WO2021247675A9 - Agents liant les coronavirus et leurs utilisations - Google Patents

Agents liant les coronavirus et leurs utilisations Download PDF

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Publication number
WO2021247675A9
WO2021247675A9 PCT/US2021/035407 US2021035407W WO2021247675A9 WO 2021247675 A9 WO2021247675 A9 WO 2021247675A9 US 2021035407 W US2021035407 W US 2021035407W WO 2021247675 A9 WO2021247675 A9 WO 2021247675A9
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coronavirus
polypeptide
ace2
seq
cov
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PCT/US2021/035407
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WO2021247675A1 (fr
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Shelley Ruth GREEN
Zhonghao LIU
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Ngm Biopharmaceuticals, Inc.
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Publication of WO2021247675A1 publication Critical patent/WO2021247675A1/fr
Publication of WO2021247675A9 publication Critical patent/WO2021247675A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present disclosure generally relates to agents that bind coronaviruses, particularly coronaviruses that use angiotensin-converting enzyme 2 (ACE2) as a receptor, as well as compositions comprising the coronavirus-binding agents and methods of using the agents and compositions.
  • ACE2 angiotensin-converting enzyme 2
  • SARS-CoV-1 Severe acute respiratory syndrome coronavirus
  • SARS-CoV-1 Severe acute respiratory syndrome coronavirus
  • SARS-CoV-2 Severe acute respiratory syndrome coronavirus
  • SARS-CoV-2 SARS-CoV-2 is responsible for a global pandemic that has resulted in almost 6 million people being infected and the deaths of more than 350,000 individuals.
  • SARS-CoV-2 causes a disease syndrome (COVID-19) similar to SARS with, in some cases, a multitude of cascading sequelae that include an immune system response that appears to be similar to cytokine release syndrome.
  • Angiotensin-converting enzyme 2 (ACE2) was identified as a functional receptor for SARS-CoV-1 (Li et al., 2003, Nature, 426:450-454). Recent studies have shown that SARS-CoV-2 also uses human ACE2 as a cellular entry receptor (Zhou et al., 2020, Nature, 579:270-275).
  • ACE2 is a potential target for therapies against SARS-CoV-1 and SARS-CoV-2.
  • the present invention provides novel agents, including, but not limited to, polypeptides, soluble proteins, fusion proteins, homodimeric molecules, and multimeric molecules that bind coronaviruses, particularly coronaviruses that use angiotensinconverting enzyme 2 (ACE2) as a receptor.
  • the agents may also be referred to herein as “coronavirus-binding agents.”
  • the agents include polypeptides comprising a first domain comprising an extracellular region of ACE2 (e.g., human ACE2) and a second domain comprising an IgM heavy chain constant region (also referred to herein as “IgM Fc region”) (e.g., a human IgM heavy chain constant region).
  • the extracellular region of ACE2 binds to coronavirus Spike protein (e.g., SARS-CoV-2 SI subunit of the Spike protein).
  • the agents also include homodimers and multimers of the polypeptides. In some embodiments, these polypeptides, homodimers, or multimers are used as coronavirus traps.
  • the coronaviruses bind ACE2 (i.e., use ACE2 as a receptor).
  • the coronaviruses can include, but are not limited to, SARS-CoV-1, SARS- CoV-2, and CoV-NL63.
  • the coronavirus-binding agents or agents described herein inhibit the interaction between a coronavirus virus and ACE2. In some embodiments, the coronavirus-binding agents described herein inhibit the interaction between a coronavirus and human ACE2. In some embodiments, the coronavirusbinding agents are used to treat a coronavirus infection. In some embodiments, the coronavirus-binding agents are used to prevent a coronavirus infection. In some embodiments, the coronavirus-binding agents are used to treat a disease caused by a coronavirus infection. In some embodiments, the coronavirus-binding agents are used in a combination therapy. In some embodiments, the coronavirus-binding agents are used in combination with at least one additional therapeutic agent.
  • the disclosure also provides compositions comprising the coronavirus-binding agents described herein.
  • the disclosure provides pharmaceutical compositions comprising the coronavirus-binding agents described herein.
  • Polynucleotides and/or vectors encoding the coronavirus-binding agents are provided.
  • Cells comprising the polynucleotides and/or the vectors described herein are also provided.
  • Cells comprising or producing the coronavirus-binding agents described herein are provided. Methods of making the binding agents described herein are also provided.
  • the present disclosure provides agents that bind coronaviruses.
  • a coronavirus-binding agent binds SARS-CoV-2.
  • a coronavirus-binding agent binds SARS-CoV-1.
  • a coronavirus-binding agent binds CoV-NL63.
  • a coronavirusbinding agent binds the Spike protein of a coronavirus.
  • a coronavirus-binding agent binds the SI subunit of a Spike protein of a coronavirus.
  • a coronavirus-binding agent binds the receptor binding domain (RBD) of the SI subunit of a Spike protein of a coronavirus. In some embodiments, a coronavirus-binding agent binds the Spike protein of SARS-CoV-2. In some embodiments, a coronavirus-binding agent binds the SI subunit of a Spike protein of SARS-CoV-2. In some embodiments, a coronavirus-binding agent binds the RBD of the SI subunit of a Spike protein of SARS-CoV-2. In some embodiments, a coronavirus-binding agent binds within amino acids 13-685 of the amino acid sequence set forth in SEQ ID NO:31.
  • a coronavirus-binding agent binds within amino acids 319-529 of the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, a coronavirus-binding agent binds the amino acid sequence set forth in SEQ ID NO:32 or SEQ ID NO:33. In some embodiments, a coronavirusbinding agent is a fusion polypeptide. In some embodiments, a coronavirus-binding agent is a fusion polypeptide that binds SARS-CoV-2. In some embodiments, a coronavirus-binding agent is a fusion polypeptide that binds SARS-CoV-1.
  • a coronavirus-binding agent is a fusion polypeptide that binds CoV- NL63. In some embodiments, a coronavirus-binding agent is a fusion polypeptide that binds SARS-CoV-2 and SARS-CoV-1.
  • the polypeptides described herein comprise a first domain comprising an extracellular region of ACE2 and a second domain comprising an IgM heavy chain constant region.
  • the extracellular region of ACE2 binds to coronavirus Spike protein (e.g., SARS-CoV-2 SI subunit of the Spike protein).
  • the polypeptides described herein comprise a first domain comprising an extracellular region of human ACE2 and a second domain comprising a human IgM heavy chain constant region.
  • Polypeptides comprising an extracellular region of a mammalian (other than human) ACE2 and a mammalian (other than human) IgM heavy chain constant region are also envisioned in this disclosure.
  • the linker is a peptide linker.
  • the polypeptides bind at least one coronavirus. In some embodiments, the polypeptides bind the Spike protein of a coronavirus. In some embodiments, the polypeptides bind the RBD of the Spike protein of a coronavirus.
  • the first domain of the polypeptide binds a coronavirus Spike protein.
  • the first domain of the polypeptide comprises the full-length extracellular domain of ACE2 (e.g., a human ACE2).
  • the first domain of the polypeptide comprises amino acids 18-740 of SEQ ID NO: 1.
  • the first domain of the polypeptide comprises the amino acid sequence of SEQ ID NO:3.
  • the first domain of the polypeptide comprises a fragment of the full-length extracellular domain of ACE2 (e.g., a human ACE2).
  • the first domain of the polypeptide comprises a fragment of the full-length extracellular domain of ACE2 (e.g., a human ACE2), wherein the fragment contains an amino acid sequence with at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) sequence identity to the full length extracellular domain of ACE2 (e.g., amino acids 18-740 of SEQ ID NO: 1 for human ACE2 or SEQ ID NO:3), wherein the first domain binds the Spike protein of a coronavirus (e.g., SARS-CoV-2 SI subunit of the Spike protein).
  • ACE2 e.g., a human ACE2
  • the fragment contains an amino acid sequence with at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) sequence identity to the full length extracellular domain of ACE2 (e.g., amino acids 18-740 of SEQ ID NO: 1 for human
  • the first domain of the polypeptide comprises amino acids 18-615 of SEQ ID NO: 1. In some embodiments, the first domain of the polypeptide comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the first domain of the polypeptide comprises a fragment of the full-length extracellular domain of ACE2 (e.g., a human ACE2), wherein the fragment contains an amino acid sequence with at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) sequence identity to the amino acid sequence of SEQ ID NO:4, wherein the first domain binds the Spike protein of a coronavirus (e.g., SARS-CoV-2 SI subunit of the Spike protein).
  • a coronavirus e.g., SARS-CoV-2 SI subunit of the Spike protein
  • the first domain of the polypeptide comprises a variant of the wild-type full-length extracellular domain of ACE2. In some embodiments, the first domain of the polypeptide comprises a variant of a wild-type fragment of the full-length extracellular domain of ACE2 (e.g., a human ACE2). In some embodiments, the first domain of the polypeptide comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:3, wherein the first domain binds the Spike protein of a coronavirus (e.g., SARS-CoV-2 SI subunit of the Spike protein).
  • a coronavirus e.g., SARS-CoV-2 SI subunit of the Spike protein
  • the first domain of the polypeptide comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:4, wherein the first domain binds the Spike protein of a coronavirus (e.g., SARS-CoV-2 SI subunit of a Spike protein).
  • the first domain of the polypeptide comprises an extracellular region of ACE2 (e.g., a human ACE2) that includes one or more mutations that reduce glycosylation of the ACE2.
  • the first domain of the polypeptide comprises an amino acid sequence of SEQ ID NO:3 containing one or more (e.g., 1, 2, 3, 4, 5, 6) mutations selected from N53S, T92A, N103S, T324A, N432S and N546S.
  • the first domain of the polypeptide comprises an amino acid sequence of SEQ ID NO:4 containing one or more (e.g., 1, 2, 3, 4, 5, 6) mutations selected from N53S, T92A, N103S, T324A, N432S and N546S.
  • the first domain of the polypeptide comprises any naturally-occurring variants of ACE2 (see e.g., Sorokina, M. et al. Structural models of human ACE2 variants with SARS-CoV-2 Spike protein for structure-based drug design. Sci Data 7, 309 (2020); Bakhshandeh B,, Infect Genet Evol. 2021 Jun; 90: 104773).
  • the second domain of the polypeptide comprises an IgM heavy chain constant region (e.g., a human IgM heavy chain constant region), which contains an IgM CH4 domain comprising the tail piece domain.
  • the second domain of the polypeptide comprises an IgM heavy chain constant region, which contains a CH3 domain and a CH4 domain comprising the tail piece domain.
  • the second domain of the polypeptide comprises an IgM heavy chain constant region, which contains a CH2 domain, a CH3 domain, and a CH4 domain comprising the tail piece domain.
  • the second domain of the polypeptide comprises a variant of an IgM heavy chain constant region, wherein the variant contains one or more mutations that abolish or reduce the IgM Fc effector functions. In some embodiments, the second domain of the polypeptide comprises a variant of an IgM heavy chain constant region, wherein the variant contains one or more mutations that increase the IgM Fc effector functions. In some embodiments, the second domain of the polypeptide comprises an IgM heavy chain constant region encompassing P311 A, P313 A or both P311 A and P313 A. In some embodiment, the IgM heavy chain constant region described herein is a human IgM heavy chain constant region.
  • the second domain of the polypeptide comprises amino acids 106-453 of SEQ ID NO:5 or amino acids 106-453 of SEQ ID NO:6. In some embodiments, the second domain of the polypeptide comprises amino acids 102-453 of SEQ ID NO:5 or amino acids 102-453 of SEQ ID NO:6.
  • the second domain of the polypeptide comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8, wherein the second domain of the polypeptide is able to multimerize as a pentamer or a hexamer.
  • at least 90% e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
  • the second domain of the polypeptide comprises an amino acid sequence having at least 90% (e.g., at least 90%, 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%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52.
  • the second domain of the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:7-14 and SEQ ID NOs:45-52.
  • the polypeptide comprises a first domain comprising an amino acid sequence with at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO:3, and a second domain comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs:7-14 and SEQ ID NOs:45-52, wherein the first domain binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein).
  • a coronavirus Spike protein e.g., SARS-CoV-2 Spike SI protein
  • the polypeptide comprises a first domain comprising an amino acid sequence with at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO:3, wherein the first domain binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein), and a second domain comprising an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to an amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8, wherein the second domain of the polypeptide is able to multimerize as a pentamer or a hexamer.
  • a coronavirus Spike protein e.g., SARS-CoV-2 Spike SI protein
  • the polypeptide comprises a first domain comprising an amino acid sequence with at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO:3, wherein the first domain binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein), and a second domain comprising an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs:9-14 and SEQ ID NOs:45-52, wherein the second domain of the polypeptide is able to multimerize as a pentamer or a hexamer.
  • the polypeptide comprises a first domain comprising an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:4, wherein the first domain binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein), and a second domain comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs:7-14 and SEQ ID NOs:45-52.
  • a coronavirus Spike protein e.g., SARS-CoV-2 Spike SI protein
  • the polypeptide comprises a first domain comprising an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:4, wherein the first domain binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein), and a second domain comprising an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to an amino acid sequence SEQ ID NO:7 or SEQ ID NO:8, wherein the second domain of the polypeptide is able to multimerize as a pentamer or a hexamer.
  • the polypeptide comprises a first domain comprising an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:4, wherein the first domain binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein), and a second domain comprising an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:9-14 and SEQ ID NOs:45-52, wherein the second domain of the polypeptide is able to multimerize as a pentamer
  • the polypeptide comprises a first domain comprising the amino acid sequence of SEQ ID NO:3 and a second domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:7-14 and SEQ ID NOs:45-52. In some embodiments, the polypeptide comprises a first domain comprising the amino acid sequence of SEQ ID NO:4 and a second domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:7-14 and SEQ ID NOs:45-52.
  • the polypeptide comprises a first domain comprising the amino acid sequence of SEQ ID NO:3 containing one or more (e.g., 1, 2, 3, 4, 5, 6) mutations selected from N53S, T92A, N103S, T324A, N432S and N546S and a second domain comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs:7-14 and SEQ ID NOs:45-52.
  • the polypeptide comprises a first domain comprising the amino acid sequence of SEQ ID NO:4 containing one or more (e.g., 1, 2, 3, 4, 5, 6) mutations selected from N53S, T92A, N103S, T324A, N432S and N546S and a second domain comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs:7-14 and SEQ ID NOs:45-52.
  • the polypeptide comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:22. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:25. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:29. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:21. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:26. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:30.
  • the present disclosure provides a coronavirus trap.
  • the coronavirus trap comprises two or more of the polypeptides described herein.
  • the first domain comprising an extracellular region of ACE2 of the polypeptides (the first domain of the polypeptide) binds free coronavirus particles.
  • the polypeptides are able to inhibit, reduce, and/or block the coronavirus particles from infecting cells via the native ACE2 receptor.
  • a coronavirus trap comprises a multimer of two or more of the polypeptides described herein.
  • a coronavirus trap comprises a multimer of two or more of the polypeptides with the identical amino acid sequence.
  • a coronavirus trap comprises a multimer of two or more of a polypeptide comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, a coronavirus trap comprises a multimer of two or more of a polypeptide comprising the amino acid sequence of SEQ ID NO:21. In some embodiments, a coronavirus trap comprises a multimer of two or more polypeptides comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, a coronavirus trap comprises a multimer of two or more of a polypeptide comprising the amino acid sequence of SEQ ID NO:29.
  • a coronavirus trap comprises a multimer of two or more of a polypeptide comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, a coronavirus trap comprises a multimer of two or more of a polypeptide comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, a coronavirus trap comprises a multimer of two or more of a polypeptide comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, a coronavirus trap comprises a multimer of two or more of a polypeptide comprising the amino acid sequence of SEQ ID NO:30.
  • the multimer is a homodimer of two identical polypeptides.
  • the multimer is a pentameric complex composed of five homodimers each comprising two identical polypeptides.
  • the pentameric complex comprises an immunoglobulin J chain, a fragment thereof, or variant thereof.
  • the multimer is a hexameric complex composed of six homodimers each comprising two identical polypeptides.
  • the present disclosure provides a multimeric binding agent.
  • the multimeric binding agent comprises two, five, or six binding units, wherein each binding unit comprises two polypeptides, wherein each polypeptide comprises a first domain comprising an extracellular region of ACE2 (e.g., human ACE2); and a second domain comprising an IgM heavy chain constant region (e.g., a human IgM heavy chain constant region) as described herein.
  • the multimeric binding molecule is a pentameric binding molecule comprising five binding units.
  • the multimeric binding molecule is a pentameric binding molecule which comprises an immunoglobulin J chain, a fragment thereof, or variant thereof.
  • the multimeric binding molecule is a hexameric binding molecule comprising six binding units.
  • the multimeric binding agent described herein binds a coronavirus.
  • the multimeric binding agent binds the Spike protein of a coronavirus.
  • the multimeric binding agent binds the receptor binding domain (RBD) of the Spike protein of a coronavirus.
