conservée de la protéine de spicule de coronavirus. Les protéines de fusion peuvent être utilisées dans le traitement et la prophylaxie d'infections à coronavirus. L'invention concerne également des procédés de fabrication et d'utilisation des protéines de fusion.
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WO2022241200A1 (fr) * 2021-05-14 2022-11-17 Vanderbilt University Anticorps anti-coronavirus à réactivité croisée
US20220403009A1 (en) * 2020-07-27 2022-12-22 Igm Biosciences, Inc. Multimeric coronavirus binding molecules and uses thereof
WO2023006850A1 (fr) * 2021-07-27 2023-02-02 Paolo Fiorina Méthodes de traitement d'une néphropathie diabétique et d'une maladie glomérulaire

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220403009A1 (en) * 2020-07-27 2022-12-22 Igm Biosciences, Inc. Multimeric coronavirus binding molecules and uses thereof
WO2022241200A1 (fr) * 2021-05-14 2022-11-17 Vanderbilt University Anticorps anti-coronavirus à réactivité croisée
WO2023006850A1 (fr) * 2021-07-27 2023-02-02 Paolo Fiorina Méthodes de traitement d'une néphropathie diabétique et d'une maladie glomérulaire

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