  • the coronavirus is a coronavirus that binds ACE2.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, and/or CoV-NL63.
  • the present disclosure provides agents that bind coronavirus and inhibit, reduce, or block the interaction between a coronavirus and ACE2.
  • the coronavirus-binding agents described herein inhibit, reduce, or block the interaction between a coronavirus and human ACE2.
  • the coronavirus-binding agents described herein inhibit, reduce, or block the interaction between the Spike protein of a coronavirus and ACE2.
  • the coronavirus-binding agents described herein inhibit, reduce, or block the interaction between the spike SI protein of a coronavirus and ACE2.
  • the coronavirus-binding agents described herein inhibit, reduce, or block the interaction between the RBD of the Spike protein of a coronavirus and ACE2.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, and/or CoV-NL63.
  • ACE2 is expressed on the surface of mammalian endothelial cells.
  • ACE2 is expressed in tissues including, but not limited to, lung, intestine, liver, heart, and kidney.
  • the ACE2 is human ACE2.
  • the present disclosure provides methods of treating a coronavirus infection.
  • a method of treating a coronavirus infection in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein, wherein the coronavirus binds ACE2.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, and/or CoV-NL63.
  • the coronavirus is a variant of SARS-CoV-2, SARS-CoV-1, or CoV-NL63, wherein the variant binds ACE2 (e.g., human ACE2).
  • the subject is human, and the ACE2 is human ACE2.
  • the subject is asymptomatic.
  • the subject is showing at least one symptom suggesting a coronavirus infection.
  • a method of treating COVID-19 in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein.
  • the subject is human.
  • the subject is asymptomatic.
  • the subject is showing at least one symptom suggesting COVID-19 infection.
  • a therapeutically effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein can be used to treat post-acute sequelae of SARS-CoV-2 infection (also known as post-acute sequelae of COVID-19 (PASC), chronic COVID syndrome (CCS) and long-haul COVID) in a subject (e.g., human) in need thereof.
  • PASC post-acute sequelae of COVID-19
  • CCS chronic COVID syndrome
  • long-haul COVID long-haul COVID
  • a method of treating SARS in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein.
  • the subject is human.
  • the subject is asymptomatic.
  • the subject is showing at least one symptom suggesting SARS infection.
  • a method of preventing a coronavirus infection in a subject comprises administering to the subject an effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein, wherein the coronavirus binds ACE2.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, and/or CoV-NL63.
  • the coronavirus is a variant of SARS-CoV-2, SARS-CoV-1, or CoV-NL63, wherein the variant binds ACE2 (e.g., human ACE2).
  • a method of preventing COVID-19 in a subject comprises administering to the subject an effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein.
  • a method of preventing SARS in a subject comprises administering to the subject an effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein.
  • the subject is a human.
  • the subject has a risk of a coronavirus infection.
  • the subject is suspected to have been infected with a coronavirus.
  • the present disclosure provides methods of inhibiting or reducing the viral replication of a coronavirus in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein, wherein the coronavirus binds ACE2.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, and/or CoV-NL63.
  • the coronavirus is a variant of SARS-CoV-2, SARS-CoV-1, or CoV- NL63, wherein the variant binds ACE2 (e.g., human ACE2).
  • a method of inhibiting or reducing the viral replication of SARS-CoV-2 in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein.
  • a method of inhibiting or reducing the viral replication of SARS-CoV-1 in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein.
  • the subject is a human.
  • viral replication is inhibited by at least 50%.
  • viral replication is inhibited by at least 60%, at least 70%, at least 80%, or at least 90%.
  • the present disclosure provides methods of inhibiting or reducing the viral replication of a coronavirus in a cell.
  • the methods comprise contacting the cell with an effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein, wherein the coronavirus binds ACE2.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, and/or CoV- NL63.
  • viral replication is inhibited by at least 50%.
  • the cell is a human cell and the ACE2 is human ACE2.
  • viral replication is inhibited by at least 60%, at least 70%, at least 80%, or at least 90%.
  • the present disclosure provides methods of preventing a coronavirus infection in a subject.
  • the methods comprise identifying a subject at risk for being exposed to a coronavirus, and administering an effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein, wherein the coronavirus binds ACE2.
  • the methods comprise identifying a subject suspected of having been exposed to a coronavirus, and administering an effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein, wherein the coronavirus binds ACE2.
  • the present disclosure provides methods of treating infection by a coronavirus in a subject.
  • the methods comprise identifying a subject as testing positive for a coronavirus infection and being asymptomatic for a coronavirus infection; and administering an effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein, wherein the coronavirus binds ACE2.
  • the methods comprise identifying a subject as being infected with a coronavirus and expressing at least one symptom of the infection; and administering an effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein, wherein the coronavirus binds ACE2.
  • the polypeptide, the coronavirus trap, or the multimeric binding agent is administered to the subject prophylactically.
  • the polypeptide, the coronavirus trap, or the multimeric binding agent is administered to the subject therapeutically.
  • the polypeptide, the coronavirus trap, or the multimeric binding agent is administered as part of a combination therapy.
  • the combination therapy includes the use of a medical device.
  • the medical device is a ventilator or an ECMO machine.
  • the combination therapy comprises at least one additional therapeutic agent.
  • the at least one additional therapeutic agent is an antiviral agent.
  • the coronavirus infection includes development of respiratory illnesses and diseases.
  • the methods relate to treating, preventing, or inhibiting the development of respiratory illnesses and diseases.
  • the respiratory illness is associated with a suspected or confirmed coronavirus infection.
  • the suspected or confirmed viral infection is, for example, a SARS-CoV-2, SARS-CoV- 1, and/or CoV-NL63 infection.
  • the suspected or confirmed viral infection is, for example, infection with a variant of SARS-CoV-2, SARS-CoV-1, or CoV-NL63, wherein the variant binds ACE2 (e.g., human ACE2).
  • a method of treating a respiratory illness in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein.
  • a method of preventing or inhibiting the development of a respiratory illness in a human subject comprises administering to the subject a therapeutically effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein.
  • the respiratory illness is selected from the group consisting of: pneumonia, acute respiratory disease (ARD), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), atypical ARDS, respiratory distress syndrome (RDS), severe acute respiratory syndrome (SARS), and coronavirus 2019 (COVID-19).
  • ARD acute respiratory disease
  • ALI acute lung injury
  • ARDS acute respiratory distress syndrome
  • RDS respiratory distress syndrome
  • SARS severe acute respiratory syndrome
  • COVID-19 coronavirus 2019
  • the subject who has been diagnosed with a coronavirus infection or suspected of having a coronavirus infection has lung damage, respiratory failure, kidney damage, kidney failure, liver damage, heart damage, vascular damage, thrombosis, stroke, central nervous system injury, and/or multiple organ failure.
  • the subject has one or more symptoms selected from the group consisting of: hypoxemia, cough, wheezing, dyspnea, hyperpnea, pulmonary/lung inflammation, shortness of breath, labored breathing, rapid breathing, accumulation of alveolar fluid, pulmonary edema, vascular leakage, lymphocyte infiltration in the lung, lymphopenia, fever, chills, shaking chills, increased heart rate, chest pain, low blood pressure, headache, confusion, seizures, extreme tiredness, sepsis, bluish coloring of nails or lips, toe rashes/redness, toe swelling, loss of sense of smell, loss of sense of taste, and diarrhea.
  • symptoms selected from the group consisting of: hypoxemia, cough, wheezing, dyspnea, hyperpnea, pulmonary/lung inflammation, shortness of breath, labored breathing, rapid breathing, accumulation of alveolar fluid, pulmonary edema, vascular leakage, lymphocyte infiltration in the lung, lymphopenia, fever, chills, shaking chills
  • the subject has mild, moderate or severe hypoxemia as determined by Partial Pressure of arterial oxygen/Fraction of inspired oxygen (PaCh/FiCh) or positive end-expiratory pressure (PEEP). In some embodiments of the methods described herein, the subject has severe hypoxemia with a PaCh/FiCh of less than 100.
  • the disclosure provides a method of treating post-acute sequelae of a SARS-CoV-2 infection in a human subject, the method comprising administering to the human subject a therapeutically effective amount of a coronavirusbinding agent (e.g., polypeptide or multimer) described herein, or a pharmaceutical composition thereof.
  • a coronavirusbinding agent e.g., polypeptide or multimer
  • the coronavirus-binding agent comprises a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17.
  • the coronavirus-binding agent comprises a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:21.
  • the coronavirus-binding agent comprises a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:25. In some instances, the coronavirus-binding agent comprises a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:29.
  • the subject is human.
  • compositions comprising a coronavirus-binding agent described herein.
  • the disclosure provides compositions comprising a polypeptide described herein.
  • the disclosure provides compositions comprising a coronavirus trap described herein.
  • the disclosure provides compositions comprising a multimeric binding agent described herein.
  • the disclosure provides pharmaceutical compositions comprising a coronavirus-binding agent described herein and a pharmaceutically acceptable carrier.
  • the disclosure provides pharmaceutical compositions comprising a polypeptide described herein and a pharmaceutically acceptable carrier.
  • the disclosure provides pharmaceutical compositions comprising a coronavirus trap described herein and a pharmaceutically acceptable carrier.
  • the disclosure provides pharmaceutical compositions comprising a multimeric binding agent described herein and a pharmaceutically acceptable carrier.
  • the coronavirus-binding agent is isolated. In some embodiments, the coronavirus-binding agent is substantially pure.
  • the disclosure provides a polynucleotide composition comprising one or more polynucleotides that encode a coronavirus-binding agent described herein. In some embodiments, a polynucleotide composition comprises one or more polynucleotides that encode a polypeptide described herein. In some embodiments, a polynucleotide composition comprises one or more polynucleotides that encode a coronavirus trap described herein.
  • a polynucleotide composition comprises one or more polynucleotides that encode a multimeric binding agent described herein. In some embodiments, the one or more polynucleotides are isolated. In some embodiments, a vector or vectors comprising the one or more polynucleotides that encode a coronavirus-binding agent described herein are provided. In some embodiments, a cell comprising the one or more polynucleotides that encode a coronavirus-binding agent described herein is provided. In some embodiments, a cell comprises the one or more vectors comprising the one or more polynucleotides that encode a coronavirus-binding agent described herein is provided.
  • the disclosure provides a cell comprising a coronavirus-binding agent described herein. In some embodiments, the disclosure provides a cell producing a coronavirus-binding agent described herein. In some embodiments, the cell produces a polypeptide described herein. In some embodiments, the cell produces a coronavirus trap described herein. In some embodiments, the cell produces a multimeric binding agent described herein. In some embodiments, the cell is a monoclonal cell line.
  • the disclosure provides a method of making a coronavirusbinding agent (e.g., a polypeptide or multimer) described herein.
  • the method comprises (a) culturing a cell (i) comprising one or more polynucleotides encoding the coronavirus-binding agent (e.g., a polypeptide or multimer), (ii) comprising one or more vectors comprising one or more polynucleotides encoding the coronavirus-binding agent (e.g., a polypeptide or multimer), or (iii) producing the coronavirus-binding agent (e.g., a polypeptide or multimer), under suitable conditions to express the coronavirus-binding agent, and (b) isolating the coronavirus-binding agent.
  • the coronavirus-binding agent e.g., a polypeptide or multimer
  • the method further comprises formulating the isolated coronavirusbinding agent as a sterile pharmaceutical composition.
  • the coronavirus-binding agent comprises a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17.
  • the coronavirusbinding agent comprises a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:21.
  • the coronavirus-binding agent comprises a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:25.
  • the coronavirus-binding agent comprises a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:29.
  • Figure 1 Binding of ACE2-IgM Fc polypeptides to SARS-CoV-2 Spike RBD protein in an ELISA format. Plates were coated with SARS-CoV-2 Spike RBD protein. Serial dilutions of ACE2-IgM Fc proteins were added to the plates. In some samples, the ACE2-IgM Fc proteins were mixed with a soluble ACE2-His protein.
  • Samples were: ACE2(740)-IgM Fc(102) (-•-); ACE2(615)-IgM Fc(102) (- ⁇ -); ACE2(740)-IgM Fc(102) + ACE2-His (-o-); ACE2(615)-IgM Fc(102) + ACE2-His (- ⁇ -); and hlgM antibody (-A-).
  • Figure 2 Binding of ACE2-IgM Fc polypeptides to SARS-CoV-2 Spike SI protein in an ELISA format. Plates were coated with ACE2(740)-IgM Fc(102) (-•-), ACE2(615)-IgM Fc(102) (- ⁇ -), or hlgM antibody (-A-). Serial dilutions of SARS- CoV-2 Spike SI protein were added to the plates.
  • Figure 3 Binding of ACE2-IgM Fc polypeptides to SARS-CoV-2 Spike SI protein in an ELISA format. Plates were coated with SARS-CoV-2 Spike SI -His protein. Serial dilutions of ACE2-IgM Fc proteins were added to the plates. In some samples, the ACE2-IgM Fc proteins were mixed with a soluble ACE2-His protein.
  • Samples were: ACE2(740)-IgM Fc(102) Prep #1 (-•-); ACE2(740)-IgM Fc(102) Prep #2 (-o-); ACE2(615)-IgM Fc(102) Prep #1 (- ⁇ -); ACE2(615)-IgM Fc(102) Prep #2 (- ⁇ - ); ACE2(740)-IgM Fc(102) Prep #2 + ACE2-His (-A-); ACE2(615)-IgM Fc(102) Prep #2 + ACE2-His (- A-); and hlgM antibody (- ⁇ -).
  • ACE2-IgGl Fc polypeptide to HCoV-NL63 Spike protein in an ELISA format.
  • AntiSpike IgM and anti-Spike IgGl are raised against the Spike protein of SARS-CoV-2.
  • FIG. 5 Binding of ACE2(740)-IgM Fc to various SARS-CoV-2 Spike RBD mutants and a Spike protein mutant (D614G) assayed in an ELISA format. EC50s of the binding are shown.
  • FIG. 6 Binding of ACE2-IgM Fc polypeptides to SARS-CoV-2 Spike protein.
  • COS7 cells were transiently transfected with a construct expressing a full- length SARS-CoV-2 Spike protein.
  • Serial dilutions of ACE2-IgM Fc proteins were added to the cells.
  • Samples were: ACE2(615)-IgM Fc(106) (-A-); ACE2(740)-IgM Fc(106) (- ⁇ -); ACE2(615)-IgGl Fc (- ⁇ -); ACE2(740)-IgGl Fc (- ⁇ -); hlgG antibody (- •-); and hlgM antibody (-o-).
  • FIG. 7 Binding of an ACE2-IgM Fc polypeptide to cells expressing polymeric Ig receptor (plgR).
  • COS7 cells were transfected with human plgR, mouse plgR, SARS-CoV-2 Spike protein, B7-H4 or ITGA5/B1.
  • Cells were incubated with ACE2(615)-IgGl Fc, ACE2(615)-IgM Fc(106), a positive control human IgM and a negative control anti-KLH-IgGl . Binding was quantified by fluorescent secondary antibodies recognizing IgGl or IgM.
  • Figure 8 Live cell imaging assaying the fusion of ACE2-expressing cells and Spike-expressing cells in the presence ACE2-IgM Fc polypeptide or ACE2-IgGl Fc polypeptide.
  • Figure 9 Inhibition of SARS-CoV-2 viral infection of Vero E6 cells expressing ACE2 by ACE2-IgM Fc or ACE2-IgGl Fc polypeptide.
  • Figure 10 Expression of ACE2-IgM Fc polypeptides containing human ACE2 extracellular region variants analyzed by SDS-PAGE and Coomassie stain. DETAILED DESCRIPTION
  • the present invention provides novel agents, including, but not limited to, polypeptides, soluble proteins, fusion proteins, homodimeric molecules, and multimeric molecules that bind to coronaviruses, particularly coronaviruses that bind to ACE2.
  • the agents comprise polypeptides comprising a first domain comprising an extracellular region of ACE2 and an IgM heavy chain constant region (also referred to herein as “IgM Fc region”).
  • IgM Fc region an IgM heavy chain constant region
  • Related multimers of the polypeptides, polynucleotides, vectors, cells, and methods of making the agents are provided.
  • Compositions comprising the agents and methods of inhibiting or reducing viral infections and treating viral diseases are also provided.
  • binding agent or “coronavirus binding agent” as used herein refers to a molecule that binds a specific antigen or target on a coronavirus, such as the Spike protein or a subdomain of the Spike protein.
  • a binding agent comprises a polypeptide.
  • a binding agent is a multimeric molecule comprising two or more polypeptides.
  • soluble protein refers to a protein or a fragment thereof that can be secreted from a cell in soluble form.
  • fusion protein refers to a hybrid protein comprising at least two domains that are encoded by a single polynucleotide. In some embodiments, the two or more of the domains are connected by one or more linkers. “Fusion protein” is used interchangeably herein with “polypeptide.”
  • linker refers to a linker inserted between a first domain (e.g., an ACE2 extracellular region) and a second domain (e.g., an IgM heavy chain constant region).
  • the linker is a peptide linker.
  • Linkers should not adversely affect the expression, secretion, or bioactivity of the domains. Preferably, linkers are not antigenic and do not elicit an immune response.
  • polypeptide”, “peptide”, and “protein” refer to polymers of amino acids of any length. The polypeptide may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art.
  • nucleic acid and nucleic acid molecule are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. A gap is considered as an unidentical nucleic acid or amino acid.
  • the percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof.
  • amino acid substitution refers to a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
  • vector means a construct, which is capable of delivering, and/or expressing, one or more gene(s) or nucleic acid sequence(s) of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.
  • isolated refers to a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature.
  • isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions are those which have been purified to a degree that they are no longer in a form in which they are found in nature.
  • a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
  • a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition may be isolated from a natural source (e.g., tissue) or from a non-natural source such as an engineered cell line.
  • substantially pure refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
  • subject refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rabbits, horses, poultry, cattle, rodents, and the like.
  • pharmaceutically acceptable refers to a substance approved or approvable by a regulatory agency or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, including humans.
  • pharmaceutically acceptable excipient, carrier, or adjuvant refers to an excipient, carrier, or adjuvant that can be administered to a subject, together with at least one therapeutic agent and which is generally safe, non-toxic, and has no effect on the pharmacological activity of the therapeutic agent.
  • pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation.
  • pharmaceutical formulation or “pharmaceutical composition” as used herein refers to a preparation which is in such form as to permit the biological activity of the agent to be effective.
  • a pharmaceutical formulation or composition generally comprises additional components, such as a pharmaceutically acceptable excipient, carrier, adjuvant, buffers, etc.
  • terapéuticaally effective amount refers to the amount of an agent which is sufficient to reduce and/or ameliorate the severity and/or duration of (i) a disease, disorder, or condition in a subject, and/or (ii) a symptom in a subject.
  • the term also encompasses an amount of an agent necessary for the (i) reduction or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction or amelioration of the recurrence, development, or onset of a given disease, disorder, or condition, and/or (iii) the improvement or enhancement of the prophylactic or therapeutic effect(s) of another agent or therapy (e.g., an agent other than the binding agents provided herein).
  • therapeutic effect refers to the effect and/or ability of an agent to reduce and/or ameliorate the severity and/or duration of (i) a disease, disorder, or condition in a subject, and/or (ii) a symptom in a subject.
  • the term also encompasses the ability of an agent to (i) reduce or ameliorate the advancement or progression of a given disease, disorder, or condition, (ii) reduce or ameliorate the recurrence, development, or onset of a given disease, disorder, or condition, and/or (iii) to improve or enhance the prophylactic or therapeutic effect(s) of another agent or therapy (e.g., an agent other than the binding agents provided herein).
  • treat or “treatment” or “treating” or “to treat” or “alleviate” or “alleviation” or “alleviating” or “to alleviate” as used herein refers to therapeutic measures that aim to cure, slow down, lessen symptoms of, and/or halt progression of a pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder.
  • prevent refers to the partial or total inhibition of the development, recurrence, onset, or spread of a disease, disorder, or condition, or a symptom thereof in a subject.
  • reference to “about” or “approximately” a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, a description referring to “about X” includes description of “X”.
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the present invention provides agents that bind members of the coronavirus family.
  • Coronaviruses are enveloped RNA viruses that causes enteric, respiratory, and central nervous systems disease in a variety of animals and in humans.
  • the family includes CoV-NL63, CoV 229E, CoV OC43, SARS-CoV-1, MERS-CoV, and SARS- CoV-2.
  • the coronavirus surface protein Spike (S) protein mediates entry into target cells by binding to a cellular receptor and subsequently fusing the viral envelope with a host cell membrane.
  • the receptor binding domain (RBD) of the S protein is located within the SI subunit, while the S2 subunit harbors the functional elements required for membrane fusion (see, e.g., Heurich et al., 2014, J. Virol., 88: 1293-1307).
  • ACE2 has been identified as the functional receptor for SARS-CoV-1, SARS-CoV-2, and CoV- NL63.
  • Soluble forms of ACE2 are known (e.g., U.S. Patent No. 8,586,319), but it is proposed that a multimeric form of ACE2 would be more effective in capturing coronavirus particles.
  • the coronavirus-binding agent is a polypeptide.
  • the polypeptide is a soluble protein.
  • the polypeptide comprises at least two domains.
  • the first domain comprises an extracellular region of ACE2, wherein the extracellular region of ACE2 binds to a coronavirus Spike protein.
  • the second domain comprises an IgM heavy chain constant region.
  • the C-terminus of the first domain is connected to the N-terminus of the second domain.
  • the first domain and second domain are connected by a linker.
  • the polypeptide comprises from the N- terminus to the C-terminus: the first domain-linker-second domain.
  • the first domain of the polypeptides comprises an extracellular region of ACE2, wherein the extracellular region of ACE2 binds to a coronavirus Spike protein.
  • the first domain comprises the full-length extracellular domain of ACE2.
  • the first domain comprises a fragment of the full- length extracellular domain of ACE2, wherein the fragment binds to a coronavirus Spike protein.
  • the first domain comprises a variant of the full- length extracellular domain of ACE2 or a variant of a fragment of the full-length extracellular domain of ACE2, wherein the variant binds to a coronavirus Spike protein.
  • the first domain comprises an extracellular region of human ACE2.
  • the first domain comprises the full-length extracellular domain of human ACE2. In some embodiments, the first domain comprises a fragment of the full-length extracellular domain of human ACE2, wherein the fragment binds to a coronavirus Spike protein. In some embodiments, the first domain comprises a variant of the full-length extracellular domain of human ACE2, or a variant of a fragment of the extracellular domain of human ACE2, wherein the variant binds to a coronavirus Spike protein.
  • the second domain of the polypeptides comprises an IgM heavy chain constant region.
  • the “IgM heavy chain constant region” referred to herein encompasses any one of the full length, a fragment of the full length, a variant of the full length, and a variant of a fragment of the full-length IgM heavy chain constant regions.
  • the variant or fragment of the IgM heavy chain constant region is able to multimerize as a pentamer or a hexamer.
  • the coronavirus-binding agent comprises a homodimer of the polypeptide described herein. In some embodiments, the agent comprises a multimeric form of the polypeptides. In some embodiments, the agent comprises a multimeric form (e.g., a hexamer or a pentamer) of the homodimer of the polypeptides.
  • the coronavirus-binding agent functions as a virus trap or viral trap. In some embodiments, the agent functions as a virus decoy or viral decoy. To those of skill in the art, a virus trap or a virus decoy acts to intercept a virus and lures the virus away from cells expressing the virus receptor.
  • a virus trap or virus decoy binds to a free virus and sequesters it, inhibiting the virus’s ability to bind to a cell expressing the virus receptor.
  • the binding agent functions as a coronavirus trap. In some embodiments, the binding agent functions as a coronavirus decoy.
  • ACE2 The amino acid (aa) sequence for human ACE2 (UniProtKB No. Q9BYF1) is provided herein as SEQ ID NO: 1. As used herein, reference to amino acid positions of ACE2 refer to the numbering of amino acid sequences including the signal sequence.
  • ACE2 is a single pass type I transmembrane protein with a predicted molecular weight of approximately 92.5 kDa. ACE2 has been observed to be predominantly expressed on endothelial cells. The tissue distribution of ACE2 includes, but may not be limited to, lung, intestine, liver, kidney, heart, and testis.
  • ACE2 is characterized by an extracellular domain comprising a peptidase catalytic domain, a transmembrane domain, and a cytoplasmic domain (see, e.g., Tipnis et al., 2000, JBC, 275:33238-33243).
  • human ACE2 is a protein of 805 amino acids (aa) - the signal sequence is aa 1-17, the extracellular domain is aa 18-740, the transmembrane region is aa 741-761, and the cytoplasmic domain is aa 762-805.
  • the peptidase catalytic domain is generally characterized as aa 147-555.
  • the first domain of the polypeptide comprises an extracellular region of ACE2, wherein the extracellular region of ACE2 binds to a coronavirus Spike protein.
  • the first domain of the polypeptide comprises an extracellular region of human ACE2, wherein the extracellular region of human ACE2 binds to a coronavirus Spike protein.
  • the first domain of the polypeptide comprises the full-length extracellular domain of ACE2. As described herein, amino acids 18-740 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 3 corresponds to the full-length of the human ACE2 extracellular domain.
  • N-terminus and/or the C-terminus of the extracellular domain described herein may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, so long as the extracellular domain retains the ability to bind to a coronavirus Spike protein.
  • the first domain of the polypeptide comprises a fragment of the full-length extracellular domain of ACE2 (e.g., human ACE2), wherein the fragment binds a coronavirus Spike protein.
  • the fragment comprises an amino acid sequence with at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:3.
  • the fragment comprises amino acids 18-615 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO:4.
  • the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4, wherein the polypeptide binds a coronavirus Spike protein.
  • the fragment comprises an amino acid sequence with at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO:4.
  • the first domain of the polypeptide comprises a variant of the full-length extracellular domain of ACE2 (e.g., human ACE2), wherein the variant binds a coronavirus Spike protein.
  • the variant has an amino acid sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:3.
  • the first domain of the polypeptide comprises a variant of a fragment of the full-length extracellular domain of ACE2 (e.g., human ACE2).
  • the variant of a fragment of the full-length extracellular domain of ACE2 has an amino acid sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:4.
  • the variant of the full-length or a fragment of the extracellular domain of ACE2 contains one or more mutations affecting glycosylation, for example one or more (e.g., 1, 2, 3, 4, 5, 6) of mutations at N53, N90, N103, N322, N432 and N546 of human ACE2.
  • the variant of the full-length or the variant of a fragment of the extracellular domain of human ACE2 contain contains one or more (e.g., 1, 2, 3, 4, 5, 6) of the following mutations including N53S, T92A, N103S, T324A, N432S and N546S.
  • the variant comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative substitutions.
  • the fragment of the full-length extracellular domain of ACE2, the variant of the full length extracellular domain of ACE2, or the variant of the fragment of the full-length extracellular domain of ACE2 described herein retain at least 50% (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100%) binding capacity to the Spike protein of a coronavirus in comparison with the full-length extracellular domain of ACE2.
  • the fragment or the variant display increased binding capacity (e.g., more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increased binding capacity) to the Spike protein of a coronavirus in comparison with the full-length extracellular domain of ACE2.
  • the fragment or the variant displays a KD for the Spike protein of a coronavirus of at least about 10' 6 M (e.g., lOOOnM, 500nM, 250nM, lOOnM, 50nM, 25nM, lOnM, 5nM, 2.5nM, InM, 0.5nM, 0.25nM or lower).
  • lOOOnM 500nM, 250nM, lOOnM, 50nM, 25nM, lOnM, 5nM, 2.5nM, InM, 0.5nM, 0.25nM or lower.
  • the fragment of the full-length extracellular domain of ACE2, the variant of the full-length extracellular domain of ACE2, or the variant of the fragment of the full-length extracellular domain of ACE2 described herein retains binding to the Spike protein of a coronavirus (e.g., SARS-CoV- 2 SI subunit of the Spike protein) if the variant or fragment binds to the Spike protein of a coronavirus (e.g., SARS-CoV-2 SI subunit of the Spike protein) at a level of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% of the binding to the Spike protein of the coronavirus (e.g., SARS-CoV-2 SI subunit of the Spike protein) by a polypeptide of amino acids 18-740 of SEQ ID NO: 1.
  • a coronavirus e.g., SARS-CoV- 2 SI subunit of the Spike protein
  • the fragment or variant retains binding to a spike protein of a coronavirus (e.g., SARS-CoV-2 SI subunit of the Spike protein) if the fragment or variant binds to the Spike protein of a coronavirus (e.g., SARS-CoV-2 SI subunit of the Spike protein) with a KD of at least about 10' 6 M (e.g., lOOOnM, 500nM, 250nM, lOOnM, 50nM, 25nM, lOnM, 5nM, 2.5nM, InM, 0.5nM, 0.25nM or lower).
  • binding is assessed by Biacore as described in Example 3.
  • the coronavirus is a SARS-CoV-1, SARS-CoV-2, or CoV-NL63.
  • the ACE2 is a human ACE2.
  • the first domain of a polypeptide described herein retains binding to the Spike protein of a coronavirus (e.g., SARS-CoV-2 SI subunit of the Spike protein). In some instances, the first domain retains binding to the Spike protein of a coronavirus (e.g., SARS-CoV-2 SI subunit of the Spike protein) if a polypeptide comprising the first domain binds to the Spike protein of a coronavirus (e.g., SARS- CoV-2 SI subunit of the Spike protein) at a level of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% of the binding to the Spike protein of the coronavirus (e.g., SARS-CoV-2 SI subunit of the Spike protein) by a polypeptide of amino acids 18-740 of SEQ ID NO: 1.
  • the first domain retains binding to the Spike protein of a coronavirus (e.g., SARS-CoV-2 SI subunit of the Spike protein) if a polypeptide comprising the first domain binds to the Spike protein of a coronavirus (e.g., SARS-CoV-2 Spike SI) with a KD of at least about 10' 6 M (e.g., lOOOnM, 500nM, 250nM, lOOnM, 50nM, 25nM, lOnM, 5nM, 2.5nM, InM, 0.5nM, 0.25nM or lower).
  • binding is assessed by Biacore as described in Example 3.
  • the second domain of the polypeptide comprises a IgM heavy chain constant region.
  • the IgM heavy chain constant region is a human IgM heavy chain constant region.
  • the “IgM heavy chain constant region” referred to herein encompasses any one of the full length, a fragment of the full length, a variant of the full length, and a variant of a fragment of the full-length IgM heavy chain constant regions.
  • the variant or fragment of an IgM heavy chain constant region is able to multimerize as a pentamer or a hexamer.
  • those of skill in the art may differ in their understanding of the exact amino acids corresponding to the full length heavy chain constant region of IgM.
  • the N- terminus and/or the C-terminus of the heavy chain constant region described herein may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.
  • human IgM heavy chain constant domains are defined as: CHI aa 1-105, CH2 aa 106-217, CH3 aa 218-323, and CH4 aa 324-452 of SEQ ID NO:5.
  • Those of skill in the art generally defined the full-length IgM heavy chain constant region (also referred to herein as “IgM Fc region”) as CH2-CH4.
  • the second domain of the polypeptide comprises a wild-type full-length IgM heavy chain constant region.
  • the second domain of the polypeptide comprises a natural variant of the IgM heavy chain constant region (e.g., human IgM Fc with G191S, SEQ ID NO:6).
  • the second domain of the polypeptide comprises a IgM heavy chain constant region containing mutations (such as mutations that abolish, reduce or increase effector functions, such as P311 A or P313 A mutation in human IgM heavy chain constant region).
  • the human IgM heavy chain constant region is modified (e.g., truncated or elongated) at the N-terminal end by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • the human IgM heavy chain constant region is truncated at the C-terminal end (e.g., the tailpiece is absent). Those of skill in the art generally define the tailpiece as the last 18 amino acids of the C-terminal end of IgM.
  • the IgM heavy chain constant region comprises SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NOTO.
  • the IgM heavy chain constant region comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 11-14 and SEQ ID NOs:45-52.
  • IgMs are polymeric antibodies that generally provide a first line defense against invading microorganisms and other pathogens.
  • IgM monomers are arranged into hexamers in the absence of an immunoglobulin J chain or into pentamers in the presence of a J chain.
  • the CH4 domain of IgM heavy chains contains a C- terminal extension, referred to as the mu tailpiece, which is essential for polymerization.
  • the polymeric structure of IgM antibodies increases the avidity for the binding target (see, e.g., Pasalic et al., 2017, PNAS, 114:E8575-E8584).
  • the characteristics of the IgM heavy chain are important to the ACE2-IgM Fc polypeptides described herein.
  • the IgM Fc region allows for formation of a multimeric binding protein comprising the polypeptides described herein.
  • the IgM Fc region allows for formation of a pentameric complex comprising the polypeptides described herein. In some embodiments, the IgM Fc region allows for formation of a hexameric complex comprising the polypeptides described herein. In some embodiments, the IgM Fc region is a fragment or variant of the Fc region, wherein the IgM Fc region fragment or variant retains the ability to polymerize (i.e., form multimeric complexes). In some embodiments, the IgM Fc region is a fragment or variant of the Fc region, wherein the IgM Fc region fragment or variant comprises the tailpiece.
  • the polypeptides comprise a first domain comprising an extracellular region of ACE2 and a second domain comprising a IgM heavy chain constant region, wherein the C-terminal end of the IgM heavy chain constant region is linked to the N-terminal end of the extracellular region of ACE2.
  • the polypeptide comprises a first domain comprising an extracellular region of ACE2 and a second domain comprising a IgM heavy chain constant region, wherein the N-terminal end of the IgM heavy chain constant region is linked to the C- terminal end of the extracellular region of ACE2.
  • the extracellular region of ACE2 is directly linked to the IgM heavy chain constant region (i.e., without an intervening peptide linker). In some embodiments, the extracellular region of ACE2 is linked to the IgM heavy chain constant region via a peptide linker.
  • linker refers to a linker inserted between a first domain (e.g., an extracellular region of ACE2) and a second domain (e.g., an IgM heavy chain constant region). In some embodiments, the linker is a peptide linker. Linkers should not adversely affect the expression, secretion, or bioactivity of the polypeptide.
  • Linkers should not be antigenic and should not elicit an immune response. Suitable linkers are known to those of skill in the art and often include mixtures of glycine and serine residues and often include amino acids that are sterically unhindered. Other amino acids that can be incorporated into useful linkers include threonine and alanine residues. Linkers can range in length, for example, from 1-50 amino acids in length, 1-22 amino acids in length, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3 amino acids in length.
  • Linkers may include, but are not limited to, SerGly, GGSG (SEQ ID NO:53), GSGS (SEQ ID NO:54), GGGS (SEQ ID NO:55), (GGGS)n wherein n to 2-7 (SEQ ID NO:56), S(GGS)n where n is 1-7 (SEQ ID NO:57), (GGGGS)s (SEQ ID NO: 37), GRA, poly(Gly), poly(Ala), GGGSGGG (SEQ ID NO: 38), ESGGGGVT (SEQ ID NO: 39), LESGGGGVT (SEQ ID NO:40), GRAQVT (SEQ ID NO:41), WRAQVT (SEQ ID NO:42), and ARGRAQVT (SEQ ID NO:43).
  • the linker may comprise a cleavage site. In some embodiments, the linker may comprise an enzyme cleavage site, so that the second polypeptide may be separated from the first polypeptide.
  • a linker is an intervening molecule (e.g., peptide) that does not include amino acid residues from either the C-terminus of the first domain (e.g., an extracellular region of ACE2) or the N-terminus of the second domain (e.g., an IgM heavy chain constant region).
  • the second domain comprises an IgM heavy chain constant region.
  • the IgM heavy chain constant region comprises an IgM CH4 domain comprising the tail piece domain.
  • the IgM heavy chain constant region comprises a CH3 domain and a CH4 domain comprising the tail piece domain.
  • the IgM heavy chain constant region comprises a CH2 domain, a CH3 domain, and a CH4 domain comprising the tail piece domain.
  • the IgM heavy chain constant region comprises a partial CHI domain (e.g., PLPV amino acid sequence), CH2 domain, a CH3 domain, and a CH4 domain comprising the tail piece domain.
  • the second domain of the polypeptide comprises amino acids 106-453 of SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the second domain of the polypeptide comprises amino acids 102- 453 of SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the second domain of the polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the second domain of the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:7-14 and SEQ ID NOs:45-52. In some embodiments, the second domain of the polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NOs:7-14 or SEQ ID NOs:45-52.
  • the first domain of the polypeptide comprises the amino acid sequence of SEQ ID NO: 3 and the second domain of the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:7-14 and SEQ ID NOs:45-52.
  • the first domain of the polypeptide comprises the amino acid sequence of SEQ ID NO:3 and the second domain of the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10.
  • the first domain of the polypeptide comprises the amino acid sequence of SEQ ID NO:3 and the second domain of the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 11-14 and SEQ ID NOs:45-52.
  • the first domain of the polypeptide comprises the amino acid sequence of SEQ ID NON and the second domain of the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:7-14 and SEQ ID NOs:45-52.
  • the first domain of the polypeptide comprises the amino acid sequence of SEQ ID NON and the second domain of the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10.
  • the first domain of the polypeptide comprises the amino acid sequence of SEQ ID NON and the second domain of the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 11-14 and SEQ ID NOs:45-52.
  • the first domain of the polypeptide comprises an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:3, wherein the first domain binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein), and the second domain of the polypeptide comprises an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:7-14 and SEQ ID NOs:45-52, wherein the second domain of the polypeptide is able to multimerize as
  • the first domain of the polypeptide comprises an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:4, wherein the first domain binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein), and the second domain of the polypeptide comprises an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:7-14 and SEQ ID NOs:45-52, wherein the second domain of the polypeptide is able to multimerize as a penta
  • the polypeptide comprises an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 17, wherein the polypeptide binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein).
  • a coronavirus Spike protein e.g., SARS-CoV-2 Spike SI protein
  • the polypeptide comprises an amino acid sequence with at least 90 % (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:21, wherein the polypeptide binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein).
  • a coronavirus Spike protein e.g., SARS-CoV-2 Spike SI protein
  • the polypeptide comprises an amino acid sequence with at least 90 % (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:25, wherein the polypeptide binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein).
  • a coronavirus Spike protein e.g., SARS-CoV-2 Spike SI protein
  • the polypeptide comprises an amino acid sequence with at least 90 % (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:29, wherein the polypeptide binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein).
  • a coronavirus Spike protein e.g., SARS-CoV-2 Spike SI protein.
  • the polypeptide is able to multermize as a pentamer or a hexamer.
  • the polypeptide comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:21. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:25. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:29.
  • the polypeptide consists of the amino acid sequence of SEQ ID NO: 17. In some embodiments, the polypeptide consists of the amino acid sequence of SEQ ID NO:21. In some embodiments, the polypeptide consists of the amino acid sequence of SEQ ID NO:25. In some embodiments, the polypeptide consists of the amino acid sequence of SEQ ID NO:29.
  • the polypeptide comprises an amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 18, wherein the polypeptide binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein).
  • a coronavirus Spike protein e.g., SARS-CoV-2 Spike SI protein
  • the polypeptide comprises an amino acid sequence with at least 90 % (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:22, wherein the polypeptide binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein).
  • a coronavirus Spike protein e.g., SARS-CoV-2 Spike SI protein
  • the polypeptide comprises an amino acid sequence with at least 90 % (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:26, wherein the polypeptide binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein).
  • a coronavirus Spike protein e.g., SARS-CoV-2 Spike SI protein
  • the polypeptide comprises an amino acid sequence with at least 90 % (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:30, wherein the polypeptide binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein).
  • a coronavirus Spike protein e.g., SARS-CoV-2 Spike SI protein.
  • the polypeptide is able to multermize as a pentamer or a hexamer.
  • the polypeptide comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:22. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:26. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:30.
  • the polypeptide consists of the amino acid sequence of SEQ ID NO: 18. In some embodiments, the polypeptide consists of the amino acid sequence of SEQ ID NO:22. In some embodiments, the polypeptide consists of the amino acid sequence of SEQ ID NO:26. In some embodiments, the polypeptide consists of the amino acid sequence of SEQ ID NO:30.
  • the present disclosure provides a coronavirus trap.
  • the coronavirus trap comprises two or more of the ACE2-IgM Fc polypeptides described herein.
  • the first domain comprising an ACE2 extracellular region of the polypeptide binds free coronavirus particles.
  • the polypeptides are able to inhibit and/or block the coronavirus particles from infecting cells via the native ACE2 receptor.
  • a coronavirus trap comprises a multimer of two or more of the polypeptides described herein. In some embodiments, the polypeptides are identical in the multimer.
  • a coronavirus trap comprises a multimer of two or more of a polypeptide comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, a coronavirus trap comprises a multimer of two or more of a polypeptide comprising the amino acid sequence of SEQ ID NO:21. In some embodiments, a coronavirus trap comprises a multimer of two or more of a polypeptide comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, a coronavirus trap comprises a multimer of two or more of a polypeptide comprising the amino acid sequence of SEQ ID NO:29. In some embodiments, two of the polypeptides associate as a homodimer.
  • five homodimers associate into a pentameric complex.
  • the pentameric complex comprises an immunoglobulin J chain, a fragment thereof, or variant thereof.
  • six homodimers associate into a hexameric complex.
  • the present disclosure provides a multimeric binding agent.
  • the multimeric binding agent comprises two, five, or six binding units, wherein each binding unit comprises two polypeptides, wherein each polypeptide comprises a first domain comprising an extracellular region of human ACE2 and a second domain comprising an IgM heavy chain constant region as described herein.
  • the multimeric binding molecule is a pentameric binding molecule comprising five binding units.
  • the multimeric binding molecule is a pentameric binding molecule which comprises an immunoglobulin J chain, a fragment thereof, or variant thereof.
  • the multimeric binding molecule is a hexameric binding molecule comprising six binding units.
  • the multimeric binding agent comprises two, five, or six binding units, wherein each binding unit comprises two polypeptides, wherein each polypeptide comprises a first domain comprising an extracellular region of human ACE2 and a second domain comprising a human IgM heavy chain constant region, and wherein the extracellular region of human ACE2 comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:3, wherein the polypeptide binds a coronavirus Spike protein (e.g., SARS-CoV-2 Spike SI protein).
  • a coronavirus Spike protein e.g., SARS-CoV-2 Spike SI protein
  • the extracellular region of human ACE2 comprises the amino acid sequence SEQ ID NO:3.
  • the first domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3 retains the ability to bind a coronavirus, e.g., SARS- CoV-2, SARS-CoV-1, and/or CoV-NL63.
  • the first domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3 retains the ability to bind the spike protein of a coronavirus, e.g., SARS-CoV-2, SARS-CoV-1, and/or CoV-NL63.
  • the first domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3 retains the ability to bind the spike SI protein of a coronavirus, e.g., SARS-CoV-2, SARS- CoV-1, and/or CoV-NL63.
  • a coronavirus e.g., SARS-CoV-2, SARS- CoV-1, and/or CoV-NL63.
  • the multimeric binding agent comprises two, five, or six binding units, wherein each binding unit comprises two polypeptides, wherein each polypeptide comprises a first domain comprising an extracellular region of human ACE2 and a second domain comprising a human IgM heavy chain constant region, and wherein the extracellular region of human ACE2 comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:4, wherein the multimeric binding agent is able to bind a coronavirus spike protein (e.g., SARS-CoV-2 SI subunit of the spike protein).
  • a coronavirus spike protein e.g., SARS-CoV-2 SI subunit of the spike protein
  • the extracellular region of human ACE2 comprises the amino acid sequence SEQ ID NO:4.
  • the first domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4 retains the ability to bind a coronavirus, e.g., SARS-CoV-2, SARS-CoV-1, and/or CoV-NL63.
  • the first domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4 retains the ability to bind the spike protein of a coronavirus, e.g., SARS-CoV-2, SARS-CoV-1, and/or CoV-NL63.
  • the first domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4 retains the ability to bind the spike SI protein of a coronavirus, e.g., SARS-CoV-2, SARS-CoV-1, and/or CoV-NL63.
  • a coronavirus e.g., SARS-CoV-2, SARS-CoV-1, and/or CoV-NL63.
  • a multimeric binding agent comprises two, five, or six binding units, wherein each binding unit comprises two polypeptides, wherein each polypeptide comprises a first domain comprising an extracellular region of human ACE2 and a second domain comprising a human IgM heavy chain constant region, and wherein the IgM heavy chain constant region comprises a CH4 domain comprising the tail piece domain.
  • the IgM heavy chain constant region comprises a CH3 domain and a CH4 domain comprising the tail piece domain.
  • the IgM heavy chain constant region comprises a CH2 domain, a CH3 domain, and a CH4 domain comprising the tail piece domain.
  • the IgM heavy chain constant region comprises a partial CHI domain, a CH2 domain, a CH3 domain, and a CH4 domain comprising the tail piece domain.
  • the IgM heavy chain constant region comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 7 or SEQ ID NO: 8, wherein the IgM heavy chain constant region is able to multimerize to form a pentamer or a hexamer.
  • the IgM heavy chain constant region comprises the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10.
  • the IgM heavy chain constant region comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:9-14 and SEQ ID NOs:45-52, wherein the IgM heavy chain constant region is able to multimerize to form a pentamer or a hexamer.
  • the IgM heavy chain constant region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:9-14 and SEQ ID NOs:45-52.
  • a multimeric binding agent comprises two, five, or six binding units, wherein each binding unit comprises two fusion polypeptides, wherein each polypeptide comprises the amino acid sequence of SEQ ID NO: 17.
  • the multimeric binding agent comprises two, five, or six binding units, wherein each binding unit comprises two polypeptides, wherein each polypeptide comprises the amino acid sequence of SEQ ID NO:21.
  • the multimeric binding agent comprises two, five, or six binding units, wherein each binding unit comprises two polypeptides, wherein each polypeptide comprises the amino acid sequence of SEQ ID NO:25. In some embodiments, the multimeric binding agent comprises two, five, or six binding units, wherein each binding unit comprises two polypeptides, wherein each polypeptide comprises the amino acid sequence of SEQ ID NO:29.
  • a coronavirus-binding agent is a bispecific molecule.
  • Bispecific molecules are capable of recognizing and binding at least two different epitopes. The different epitopes can either be within the same molecule (e.g., two binding sites on a coronavirus) or on different molecules (e.g., one binding site on a coronavirus and a second binding site on a different target).
  • a bispecific molecule has enhanced potency as compared to a monospecific molecule or to a combination of more than one molecule.
  • a bispecific molecule has reduced toxicity as compared to an individual molecule or to a combination of more than one molecule.
  • a bispecific molecule has the ability to synchronize the PK of two active binding agents wherein the two individual binding agents have different PK profiles.
  • a bispecific molecule has the ability to concentrate the actions of two agents in a common area (e.g., tissue) in a subject.
  • a bispecific molecule has the ability to concentrate the actions of two agents to a common target (e.g, a specific cell type).
  • a bispecific molecule has the ability to target the actions of two agents to more than one biological pathway or function.
  • a bispecific molecule has the ability to target two different cells and bring them closer together.
  • a bispecific molecule has decreased toxicity and/or side effects. In some embodiments, a bispecific molecule has decreased toxicity and/or side effects as compared to a mixture of the two individual molecules or the molecules as single agents. In some embodiments, a bispecific molecule has an increased therapeutic index. In some embodiments, a bispecific molecule has an increased therapeutic index as compared to a mixture of the two individual molecules or the molecules as single agents.
  • the bispecific molecules comprise heavy chain constant regions with modifications in the amino acids that are part of the interface between the two heavy chains. These modifications are made to enhance heterodimer formation and generally reduce or eliminate homodimer formation.
  • the bispecific molecules are generated using a knobs-into-holes (KIH) strategy.
  • the bispecific molecules comprise variant hinge regions incapable of forming disulfide linkages between identical heavy chains (e.g., reduce homodimer formation).
  • the bispecific molecules comprise heavy chains with changes in amino acids that result in altered electrostatic interactions.
  • a coronavirus-binding agent is a variant of the polypeptide, the coronavirus trap, or the multimer binding agent described herein.
  • a variant of a coronavirus trap described herein must still bind a coronavirus.
  • the variant of a coronavirus trap described herein must still bind SARS- CoV-2.
  • the variant of a coronavirus trap described herein must still bind SARS-CoV-1.
  • a variant of the polypeptide, the coronavirus trap, or the multimeric coronavirus-binding agent described herein comprises one to thirty amino acid substitutions. In some embodiments, a variant of the polypeptide, the coronavirus trap, or the multimer binding agent comprises one to twenty-five amino acid substitutions. In some embodiments, a variant of the polypeptide, the coronavirus trap, or the multimer binding agent comprises one to twenty amino acid substitutions. In some embodiments, a variant of the polypeptide, the coronavirus trap, or the multimer binding agent comprises one to fifteen amino acid substitutions. In some embodiments, a variant of the polypeptide, the coronavirus trap, or the multimer binding agent comprises one to ten amino acid substitutions.
  • a variant of the polypeptide, the coronavirus trap, or the multimer binding agent comprises one to five amino acid substitutions. In some embodiments, a variant of the polypeptide, the coronavirus trap, or the multimer binding agent comprises one to three amino acid substitutions. In some embodiments, the amino acid substitutions are conservative substitutions.
  • a coronavirus-binding agent described herein comprises a polypeptide in which at least one or more of the constant domains (i.e., CH2, CH3, and/or CH4) of IgM Fc region has been modified or deleted.
  • the IgM Fc region of the modified binding agent comprises at least one human constant domain.
  • the IgM Fc region of the modified binding agent comprises more than one human constant domain.
  • modifications to the IgM Fc region comprise additions, deletions, or substitutions of one or more amino acids in one or more domains.
  • one or more domains are partially or entirely deleted from the IgM Fc region of the modified binding agent.
  • the entire CH2 domain has been removed from a binding agent (ACH2 constructs). In some embodiments, the entire CH3 domain has been removed from a binding agent (ACH3 constructs). In some embodiments, the tail piece has been removed from the CH4 domain of a binding agent. In some embodiments, the majority of the CH4 domain has been removed from a binding agent while retaining the tail piece. In some embodiments, a deleted constant domain is replaced by a short amino acid spacer that provides some of the molecular flexibility typically imparted by the absent constant domain. In some embodiments, a modified binding agent comprises a CH3 domain directly fused to the ACE2 polypeptide. In some embodiments, a modified binding agent comprises a peptide spacer inserted between a CH2 domain and a modified CH3 and/or a CH4 domain.
  • the constant domains of an antibody mediate several effector functions and these effector functions can vary depending on the isotype of the antibody.
  • binding of the Clq component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system.
  • Activation of complement is important in the opsonization and lysis of cell pathogens.
  • the activation of complement also stimulates the inflammatory response and can be involved in autoimmune hypersensitivity.
  • the Fc region of an antibody can bind a Fc receptor (FcR) on the surface of a cell.
  • Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.
  • IgG gamma receptors
  • IgE epsilon receptors
  • IgA alpha receptors
  • IgM mi receptors
  • a coronavirus-binding agent comprises a variant IgM Fc region.
  • the amino acid sequence of the Fc region of human IgM is known to those of ordinary skill in the art (e.g., a representative human IgM constant region (CH1- CH4) and Fc region (CH2-CH4) are SEQ ID NO:5 and SEQ ID NO:7, respectively).
  • IgM antibodies with amino acid variations in the constant region have been identified in native antibodies (see, e.g., SEQ ID NO:6; G191S).
  • a variant Fc region is engineered with substitutions at specific amino acid positions as compared to a native Fc region (e.g., SEQ ID NOs: 11-14 and SEQ ID NOs:45-52).
  • the modified Fc region provides for altered effector functions that, in turn, affect the biological profile of the coronavirus-binding agent.
  • the deletion or inactivation (through point mutations or other means) of a Fc region reduces Fc receptor binding of the modified binding agent as it circulates.
  • the Fc region modifications increase the serum half-life of the binding agent.
  • the Fc region modifications reduce the serum half-life of the binding agent.
  • the Fc region modifications decrease or remove ADCC and/or complement dependent cytotoxicity (CDC) of the binding agent.
  • a binding agent does not have one or more effector functions.
  • a binding agent does not bind an Fc receptor and/or complement factors. In some embodiments, a binding agent has no effector function(s) associated with an intact IgM antibody (e.g., “effectorless” agent). In some embodiments, the Fc region modifications increase or enhance ADCC and/or CDC of the binding agent. In some embodiments, the Fc region is modified to eliminate disulfide linkages or oligosaccharide moieties. In some embodiments, the Fc region is modified to add/substitute one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites.
  • binding agent variants are prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired polypeptide. Using these molecular techniques it may be possible to disrupt the activity or effector function provided by a specific sequence or region while substantially maintaining the structure, binding activity, and other desired characteristics of the modified binding agent.
  • the present disclosure further embraces additional variants and equivalents that are substantially homologous to the polypeptide described herein.
  • it is desirable to improve the binding affinity of the polypeptide or coronavirus-binding agent.
  • it is desirable to modulate biological properties of the polypeptide or coronavirus-binding agent, including but not limited to, specificity, thermostability, expression level, effector function(s), glycosylation, immunogenicity, or solubility.
  • specificity, thermostability, expression level, effector function(s), glycosylation, immunogenicity, or solubility include but not limited to, specificity, thermostability, expression level, effector function(s), glycosylation, immunogenicity, or solubility.
  • amino acid changes may alter post-translational processes of a protein, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.
  • Variations may be a substitution, deletion, or insertion of one or more nucleotides encoding the polypeptide that results in a change in the amino acid sequence as compared with the parent polypeptide sequence or for example, the wildtype ACE2 amino acid sequence.
  • amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid substitutions.
  • Insertions or deletions may optionally be in the range of about 1 to 5 amino acids.
  • the substitution, deletion, or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the parent molecule.
  • variations in the amino acid sequence that are biologically useful and/or relevant are determined by systematically making insertions, deletions, or substitutions in the sequence and testing the resulting variant proteins for activity as compared to the parental polypeptide or to wild-type ACE2.
  • variants may include addition of amino acid residues at the amino- and/or carboxyl-terminal end of the extracellular domain of ACE2 or a fragment thereof. In some embodiments, variants may include addition of amino acid residues at the amino- and/or carboxyl-terminal end of the IgM Fc region or a fragment thereof. The length of additional amino acids residues may range from one residue to a hundred or more residues. In some embodiments, a variant comprises an N-terminal methionyl residue. In some embodiments, a variant does not comprise an N-terminal methionyl residue.
  • a variant is engineered to be detectable and may comprise a detectable label and/or protein (e.g., a fluorescent label).
  • a cysteine residue not involved in maintaining the proper conformation of the coronavirus-binding agent may be substituted or deleted to modulate the binding agent’s characteristics, for example, to improve oxidative stability and/or prevent aberrant disulfide crosslinking.
  • one or more cysteine residues may be added to create disulfide bond(s) to improve stability.
  • a polypeptide of the present disclosure is “deimmunized”.
  • the deimmunization of proteins generally consists of introducing specific amino acid mutations (e.g., substitutions, deletions, additions) that result in removal of T-cell epitopes without significantly reducing the binding affinity or other desired activities of the coronavirus-binding agent.
  • variant polypeptides and/or coronavirus-binding agents described herein may be generated using methods known in the art, including but not limited to, site- directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.
  • coronavirus-binding agents described herein are chemically modified.
  • the coronavirus-binding agents are polypeptides that have been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of numerous chemical modifications may be carried out by known techniques.
  • a coronavirus-binding agent is attached, either directly or indirectly, to a half-life extending moiety including, but not limited to, polyethylene glycol (PEG), a PEG mimetic, XTEN®, serum albumin, polysialic acid, N-(2- hydroxypropyl)methacrylamide, or dextran.
  • PEG polyethylene glycol
  • XTEN® serum albumin
  • serum albumin polysialic acid
  • N-(2- hydroxypropyl)methacrylamide or dextran.
  • binding agent interactions are non-covalent and reversible, formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic and van der Waals forces.
  • affinity and/or avidity are commonly used.
  • the binding of a binding agent to its target is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (KD).
  • KD is the ratio of a dissociation rate (k O ff) (how quickly binding agent dissociates from its target) to the association rate (kon) (how quickly the binding agent binds to its target).
  • KD values are determined by measuring the k on and k O ff rates of a specific binding agent/target interaction and then using a ratio of these values to calculate the KD value.
  • KD values may be used to evaluate and rank order the strength of individual binding agent/target interactions. The lower the KD of a binding agent, the higher the affinity of the binding agent for its target.
  • affinity is measured using SPR technology in a Biacore system. Avidity gives a measure of the overall strength of a binding agent/target complex. It is dependent on three major parameters: (i) affinity of the binding agent for the target, (ii) valency of both the binding agent and target, and (iii) structural arrangement of the parts that interact.
  • a multimeric binding agent that has a higher valency compared to a dimeric binding agent (e.g., decavalent- IgM-based vs bivalent-IgG-based valency) would have a higher avidity if the affinity of the binding site for each binding agent was equivalent.
  • a coronavirus-binding agent binds a coronavirus (e.g., SARS-CoV-1, SARS-CoV-2, or CoV-NL63) with a dissociation constant (KD) of 1 pM or less, 100 nM or less, 40 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less.
  • KD dissociation constant
  • a coronavirus-binding agent binds a coronavirus with a KD of 20 nM or less.
  • a coronavirus-binding agent binds a coronavirus with a KD of 10 nM or less. In some embodiments, a coronavirus-binding agent binds a coronavirus with a KD of 1 nM or less. In some embodiments, a coronavirus-binding agent binds a coronavirus with a KD of 0.5 nM or less. In some embodiments, a coronavirus-binding agent binds a coronavirus with a KD of 0.1 nM or less. In some embodiments, a coronavirus-binding agent binds a coronavirus with a KD of 50 pM or less.
  • a coronavirus-binding agent binds a coronavirus with a KD of 25 pM or less. In some embodiments, a coronavirus-binding agent binds a coronavirus with a KD of 10 pM or less. In some embodiments, a coronavirus-binding agent binds a coronavirus with a KD of 1 pM or less. In some embodiments, the dissociation constant of the binding agent for a coronavirus is the dissociation constant determined using a coronavirus spike protein or SI protein immobilized on a Biacore chip and the binding agent flowed over the chip.
  • the dissociation constant of the binding agent for a coronavirus is the dissociation constant determined using the binding agent captured by an anti-human Fc antibody on a Biacore chip and soluble coronavirus spike protein or SI protein flowed over the chip.
  • a coronavirus-binding agent binds a coronavirus with a half maximal effective concentration (EC50) of 1 pM or less, 100 nM or less, 40 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less.
  • EC50 half maximal effective concentration
  • a coronavirus-binding agent binds a coronavirus with an EC50 of 1 pM or less, 100 nM or less, 40 nM or less, 20 nM or less, 10 nM or less, 7 nM or less, 5 nM or less, 3 nM or less, 1 nM or less, or 0.1 nM or less.
  • the coronavirus-binding agents (e.g., polypeptides) described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host.
  • a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest.
  • the sequence can be mutagenized by site-specific mutagenesis to provide functional variants thereof.
  • a DNA sequence encoding a polypeptide of interest is constructed by chemical synthesis using an oligonucleotide synthesizer.
  • Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or expression of a biologically active polypeptide in a suitable host. As is well-known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.
  • recombinant expression vectors are used to amplify and express DNA encoding the polypeptides and/or coronavirus-binding agents described herein.
  • recombinant expression vectors can be replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a polypeptide comprising a first domain comprising an extracellular region of ACE2 and a second domain comprising an IgM heavy chain constant region, operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes.
  • a transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are “operatively linked” when they are functionally related to each other.
  • DNA for a signal peptide is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.
  • structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • a polypeptide may include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coh. including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as Ml 3 and other filamentous single-stranded DNA phages.
  • Suitable host cells for expression of a coronavirus-binding agent include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters.
  • Prokaryotes include gram-negative or gram-positive organisms, for example E. coli o Bacillus.
  • Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Cell-free translation systems may also be employed.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including polypeptide production are well known in the art.
  • Suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived), L-929 (murine fibroblast-derived), Cl 27 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney-derived) cell lines and variants thereof.
  • Mammalian expression vectors can comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • insect cell culture systems e.g., baculovirus
  • Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.
  • the present disclosure provides cells comprising the coronavirus-binding agents described herein.
  • the cells produce the coronavirusbinding agents described herein.
  • the cells produce a polypeptide.
  • the cells produce a polypeptide that binds SARS- CoV-2, SARS-CoV-1, and/or CoV-NP63.
  • the cells produce a polypeptide comprising a first domain comprising an extracellular region of human ACE2 and a second domain comprising an IgM Fc region.
  • the cells produce a polypeptide designated ACE2(740)-IgM Fc(102) (SEQ ID NO: 17).
  • the cells produce a polypeptide designated ACE2(740)-IgM Fc(106) (SEQ ID NO:21). In some embodiments, the cells produce a polypeptide designated ACE2(615)-IgM Fc(102) (SEQ ID NO:25). In some embodiments, the cells produce a polypeptide designated ACE2(615)-IgM Fc(106) (SEQ ID NO:29). In some embodiments, the polypeptides associate into multimeric complexes. In some embodiments, the polypeptides associated into a pentameric complex. In some embodiments, the polypeptides associate into homodimer. In some embodiments, five homodimers associated into a pentameric complex.
  • the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a hybridoma cell.
  • Proteins produced by a host cell can be purified according to any suitable method.
  • Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
  • Affinity tags such as hexa-histidine (SEQ ID NO:36), maltose binding domain, influenza coat sequence, and glutathione-S- transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column.
  • Affinity chromatography used for purifying immunoglobulins can include Protein A, Protein G, and Protein L chromatography.
  • Isolated proteins can be physically characterized using such techniques as proteolysis, size exclusion chromatography (SEC), mass spectrometry (MS), nuclear magnetic resonance (NMR), isoelectric focusing (IEF), high performance liquid chromatography (HPLC), and x-ray crystallography.
  • SEC size exclusion chromatography
  • MS mass spectrometry
  • NMR nuclear magnetic resonance
  • IEF isoelectric focusing
  • HPLC high performance liquid chromatography
  • x-ray crystallography x-ray crystallography
  • supernatants from expression systems which secrete recombinant protein into culture media are first concentrated using a commercially available protein concentration filter, for example, an Amicon® or Millipore Pellicon® ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix.
  • a suitable purification matrix for example, an anion exchange resin is employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification.
  • a cation exchange step is employed.
  • Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • a hydroxyapatite media is employed, including but not limited to, ceramic hydroxyapatite (CHT).
  • CHT ceramic hydroxyapatite
  • one or more reverse-phase HPLC steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, are employed to further purify a recombinant protein.
  • hydrophobic interaction chromatography HIC is used to separate recombinant proteins based on their hydrophobicity.
  • HIC is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions than some other techniques.
  • polypeptides of the present disclosure may be analyzed for their physical/chemical properties and/or biological activities by various assays known in the art.
  • a polypeptide described herein is tested for its ability to bind a coronavirus spike protein or SI protein.
  • a polypeptide described herein is tested for its ability to bind a SARS-CoV-2 spike protein or SI protein.
  • a polypeptide described herein is tested for its ability to bind a SARS-CoV-1 spike protein or SI protein.
  • Binding assays include, but are not limited to, SPR (e.g., Biacore), ELISA, and FACS.
  • a polypeptide described herein is tested for its ability to inhibit, reduce, or block the interaction between a coronavirus and human ACE2. In some embodiments, a polypeptide described herein is tested for its ability to inhibit, reduce, or block the interaction between SARS-CoV-2 and human ACE2. In some embodiments, a polypeptide described herein is tested for its ability to inhibit, reduce, or block the interaction between SARS-CoV-1 and human ACE2. In some embodiments, a polypeptide described herein is tested for its ability to inhibit, reduce, or block coronavirus replication. In some embodiments, a polypeptide described herein is tested for its ability to inhibit, reduce, or block SARS-CoV-2 replication.
  • a polypeptide described herein is tested for its ability to inhibit, reduce, or block SARS-CoV-1 replication.
  • Viral replication assays and viral titer assays are known to those of skill in the art (see, e.g., Schneider et al., 2012, J. Virol., 86: 10112- 10122).
  • polypeptides described herein may be evaluated for solubility, stability, thermostability, viscosity, expression levels, expression quality, and/or purification efficiency.
  • Epitope mapping is the process of identifying the binding site, or epitope on a target protein/antigen where a binding agent binds.
  • a variety of methods are known in the art for mapping epitopes on target proteins. These methods include mutagenesis, including but not limited to, shotgun mutagenesis, site-directed mutagenesis, and alanine scanning; domain or fragment scanning, peptide scanning (e.g., Pepscan technology); display methods (e.g., phage display, microbial display, and ribosome/mRNA display); methods involving proteolysis and mass spectroscopy; and structural determination (e.g., x-ray crystallography and NMR).
  • mutagenesis including but not limited to, shotgun mutagenesis, site-directed mutagenesis, and alanine scanning
  • domain or fragment scanning peptide scanning (e.g., Pepscan technology)
  • display methods e.g., phage display, microbial display, and ribosome
  • purified polypeptides described herein are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.
  • assays including, but not limited to, N-terminal sequencing, amino acid analysis, high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.
  • the present disclosure also provides conjugates comprising a coronavirusbinding agent described herein.
  • the binding agent is attached to a second molecule.
  • the binding agent is conjugated to a cytotoxic agent or moiety.
  • the binding agent is conjugated to a cytotoxic agent.
  • the cytotoxic agent is a chemotherapeutic agent including, but not limited to, methotrexate, adriamycin/doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrol Whyzodi azepines (PBDs), or other intercalating agents.
  • the cytotoxic agent is a microtubule inhibitor including, but not limited to, auristatins, maytansinoids (e.g., DM1 and DM4), and tubulysins.
  • the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tric
  • a binding agent is conjugated to one or more small molecule toxins, such as calicheamicins, maytansinoids, trichothenes, and CC1065.
  • small molecule toxins such as calicheamicins, maytansinoids, trichothenes, and CC1065.
  • a derivative of any one of these toxins may be used as long as the derivative retains the cytotoxic activity of the parent molecule.
  • Conjugates comprising a coronavirus-binding agent described herein may be made using any suitable method known in the art.
  • conjugates are made using a variety of bifunctional protein-coupling agents such as N- succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6- diisocyanate), and bis-active fluorine compounds (such as l,5-d
  • SPDP N-
  • a coronavirus-binding agent described herein is conjugated to a detectable substance or molecule that allows the agent to be used for diagnosis and/or detection.
  • a detectable substance can include but is not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavine(s); fluorescent materials, such as, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), and phycoerythrin; bioluminescent materials, such as luciferase; radioactive materials, such as 212 Bi, 14 C, 57 Co, 51 Cr, 67 Cu, 18 F
  • a coronavirus-binding agent described herein can also be conjugated to an antibody to form an antibody conjugate.
  • a coronavirus-binding agent described herein may be attached to a solid support.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
  • immobilized polypeptides described herein are used in immunoassays. In some embodiments, immobilized polypeptides described herein are used in purification of the target antigen.
  • the disclosure encompasses a polynucleotide composition comprising one or more polynucleotides that encode a polypeptide or protein (e.g., a coronavirus-binding agent) described herein.
  • polynucleotides that encode a polypeptide encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences.
  • the polynucleotides of the disclosure can be in the form of RNA or in the form of DNA.
  • DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.
  • a polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 15-30.
  • the polynucleotide composition comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, 97%, 98%, or 99% identical to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NOs: 15-30.
  • a polynucleotide composition that comprises one or more polynucleotides that hybridize to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NOs: 15-30.
  • the hybridization is under conditions of high stringency as is known to those skilled in the art.
  • the polynucleotide composition comprises one or more polynucleotides encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 15-30. In some embodiments, the polynucleotide composition comprises one or more polynucleotides encoding an amino acid sequence of SEQ ID NO: 17. In some embodiments, the polynucleotide composition comprises one or more polynucleotides encoding an amino acid sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide composition comprises one or more polynucleotides encoding an amino acid sequence of SEQ ID NO:21.
  • the polynucleotide composition comprises one or more polynucleotides encoding an amino acid sequence of SEQ ID NO:22. In some embodiments, the polynucleotide composition comprises one or more polynucleotides encoding an amino acid sequence of SEQ ID NO:25. In some embodiments, the polynucleotide composition comprises one or more polynucleotides encoding an amino acid sequence of SEQ ID NO:26. In some embodiments, the polynucleotide composition comprises one or more polynucleotide encoding an amino acid sequence of SEQ ID NO:29. In some embodiments, the polynucleotide composition comprises one or more polynucleotides encoding an amino acid sequence of SEQ ID NO:30.
  • a polynucleotide comprises the coding sequence for a polypeptide fused in the same reading frame to a polynucleotide which aids in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide).
  • the polypeptide can have the leader sequence cleaved by the host cell to form a “mature” form of the polypeptide.
  • a polynucleotide comprises the coding sequence for a polypeptide fused in the same reading frame to a marker or tag sequence.
  • a marker sequence is a hexa-histidine peptide (SEQ ID NO:36) (His-tag) that allows for efficient purification of the polypeptide fused to the marker.
  • a marker sequence is a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host is used.
  • the marker sequence is a FLAGTM tag.
  • a marker may be used in conjunction with other markers or tags.
  • the present disclosure also provides variants of the polynucleotides described herein, wherein the variant encodes, for example, fragments, analogs, and/or derivatives of a polypeptide described herein.
  • the present disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a polypeptide described herein.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a polynucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to a reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. It is understood by those of skill in the art that an appropriate calculation would be made for other “% identical” statements, for example, 90% identical or 85% identical. These mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the polynucleotide variants can contain alterations in the coding regions, noncoding regions, or both.
  • a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide.
  • a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code).
  • a polynucleotide variant comprises one or more mutated codons comprising one or more (e.g., 1, 2, or 3) substitutions to the codon that change the amino acid encoded by that codon.
  • polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by a bacterial host such as E. colt).
  • a polynucleotide variant comprises at least one silent mutation in a noncoding or a coding region of the sequence.
  • a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.
  • a polynucleotide is isolated. In some embodiments, a polynucleotide is substantially pure.
  • Vectors and cells comprising each and every one of the polynucleotides described herein are also provided.
  • a vector comprises one or more polynucleotides encoding a coronavirus-binding agent described herein.
  • a vector comprises one or more polynucleotides encoding an ACE2-IgM Fc polypeptide described herein.
  • a cell comprises one or more vectors encoding a coronavirus-binding agent described herein.
  • a cell comprises one or more vectors encoding an ACE2-IgM Fc polypeptide described herein. In some embodiments, a cell comprises one or more polynucleotides encoding a coronavirus-binding agent described herein. In some embodiments, a cell comprises one or more polynucleotides encoding an ACE2-IgM Fc polypeptide described herein.
  • a method comprises providing a cell expressing a polypeptide described herein, culturing the cell under conditions that permit the expression of the polypeptide, and isolating the polypeptide. In some embodiments, a method comprises providing a cell expressing a polypeptide described herein, culturing the cell under conditions that permit the expression and association of the polypeptides into a multimeric binding agent, and isolating the multimeric binding agent.
  • a polynucleotide encoding a polypeptide described herein is transiently transfected into a cell. In some embodiments, a polynucleotide encoding a polypeptide described herein is stably transfected into a cell. In some embodiments, a vector comprising a polynucleotide encoding a polypeptide described herein is transiently transfected into a cell. In some embodiments, a vector comprising a polynucleotide encoding a polypeptide described herein is stably transfected into a cell.
  • the cell used to make a polypeptide is a bacterial cell. In some embodiments, the cell used to make a polypeptide is a yeast cell. In some embodiments, the cell used to make a polypeptide is a CHO cell. In other embodiments, the cell used to make a polypeptide is a HEK-293 cell.
  • a coronavirus-binding agent may be a polypeptide (e.g., an ACE2-IgM Fc polypeptide) described herein, a coronavirus trap comprising a multimer of the polypeptides described herein, or a multimeric binding agent comprising the polypeptides described herein.
  • the coronavirus-binding agent comprises a polypeptide having at least 90% (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%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 17.
  • the coronavirus-binding agent comprises a polypeptide having at least 90% (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%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:21.
  • the coronavirus-binding agent comprises a polypeptide having at least 90% (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%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:25.
  • the coronavirus-binding agent comprises a polypeptide having at least 90% (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%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:29.
  • the coronavirus-binding agent comprises the amino acid sequence of SEQ ID NO: 17.
  • the coronavirus- binding agent comprises the amino acid sequence of SEQ ID NO:21.
  • the coronavirus-binding agent comprises the amino acid sequence of SEQ ID NO:25.
  • the coronavirus-binding agent comprises the amino acid sequence of SEQ ID NO:29.
  • the coronavirus-binding agent comprises a multimer of two or more polypeptides, wherein each of the two or more polypeptides are the same (i.e., the two or more polypeptides are identical to each other), and wherein the two or more polypeptides comprise an amino acid sequence having at least 90% (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%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 17.
  • the coronavirus-binding agent comprises a multimer of two or more polypeptides, wherein each of the two or more polypeptides are the same (i.e., the two or more polypeptides are identical to each other), and wherein the two or more polypeptides comprise an amino acid sequence having at least 90% (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%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:21.
  • the coronavirus-binding agent comprises a multimer of two or more polypeptides, wherein each of the two or more polypeptides are the same (i.e., the two or more polypeptides are identical to each other), and wherein the two or more polypeptides comprise an amino acid sequence having at least 90% (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%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:25.
  • the coronavirus-binding agent comprises a multimer of two or more polypeptides, wherein each of the two or more polypeptides are the same (i.e., the two or more polypeptides are identical to each other), and wherein the two or more polypeptides comprise an amino acid sequence having at least 90% (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%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:29.
  • the coronavirus-binding agent comprises a multimer of two or more polypeptides, wherein each of the two or more polypeptides are the same (i.e., the two or more polypeptides are identical to each other), and wherein the two or more polypeptides consist of the amino acid sequence of SEQ ID NO: 17.
  • the coronavirusbinding agent comprises a multimer of two or more polypeptides, wherein each of the two or more polypeptides are the same (i.e., the two or more polypeptides are identical to each other), and wherein the two or more polypeptides consist of the amino acid sequence of SEQ ID NO:21.
  • the coronavirus-binding agent comprises a multimer of two or more polypeptides, wherein each of the two or more polypeptides are the same (i.e., the two or more polypeptides are identical to each other), and wherein the two or more polypeptides consist of the amino acid sequence of SEQ ID NO:25.
  • the coronavirus-binding agent comprises a multimer of two or more polypeptides, wherein each of the two or more polypeptides are the same (i.e., the two or more polypeptides are identical to each other), and wherein the two or more polypeptides consist of the amino acid sequence of SEQ ID NO:29.
  • a coronavirus-binding agent described herein is useful in methods for inhibiting or reducing coronavirus infection, wherein the coronavirus binds ACE2 (e.g., human ACE2).
  • ACE2 e.g., human ACE2
  • SARS-CoV-1, SARS-CoV-2, and CoV-NL63 are coronaviruses that have been identified to bind human ACE2.
  • the coronavirus-binding agents described herein may be effective against any coronavirus that binds ACE2.
  • viruses characteristically mutate in an attempt to evade the host immune response, while still retaining the ability to infect and replicate within the host.
  • the coronavirus-binding agents described herein are also useful in methods for, e.g., inhibiting or reducing coronavirus infection, treating a coronavirus infection, or preventing a coronavirus infection, wherein the coronavirus infection is with a variant of SARS-CoV-1, SARS-CoV-2, or CoV-NL63, wherein said variant bind to ACE2.
  • a coronavirus-binding agent described herein is useful in methods for inhibiting or reducing SARS-CoV-2 infection.
  • a coronavirus-binding agent described herein is useful in methods for inhibiting or reducing SARS-CoV-1 infection.
  • a coronavirus-binding agent described herein is useful in methods for inhibiting or reducing coronavirus replication, wherein the coronavirus binds ACE2.
  • a coronavirus-binding agent described herein is useful in methods for inhibiting or reducing SARS-CoV-2 replication.
  • a coronavirus-binding agent described herein is useful in methods for inhibiting or reducing SARS-CoV-1 replication.
  • a coronavirus-binding agent described herein is useful in methods for preventing COVID-19.
  • a coronavirus-binding agent described herein is useful in methods for preventing SARS.
  • a coronavirusbinding agent described herein is useful in methods for treating COVID-19. In some embodiments, a coronavirus-binding agent described herein is useful in methods for treating SARS. In some embodiments, a coronavirus-binding agent described herein is useful in methods of preventing, reducing, or inhibiting the development of respiratory illness, wherein the respiratory illness is associated with a coronavirus infection. In some embodiments, a coronavirus-binding agent described herein is useful in methods of preventing, reducing, or inhibiting the development of severe respiratory illness, wherein the respiratory illness is associated with a coronavirus infection.
  • a method of inhibiting, reducing, or blocking the interaction of a coronavirus with ACE2 on the surface of a cell comprises contacting the coronavirus with an effective amount of a coronavirus-binding agent described herein. In some embodiments, a method of inhibiting, reducing, or blocking the interaction of a coronavirus with ACE2 on the surface of a cell comprises contacting the coronavirus with an effective amount of an ACE2-IgM Fc polypeptide described herein.
  • a method of inhibiting, reducing, or blocking the interaction of a coronavirus with ACE2 on the surface of a cell comprises contacting the coronavirus with an effective amount of a coronavirus trap described herein. In some embodiments, a method of inhibiting, reducing, or blocking the interaction of a coronavirus with ACE2 on the surface of a cell comprises contacting the coronavirus with an effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein. In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV-1, or CoV- NL63.
  • a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-2 with ACE2 on the surface of a cell comprises contacting SARS-CoV-2 with an effective amount of a coronavirus-binding agent described herein. In some embodiments, a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-2 with ACE2 on the surface of a cell comprises contacting SARS-CoV-2 with an effective amount of an ACE2-IgM Fc polypeptide described herein.
  • a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-2 with ACE2 on the surface of a cell comprises contacting SARS-CoV-2 with an effective amount of a coronavirus trap described herein. In some embodiments, a method of inhibiting, reducing, or blocking the interaction of SARS- CoV-2 with ACE2 on the surface of a cell comprises contacting SARS-CoV-2 with an effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-1 with ACE2 on the surface of a cell comprises contacting SARS-CoV-1 with an effective amount of a coronavirus-binding agent described herein. In some embodiments, a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-1 with ACE2 on the surface of a cell comprises contacting SARS-CoV-1 with an effective amount of an ACE2-IgM Fc polypeptide described herein.
  • a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-1 with ACE2 on the surface of a cell comprises contacting SARS-CoV-1 with an effective amount of a coronavirus trap described herein. In some embodiments, a method of inhibiting, reducing, or blocking the interaction of SARS- CoV-1 with ACE2 on the surface of a cell comprises contacting SARS-CoV-1 with an effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • a method of inhibiting or reducing the viral replication of a coronavirus in a cell comprises contacting the cell with an effective amount of a coronavirus-binding agent described herein, wherein the coronavirus binds ACE2.
  • a method of inhibiting or reducing the viral replication of a coronavirus in a cell comprises contacting the cell with an effective amount of an ACE2-IgM Fc polypeptide described herein, wherein the coronavirus binds ACE2.
  • a method of inhibiting or reducing the viral replication of a coronavirus in a cell comprises contacting the cell with an effective amount of a coronavirus trap described herein, wherein the coronavirus is binds ACE2.
  • a method of inhibiting or reducing the viral replication of a coronavirus in a cell comprises contacting the cell with an effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein, wherein the coronavirus is binds ACE2.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, or CoV-NL63.
  • the coronavirus is SARS-CoV-2.
  • the viral replication is inhibited by at least 50%.
  • the viral replication is inhibited by at least 60%, at least 70%, or at least 80%.
  • the viral replication is inhibited by at least 90%.
  • a method of inhibiting or reducing the viral replication of SARS-CoV-2 in a cell comprises contacting the cell with an effective amount of a coronavirus-binding agent described herein. In some embodiments, a method of inhibiting or reducing the viral replication of SARS-CoV-2 in a cell comprises contacting the cell with an effective amount of an ACE2-IgM Fc polypeptide described herein. In some embodiments, a method of inhibiting or reducing the viral replication of SARS-CoV-2 in a cell comprises contacting the cell with an effective amount of a coronavirus trap described herein.
  • a method of inhibiting or reducing the viral replication of SARS-CoV-2 in a cell comprises contacting the cell with an effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • the viral replication is inhibited by at least 50%.
  • the viral replication is inhibited by at least 60%, at least 70%, or at least 80%.
  • the viral replication is inhibited by at least 90%.
  • a method of inhibiting or reducing the viral replication of SARS-CoV-1 in a cell comprises contacting the cell with an effective amount of a coronavirus-binding agent described herein. In some embodiments, a method of inhibiting or reducing the viral replication of SARS-CoV-1 in a cell comprises contacting the cell with an effective amount of an ACE2-IgM Fc polypeptide described herein. In some embodiments, a method of inhibiting or reducing the viral replication of SARS-CoV-1 in a cell comprises contacting the cell with an effective amount of a coronavirus trap described herein.
  • a method of inhibiting or reducing the viral replication of SARS-CoV-1 in a cell comprises contacting the cell with an effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • the viral replication is inhibited by at least 50%.
  • the viral replication is inhibited by at least 60%, at least 70%, or at least 80%.
  • the viral replication is inhibited by at least 90%.
  • a method of preventing a coronavirus infection in a human subject comprises administering to the subject an effective amount of a coronavirus-binding agent described herein, wherein the coronavirus binds ACE2.
  • a method of preventing coronavirus infection in a human subject comprises administering to the subject an effective amount of an ACE2-IgM Fc polypeptide described herein, wherein the coronavirus binds ACE2.
  • a method of preventing coronavirus infection in a human subject comprises administering to the subject an effective amount of a coronavirus trap described herein, wherein the coronavirus binds ACE2.
  • a method of preventing coronavirus infection in a human subject comprises administering to the subject an effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein, wherein the coronavirus binds ACE2.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, or CoV-NL63.
  • the coronavirus is SARS-CoV-2.
  • the subject receiving a coronavirus-binding agent described herein for preventing a coronavirus infection has a higher risk of or is at risk of being infected with a coronavirus, for example, healthcare workers, passengers onboarding a plane, attendees of in-door gatherings etc.
  • the subject receiving a coronavirus-binding agent described herein for preventing a coronavirus infection is suspected of having been exposed to a coronavirus within about the past 7 days before testing positive for a coronavirus infection.
  • the present disclosure provides methods of preventing a coronavirus infection in a subject, wherein the methods comprise identifying a subject at risk for being exposed to a coronavirus, and administering an effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein. Further, the present disclosure provides methods of preventing a coronavirus infection in a subject, wherein the methods comprise identifying a subject suspected of having been exposed to a coronavirus, and administering an effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, or CoV-NL63.
  • a method of treating a coronavirus infection in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus-binding agent described herein, wherein the coronavirus binds ACE2.
  • a method of treating a coronavirus infection in a human subject comprises administering to the subject a therapeutically effective amount of an ACE2- IgM Fc polypeptide described herein, wherein the coronavirus binds ACE2.
  • a method of treating a coronavirus infection in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus trap described herein, wherein the coronavirus is binds ACE2.
  • a method of treating a coronavirus infection in a human subject comprises administering to the subject a therapeutically effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein, wherein the coronavirus is binds ACE2.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, or CoV-NL63.
  • the coronavirus is SARS-CoV-2.
  • the subject receiving a coronavirus-binding agent described herein for treating a coronavirus infection has tested positive for the viral infection but is asymptomatic.
  • the subject receiving a coronavirus-binding agent described herein for treating a coronavirus infection has tested positive for the viral infection and expressing at least one symptom of the infection.
  • the present disclosure provides methods of treating infection by a coronavirus in a subject, wherein the methods comprise identifying a subject as testing positive for a coronavirus infection and being asymptomatic for a coronavirus infection; and administering an effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein.
  • the present disclosure provides methods of treating infection by a coronavirus in a subject, wherein the methods comprise identifying a subject identifying a subject as being infected with a coronavirus and expressing at least one symptom of the infection; and administering an effective amount of a polypeptide described herein, a coronavirus trap described herein, or a multimeric binding agent described herein.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, or CoV-NL63.
  • a method of preventing COVID-19 in a human subject comprises administering to the subject an effective amount of a coronavirus-binding agent described herein. In some embodiments, a method of preventing CO VID-19 in a human subject comprises administering to the subject an effective amount of an ACE2- IgM Fc polypeptide described herein. In some embodiments, a method of preventing COVID-19 in a human subject comprises administering to the subject an effective amount of a coronavirus trap described herein. In some embodiments, a method of preventing COVID-19 in a human subject comprises administering to the subject an effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • a method of treating COVID-19 in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus-binding agent described herein. In some embodiments, a method of treating COVID-19 in a human subject comprises administering to the subject a therapeutically effective amount of an ACE2-IgM Fc polypeptide described herein. In some embodiments, a method of treating COVID-19 in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus trap described herein.
  • a method of treating CO VID-19 in a human subject comprises administering to the subject a therapeutically effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • a method of preventing SARS in a human subject comprises administering to the subject an effective amount of a coronavirus-binding agent described herein.
  • a method of preventing SARS in a human subject comprises administering to the subject an effective amount of an ACE2- IgM Fc polypeptide described herein.
  • a method of preventing SARS in a human subject comprises administering to the subject an effective amount of a coronavirus trap described herein.
  • a method of preventing SARS in a human subject comprises administering to the subject an effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • a method of treating SARS in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus-binding agent described herein.
  • a method of treating SARS in a human subject comprises administering to the subject a therapeutically effective amount of an ACE2-IgM Fc polypeptide described herein.
  • a method of treating SARS in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus trap described herein.
  • a method of treating SARS in a human subject comprises administering to the subject a therapeutically effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • a method of inhibiting, reducing, or blocking the interaction of a coronavirus with ACE2 on the surface of a cell in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus-binding agent described herein.
  • a method of inhibiting, reducing, or blocking the interaction of a coronavirus with ACE2 on the surface of a cell in a human subject comprises administering to the subject a therapeutically effective amount of an ACE2-IgM Fc polypeptide described herein. In some embodiments, a method of inhibiting, reducing, or blocking the interaction of a coronavirus with ACE2 on the surface of a cell in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus trap described herein.
  • a method of inhibiting, reducing, or blocking the interaction of a coronavirus with ACE2 on the surface of a cell in a subject comprises administering to the subject a therapeutically effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, or CoV-NL63.
  • a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-2 with ACE2 on the surface of a cell in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus-binding agent described herein.
  • a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-2 with ACE2 on the surface of a cell in a subject comprises administering to the subject a therapeutically effective amount of an ACE2-IgM Fc polypeptide described herein.
  • a method of inhibiting, reducing, or blocking the interaction of SARS- CoV-2 with ACE2 on the surface of a cell in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus trap described herein.
  • a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-2 with ACE2 on the surface of a cell in a human subject comprises administering to the subject a therapeutically effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-1 with ACE2 on the surface of a cell in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus-binding agent described herein.
  • a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-1 with ACE2 on the surface of a cell in a human subject comprises administering to the subject a therapeutically effective amount of an ACE2-IgM Fc polypeptide described herein.
  • a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-1 with ACE2 on the surface of a cell in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus trap described herein. In some embodiments, a method of inhibiting, reducing, or blocking the interaction of SARS-CoV-1 with ACE2 on the surface of a cell in a human subject comprises administering to the subject a therapeutically effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • a method of inhibiting or reducing the viral replication of a coronavirus in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus-binding agent described herein, wherein the coronavirus binds ACE2.
  • a method of inhibiting or reducing the viral replication of a coronavirus in a human subject comprises administering to the subject a therapeutically effective amount of an ACE2-IgM Fc polypeptide described herein, wherein the coronavirus binds ACE2.
  • a method of inhibiting or reducing the viral replication of a coronavirus in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus trap described herein, wherein the coronavirus is binds ACE2.
  • a method of inhibiting or reducing the viral replication of a coronavirus in a human subject comprises administering to the subject a therapeutically effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein, wherein the coronavirus is binds ACE2.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, or CoV-NL63.
  • the coronavirus is SARS-CoV-2.
  • the viral replication is inhibited by at least 50%. In some embodiments, the viral replication is inhibited by at least 60%, at least 70%, or at least 80%. In some embodiments, the viral replication is inhibited by at least 90%.
  • a method of inhibiting or reducing the viral replication of SARS-CoV-2 in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus-binding agent described herein. In some embodiments, a method of inhibiting or reducing the viral replication of SARS- CoV-2 in a human subject comprises administering to the subject a therapeutically effective amount of an ACE2-IgM Fc polypeptide described herein. In some embodiments, a method of inhibiting or reducing the viral replication of SARS-CoV-2 in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus trap described herein.
  • a method of inhibiting or reducing the viral replication of SARS-CoV-2 in a human subject comprises administering to the subject a therapeutically effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • the viral replication is inhibited by at least 50%.
  • the viral replication is inhibited by at least 60%, at least 70%, or at least 80%.
  • the viral replication is inhibited by at least 90%.
  • a method of inhibiting or reducing the viral replication of SARS-CoV-1 in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus-binding agent described herein. In some embodiments, a method of inhibiting or reducing the viral replication of SARS- CoV-1 in a human subject comprises administering to the subject a therapeutically effective amount of an ACE2-IgM Fc polypeptide described herein. In some embodiments, a method of inhibiting or reducing the viral replication of SARS-CoV-1 in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus trap described herein.
  • a method of inhibiting or reducing the viral replication of SARS-CoV-1 in a human subject comprises administering to the subject a therapeutically effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein.
  • the viral replication is inhibited by at least 50%.
  • the viral replication is inhibited by at least 60%, at least 70%, or at least 80%.
  • the viral replication is inhibited by at least 90%.
  • a method of treating a respiratory illness in a subject comprises administering to the subject a therapeutically effective amount of a coronavirus-binding agent described herein, wherein the respiratory illness is associated with a coronavirus infection.
  • a method of treating a respiratory illness in a human subject comprises administering to the subject a therapeutically effective amount of an ACE2-IgM Fc polypeptide described herein, wherein the respiratory illness is associated with a coronavirus infection.
  • a method of treating a respiratory illness in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus trap described herein, wherein the respiratory illness is associated with a coronavirus infection.
  • a method of treating a respiratory illness in a human subject comprises administering to the subject a therapeutically effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein, wherein the respiratory illness is associated with a coronavirus infection.
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, or CoV-NL63. In some embodiments, the coronavirus is SARS-CoV-2.
  • a method of preventing or inhibiting development of a respiratory illness in a subject comprises administering to the subject a therapeutically effective amount of a coronavirus-binding agent described herein, wherein the respiratory illness is associated with a coronavirus infection.
  • a method of preventing or inhibiting development of a respiratory illness in a human subject comprises administering to the subject a therapeutically effective amount of an ACE2-IgM Fc polypeptide described herein, wherein the respiratory illness is associated with a coronavirus infection.
  • a method of preventing or inhibiting development of a respiratory illness in a human subject comprises administering to the subject a therapeutically effective amount of a coronavirus trap described herein, wherein the respiratory illness is associated with a coronavirus infection.
  • a method of preventing or inhibiting development of a respiratory illness in a human subject comprises administering to the subject a therapeutically effective amount of a multimeric binding agent comprising ACE2-IgM Fc polypeptides described herein, wherein the respiratory illness is associated with a coronavirus infection.
  • the coronavirus is SARS-CoV-2, SARS- CoV-1, or CoV-NL63. In some embodiments, the coronavirus is SARS-CoV-2.
  • the respiratory illness is selected from the group consisting of: pneumonia, acute respiratory disease (ARD), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), atypical ARDS, respiratory distress syndrome (RDS), severe acute respiratory syndrome (SARS), or coronavirus 2019 (CO VID-19).
  • the respiratory illness is pneumonia.
  • the respiratory illness is ARD.
  • the respiratory illness is ALI.
  • the respiratory illness is ARDS.
  • ARDS is an acute diffuse, inflammatory lung injury, leading to increased pulmonary vascular permeability, increased lung weight, and loss of aerated lung tissue with hypoxemia and bilateral radiographic opacities, associated with increased venous admixture, increased physiological dead space and decreased lung compliance.
  • the definition identifies three mutually exclusive categories of increasingly severe ARDS based on the degree of arterial hypoxemia as measured by the PaCh/FiCh ratio (P/F): (i) mild - P/F 201 to 300 mm Hg; (ii) moderate - P/F 101 to 200 mm Hg; and (iii) severe - P/F ⁇ 100 mm Hg.
  • the respiratory illness is atypical ARDS. Symptoms include hypoxia, shortness of breath, rapid breathing, and bluish skin coloration. For those who survive, a decreased quality of life is common. Atypical ARDS has been observed in some subjects infected with SARS-CoV-2. As used herein, “atypical ARDS” is characterized by moderate or severe hypoxia, without the feeling of shortness of breath. It is also characterized by severe hypoxia but well preserved lung gas volume, which is not seen in ARDS. In some embodiments of the methods described herein, the respiratory illness is RDS. In some embodiments of the methods described herein, the respiratory illness is SARS. In some embodiments of the methods described herein, the respiratory illness is MERS. In some embodiments of the methods described herein, the respiratory illness is COVID- 19.
  • the coronavirus infection is associated with a dysregulated pro-inflammatory cytokine response, which may be referred to as “severe cytokine release syndrome” or “cytokine storm”.
  • the pro-inflammatory cytokine response includes, but is not limited to, TNF-a, IL-6, IL-10, IL-la, IL-ip, and IL-12.
  • there is an elevated level of cytokines in a sample obtained from a subject wherein the cytokines include, but are not limited to, TNF-a, IL-6, IL-10, IL-la, IL-ip, and IL-12.
  • the subject has lung damage, respiratory failure, kidney damage, kidney failure, liver damage, heart damage, vascular damage, thrombosis, stroke, central nervous system injury, and/or multiple organ failure.
  • the subject has one or more symptoms selected from the group consisting of: hypoxemia, cough, wheezing, dyspnea, hyperpnea, pulmonary /lung inflammation, shortness of breath, labored breathing, rapid breathing, accumulation of alveolar fluid, pulmonary edema, vascular leakage, lymphocyte infiltration in the lung, lymphopenia, fever, chills, shaking chills, increased heart rate, chest pain, low blood pressure, headache, confusion, seizures, extreme tiredness, sepsis, bluish coloring of nails or lips, toe rashes/redness, toe swelling, loss of sense of smell, loss of sense of taste, and diarrhea.
  • the subject has mild, moderate or severe hypoxemia as determined by Partial Pressure of arterial oxygen/Fraction of inspired oxygen (PaCh/FiCh) or positive end-expiratory pressure (PEEP).
  • PaCh/FiCh Partial Pressure of arterial oxygen/Fraction of inspired oxygen
  • PEEP positive end-expiratory pressure
  • the subject has severe hypoxemia with a PaO2/FiO2 of less than 100.
  • a method comprises using a coronavirus-binding agent described herein in combination therapy.
  • combination therapy includes, but is not limited to, (i) combination with a medical device, for example, a ventilator or ECMO machine and/or (ii) combination with at least one additional therapeutic agent.
  • combination therapy comprises administration of a coronavirus-binding agent described herein in combination with at least one additional therapeutic agent and the usage of a medical device.
  • a method comprises administering a coronavirus-binding agent described herein in combination with usage of a medical device.
  • a method comprises administering a coronavirus-binding agent described herein in combination with at least one additional therapeutic agent.
  • Treatment with two or more therapeutic agents often uses agents that work by different mechanisms of action, although this is not required.
  • Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects.
  • Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent(s).
  • Combination therapy may decrease the likelihood that resistance to an agent will develop.
  • the combination of a coronavirus-binding agent described herein and at least one additional therapeutic agent results in additive or synergistic results.
  • the combination therapy results in an increase in the therapeutic index of the coronavirus-binding agent. In some embodiments, the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s). In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the coronavirus-binding agent. In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the additional therapeutic agent(s).
  • a combination treatment comprises one additional therapeutic agent or two or more additional therapeutic agents.
  • treatment with a coronavirus-binding agent described herein can occur prior to, concurrently with, or subsequent to administration of the additional therapeutic agents.
  • combined administration includes co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities.
  • preparation of agents and/or dosing schedules for additional therapeutic agents are according to manufacturers' instructions or as determined empirically by the skilled practitioner.
  • a combination treatment or combination therapy comprises a coronavirus-binding agent described herein and at least one additional therapeutic agent, wherein the additional therapeutic agent is antiviral agent.
  • Antiviral drugs include but are not limited to, nucleosides analogues, pyrophosphate analogues, acyclic nucleoside phosphonate analogues, protease inhibitors, entry blockers, and interferons.
  • the antiviral drug is a nucleoside analogue.
  • the antiviral drug is remdesivir.
  • a combination treatment or combination therapy comprises a coronavirus-binding agent described herein and at least one additional therapeutic agent, wherein the additional therapeutic agent is a kinase signaling inhibitor.
  • the kinase signaling inhibitor is imatinib mesylate (GLEEVEC), nilotinib (TASIGNA), or dasatinib (SPRYCEL).
  • the appropriate dosage of a coronavirus-binding agent of the present disclosure depends on the disorder or disease to be treated, the severity and course of the disorder or disease, the responsiveness of the disorder or disease, whether the agent is administered for therapeutic or preventative purposes, previous therapy, the subject’s condition, the subject's clinical history, and so on.
  • a coronavirus-binding agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • the dose of a coronavirus-binding agent depends on the site of administration.
  • the dose of a coronavirus-binding agent depends on the delivery system.
  • dosage of the coronavirus-binding agent is from 0.01 pg/kg to 100 mg/kg of body weight, from 0.1 pg/kg to 100 mg/kg of body weight, from 1 pg/kg to 100 mg/kg of body weight, from 1 mg/kg to 100 mg/kg of body weight, 1 mg/kg to 80 mg/kg of body weight, from 1 mg/kg to 50 mg/kg of body weight, from 1 mg/kg to 25 mg/kg of body weight, from 1 mg/kg to 15 mg/kg of body weight, from 10 mg/kg to 100 mg/kg of body weight, from 10 mg/kg to 75 mg/kg of body weight, or from 10 mg/kg to 50 mg/kg of body weight.
  • dosage of the coronavirus-binding agent is from about 0.1 mg to about 20 mg/kg of body weight. In some embodiments, dosage of the coronavirus-binding agent is about 0.5 mg/kg of body weight. In some embodiments, dosage of the LAIR- 1 -binding agent is about 1 mg/kg of body weight. In some embodiments, dosage of the coronavirus-binding agent is about 1.5 mg/kg of body weight. In some embodiments, dosage of the coronavirus-binding agent is about 2 mg/kg of body weight. In some embodiments, dosage of the agent is about 2.5 mg/kg of body weight. In some embodiments, dosage of the coronavirusbinding agent is about 5 mg/kg of body weight.
  • dosage of the coronavirus-binding agent is about 7.5 mg/kg of body weight. In some embodiments, dosage of the coronavirus-binding agent is about 10 mg/kg of body weight. In some embodiments, dosage of the coronavirus-binding agent is about 12.5 mg/kg of body weight. In some embodiments, dosage of the coronavirus-binding agent is about 15 mg/kg of body weight.
  • compositions comprising a coronavirusbinding agent described herein.
  • a composition comprises an ACE2-IgM Fc polypeptide described herein.
  • a composition comprises a coronavirus trap describe herein.
  • a composition comprises a multimeric binding agent comprising the ACE2-IgM Fc polypeptides described herein.
  • the present disclosure also provides pharmaceutical compositions comprising a coronavirus-binding agent described herein and a pharmaceutically acceptable vehicle.
  • the present disclosure also provides pharmaceutical compositions comprising an ACE2-IgM Fc polypeptide described herein and a pharmaceutically acceptable vehicle.
  • the present disclosure also provides pharmaceutical compositions comprising a coronavirus trap described herein and a pharmaceutically acceptable vehicle.
  • the present disclosure also provides pharmaceutical compositions comprising a multimeric binding agent comprising the ACE2-IgM Fc polypeptides described herein and a pharmaceutically acceptable vehicle.
  • Formulations are prepared for storage and use by combining a binding agent of the present disclosure with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient).
  • a pharmaceutically acceptable vehicle e.g., a carrier or excipient.
  • pharmaceutically acceptable carriers, excipients, and/or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.
  • Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3- pentanol, and m-cresol; low molecular weight polypeptides (e.g, less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine
  • the formulation is in the form of an aqueous solution. In some embodiments, the formulation is stored in a lyophilized or in an alternative dried form.
  • the binding agents of the present disclosure can be formulated in any suitable form for delivery to a target cell/tissue.
  • a coronavirus-binding agent can be formulated as a liposome, microparticle, microcapsule, albumin microsphere, microemulsion, nanoparticle, nanocapsule, or macroemulsion.
  • the pharmaceutical formulation includes an agent of the present disclosure complexed with liposomes.
  • liposomes are known to those of skill in the art. For example, some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).
  • PEG-PE PEG-derivatized phosphatidylethanolamine
  • a coronavirus-binding agent is formulated as a sustained-release preparation.
  • sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing an agent, where the matrices are in the form of shaped articles (e.g., films or microcapsules).
  • Sustained-release matrices include but are not limited to polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3 -hydroxybutyric acid.
  • polyesters such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid
  • compositions or formulations of the present disclosure can be administered in any number of ways for either local or systemic treatment. Administration can be (i) topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders, (ii) pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal, (iii) oral, or (iv) parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).
  • parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).
  • a series of soluble ACE2-IgM Fc constructs were generated using the extracellular domain of human ACE2 (amino acids 18-740 of SEQ ID NO: 1; also SEQ ID NO:3) or a fragment thereof (amino acids 18-615 of SEQ ID NO: 1; also SEQ ID NO:4) and a human IgM Fc region (SEQ ID NO:9 or SEQ ID NO: 10).
  • Constructs were generated with the native ACE2 signal sequence, MSSSSWLLLSLVAVTAA (SEQ ID NO:34) or the human IgK signal sequence MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO:35).
  • the constructs include:
  • ACE2(740)-IgM Fc(102) This construct is set forth as SEQ ID NO: 15 and after cleavage of the signal sequence - SEQ ID NO: 17 and referred to as ACE2(740)-IgM Fc(102).
  • This construct is set forth as SEQ ID NO:28 and after cleavage of the signal sequence - SEQ ID NO:29 and referred to as ACE2(615)-IgM Fc(106).
  • ACE2(615)-IgM Fc(102) This construct is set forth as SEQ ID NO:23 and after cleavage of the signal sequence - SEQ ID NO:25 and referred to as ACE2(615)-IgM Fc(102).
  • ACE2(615)-IgM Fc(102) human IgK signal sequence, extracellular domain of human ACE2 (aa 18-615 of SEQ ID NO: 1) and human IgM Fc region PLPV-CH2-CH4 (SEQ ID NO: 10) - referred to herein as “IgK-ACE2(615)-IgM Fc(102)”.
  • This construct is set forth as SEQ ID NO:24 and after cleavage of the signal sequence - SEQ ID NO:25 and referred to as ACE2(615)-IgM Fc(102).
  • Each of these ACE2-IgM Fc constructs were transfected together a vector encoding the J-chain into Expi293FTM cells using standard transient transfection methods.
  • the polypeptides were expressed and then purified by a variety of methods including affinity chromatography, size exclusion chromatography, and anion-exchange chromatography.
  • ACE2-IgM Fc polypeptides were assayed for binding to SARS-CoV-2 Spike SI or SARS-CoV-2 Spike receptor binding domain (RBD) using ELISAs.
  • the ACE2- IgM Fc polypeptides were purified using an anti-IgM resin (POROSTM Capture SelectTM IgM Affinity Matrix; ThermoFisher Scientific). Conditioned media containing each polypeptide was passed over a column of the anti-IgM resin at 4° C and the column was washed with PBS to remove unbound material.
  • ACE2-IgM Fc polypeptides were eluted with elution buffer (0.1 M glycine, pH 3.0) and the pH of the eluate was adjusted with 1 M Tris-CL, pH 8.0.
  • ELISA plates were coated with SARS-CoV-2 Spike RBD-mFc protein (Sino Biological) at 2 pg/mL overnight at 4° C. Plates were washed and incubated with an ELISA blocking reagent following manufacturer’s recommendations (ThermoFisher Scientific). After washing, 2-fold serial dilutions of ACE2-IgM Fc polypeptides were added to the plates starting at 10 pg/mL and incubated for 2 hours at room temperature. The polypeptides were (i) ACE2(740)-IgM Fc(102) and (ii) ACE2(615)-IgM Fc(102) and an IgM antibody control.
  • the ACE2-IgM Fc polypeptides were mixed with a soluble human ACE2-His protein (Sino Biological) at 10 pg/mL. Plates were washed and anti-hlgM Fc-HRP antibody conjugate was added to the wells to detect bound ACE2-hIgM Fc. TMB (3, 3'5,5'- tetramethylbenzidine) chromogen solution was used as the substrate for HRP. After adding stop solution, the plates were analyzed on a SpectraMax® plate reader (Molecular Devices).
  • ACE2(740)-IgM Fc(102) bound to SARS-CoV-2 Spike RBD protein in a dose-dependent manner.
  • the binding affinity of the polypeptide containing the full-length extracellular domain, ACE2(740)-IgM Fc(102) was stronger than the binding of the polypeptide with a truncated extracellular domain, ACE2(615)- IgM Fc(102).
  • the ACE2-IgM Fc polypeptides had low nM EC50s - ACE2(740)-IgM Fc(102) had an EC50 of 2.2 nM and ACE2(615)-IgM Fc(102) had an EC50 of 14.7 nM.
  • a soluble ACE2 protein competed with the polypeptides, demonstrating the specificity of the ACE2-IgM Fc polypeptide binding to the coronavirus RBD protein.
  • SARS-CoV-2 Spike SI protein bound to ACE2(740)- IgM Fc(102) and ACE2(615)-IgM Fc(106) in a dose-dependent manner.
  • the binding affinity of the SI protein for the polypeptide containing the full-length extracellular domain, ACE2(740)-IgM Fc(102) was stronger than the binding of the SI protein for the polypeptide with a truncated extracellular domain, ACE2(615)-IgM Fc(102).
  • the Spike SI protein had an EC50 of 1.1 to ACE2(740)-IgM Fc(102) and had an EC50 of 1.2 nM to ACE(615)-IgM Fc(102).
  • ELISA plates were coated with SARS-CoV-2 Spike Sl-His protein (Sino Biological) at 2 pg/mL for 2 hours at 37° C. Plates were washed and incubated with an ELISA blocking reagent following manufacturer’s recommendations (ThermoFisher Scientific).
  • ACE2-IgM Fc polypeptides were added to the plates starting at 10 pg/mL and incubated for 2 hours at room temperature.
  • the polypeptides were (i) ACE2(740)-IgM Fc(102) - 2 different preparations and (ii) ACE2(615)-IgM Fc(102) - 2 different preparations.
  • the ACE2-IgM Fc polypeptides were mixed with a soluble human ACE2-His protein (Sino Biological) at 10 pg/mL.
  • ACE2(740)-IgM Fc(102) bound to SARS-CoV-2 Spike SI protein in a dose-dependent manner.
  • the binding of the polypeptide containing the full-length extracellular domain, ACE2(740)-IgM Fc(102) was significantly stronger than the binding of the polypeptide with a truncated extracellular domain, ACE2(615)-IgM Fc(102).
  • the ACE2-IgM Fc polypeptides had low nM EC50s - ACE2(740)-IgM Fc(102) had EC50s of 3.2 and 3.1 nM and ACE2(615)-IgM Fc(102) had EC50s of 29.4 and 23.8 nM. Similar to results above, a soluble ACE2 protein competed with the polypeptides for binding to the SARS-CoV-2 Spike 1 protein.
  • ELISA was performed.
  • the ACE2-IgM Fc polypeptide tested was ACE2(740)-IgM Fc(106).
  • the ACE2-IgGl Fc polypeptide tested was composed of extracellular domain of human ACE2 (SEQ ID NO:3), and human IgGl Fc region hinge-CH2-CH3, referred to herein as “ACE2(740)-IgGl Fc.”
  • ELISA plates were coated with the HCoV-NL63 Spike protein at 2 pg/mL for 2 hours at 37° C. Plates were washed and incubated with an ELISA blocking reagent following manufacturer’s recommendations.
  • anti-Spike IgM IgM against the Spike protein of SARS-CoV-2
  • IgGl against the Spike protein of SARS-CoV-2 IgGl against the Spike protein of SARS-CoV-2
  • Anti-hlgM Fc-HRP or anti-hlgGl Fc-HRP antibody conjugate was added to the wells to detect bound ACE2(740)-IgM Fc(106) or ACE2(740)-IgGl Fc.
  • TMB (3,3',5,5'-tetramethylbenzidine) chromogen solution was used as the substrate for HRP. After adding stop solution, the plates were analyzed on a SpectraMax® plate reader (Molecular Devices).
  • ACE2(740)-IgM Fc(106) binds the Spike protein of HCoV-NL63 more strongly than ACE2(740)-IgGl Fc, while the monoclonal anti-Spike IgM and anti-Spike IgGl do not bind to the Spike protein of HCoV-NL63.
  • ACE2-IgM Fc polypeptides have advantage over anti-Spike IgM and anti-Spike IgG since ACE2-IgM Fc polypeptides are able to bind and neutralize coronavirus possessing variations/mutations in the Spike protein, for example when undergoing viral drifting, whereas anti-Spike IgM and anti-Spike IgG lose the binding capacity under these circumstances.
  • Coronaviruses constantly undergo various mutations, such as mutations in the Spike protein.
  • the ability of ACE2-IgM Fc polypeptides to bind to various mutant Spike proteins of SARS-CoV-2 was tested.
  • ELISA plates were coated with the wildtype RBD (SEQ ID NO:33), various RBD mutants (including K444R, K458R, I472V, S477N, S477R, T478I, P479S, V483I, G485S, and S494P) and Spike protein mutant (D614G) at 2 pg/mL for 2 hours at 37° C. Plates were washed and incubated with an ELISA blocking reagent following manufacturer’s recommendations.
  • ACE2(740)-IgM Fc(106) was added to the plates starting at 5 nM and incubated for 2 hours at room temperature. Irrelevant IgM was used as the negative control. Binding of ACE2(740)-IgM Fc(106) to the plates was detected by anti-hlgM Fc-HRP conjugate and TMB (3,3',5,5'-tetramethylbenzidine) chromogen solution as the substrate for HRP. EC50s were calculated.
  • the ACE2-IgM Fc polypeptide binds to a variety of RBD mutants at a comparable level to wild-type RBD (EC50s shown in the table). Further, D614G mutation does not affect the binding of the ACE2-IgM Fc polypeptide to the Spike protein. This suggests that an advantage of using ACE2-IgM Fc polypeptides as therapeutics is that they retain the binding capacity to Spike protein mutations, whereas anti-Spike antibodies may no longer effectively recognize the Spike protein variants after viral escape, leading to therapeutic resistance.
  • ACE2-hIgM Fc polypeptides were assayed for binding to SARS-CoV-2 Spike SI using a Biacore-based assay. Two additional polypeptides were generated for these studies using an IgGl Fc as the fusion partner in place of IgM Fc.
  • constructs are: (i) native signal sequence of human ACE2, extracellular domain of human ACE2 (aa 18-740 of SEQ ID NO: 1; and SEQ ID NO:3), and human IgGl Fc region hinge-CH2- CH3 - referred to herein as “native-ACE2(740)-IgGl Fc” and after signal sequence cleavage “ACE2(740)-IgGl Fc” and (ii) native signal sequence of human ACE2, extracellular domain of human ACE2 (aa 18-615 of SEQ ID NO: 1, and SEQ ID NO:4), and human IgGl Fc region hinge-CH2-CH3 - referred to herein as “native-ACE2(615)- IgGl Fc” after signal sequence cleavage “ACE2(615)-IgGl Fc”.
  • Equilibrium dissociation constant (KD) measurements were carried out using Biacore system (GE Healthcare LifeSciences) with the purified ACE2-IgGl and ACE2- IgM constructs to evaluate their binding affinity to SARS-CoV-2 SI protein. Briefly, using a CM5 chip coated with an anti-human Fc antibody, purified ACE2(740)-IgGl Fc or ACE2(615)-IgGl Fc was captured (100-150 RUs) on flow cell 2 and flow cell 1 was used as a reference.
  • ACE2(740)-IgM Fc(106) or ACE2(615)-IgM Fc(106) was captured (50 RUs) on flow cell 4 coated with an anti-IgM Fc antibody (using flow cell 3 as a reference).
  • SARS-CoV-2-Sl-His protein (Sino Biologic) was injected at different concentrations (11-100 nM, 3 -fold dilution) at a flow rate of 30 pL/min at 25° C.
  • Kinetic data were collected over time and fit using the simultaneous global fit equation to yield affinity constants (KD values). The binding affinities are shown in Table 1.
  • Binding assays - Cell-surfaced expressed SARS-CoV-2 Spike protein
  • C0S7 cells were transiently transfected with a construct expressing a full- length SARS-CoV-2 Spike protein.
  • the Spike protein is expressed at the COS7 cell surface as a trimer mimicking what is displayed on the virus surface.
  • Spike proteinexpressing cells were incubated with ACE2-IgM Fc and ACE2-IgGl Fc polypeptides.
  • the polypeptides were (i) ACE2(740)-IgM Fc(106), (ii) ACE2(615)-IgM Fc(106), (iii) ACE2(740)-IgGl Fc, (iv) ACE2(615)-IgGl Fc, and IgM and IgG controls.
  • the polypeptides were added to the cells at different concentrations (2 -fold serial dilutions, starting at lug/mL for ACE2-IgGl Fc polypeptides and 5ug/mL ACE2-IgM Fc polypeptide).
  • the cells were analyzed using a Celllnsight CX5 instrument (ThermoFisher Scientific).
  • ACE2(740)-IgM Fc(106) and ACE2(615)-IgM Fc(106) bound to SARS-CoV-2 Spike SI protein in a dose-dependent manner.
  • the multimeric ACE2-IgM Fc polypeptides bound to SARS-CoV-2 SI protein with a higher affinity than the ACE2-IgGl Fc polypeptides.
  • the ACE2-IgM Fc polypeptides’ EC50s were: ACE2(740)-IgM Fc(106) - EC50 of 0.54 nM and ACE2(615)-IgM Fc(106) - EC50 of 0.33 nM.
  • the ACE2-IgGl polypeptides’ EC50s were > 5 nM. For this experiment and the concentrations of the ACE2-IgGl Fc polypeptides used, more specific EC50s could not be calculated. These results demonstrate that the ACE2-IgM Fc polypeptides have higher binding affinities for the SARS-CoV-2 Spike protein in its nature trimer conformation than ACE2-IgG Fc polypeptides. These results suggest that ACE2-IgM Fc polypeptides would be better coronavirus traps than ACE2-IgG Fc molecules. These results support the use of an ACE2-IgM Fc polypeptide as a therapeutic agent for controlling and alleviating coronavirus infections.
  • Binding of ACE2-IgM Fc polypeptides to plgR was assayed.
  • COS7 cells were transfected with human plgR, mouse plgR, or SARS-CoV-2 Spike protein. Cells were incubated with ACE2(615)-IgM Fc(106) or ACE2(615)-IgGl Fc.
  • Human IgM was used as a positive control, and anti- KLH IgGl was used as a negative control.
  • IgM molecules were used at 5 pg/mL, and IgG molecules were used at 1 pg/mL.
  • IgGl or IgM was detected with a secondary fluorescent goat Fab2' anti -human Fc and anti-human IgM and analyzed using a Celllnsight CX5 instrument (ThermoFisher Scientific).
  • ACE2(615)-IgM Fc(106) binds cells expressing human plgR and mouse plgR, suggesting that ACE2(615)-IgM Fc(106) forms pentamers in the presence of J-chain sufficient for binding to plgR. Similar to ACE2(740)-IgM Fc polypeptides, ACE2(615)-IgM Fc(106) shows stronger binding to the Spike protein than ACE2(615)-IgGl Fc polypeptide.
  • ACE2-IgM Fc polypeptides The ability of ACE2-IgM Fc polypeptides to trap coronavirus and reduce viral infection was assayed in a cell-cell fusion experiment.
  • a batch of HEK293 cells were co-transfected with SARS-CoV-2 Spike protein and GFP.
  • Another batch of HEK293 cells were transfected with human ACE2 and stained with Cell Tracker Deep Red (Thermofi lier). Cells transfected with CD276 and stained with Cell Tracker Deep Red were used as a negative control.
  • ACE2-IgM Fc polypeptides block interaction between ACE2 and the Spike protein more effectively than ACE2-IgGl Fc polypeptides.
  • ACE2-IgM Fc polypeptides would act as a better coronavirus trap than ACE2-IgG Fc molecules.
  • ACE2-IgM Fc polypeptides were assayed for their ability to trap live coronavirus.
  • ACE2(615)-IgM Fc(106) and ACE2(615)-IgGl Fc each was incubated with SARS-CoV-2 viral particles (strain 2019-nCoV/USA_WAl/2020) to form viral- polypeptide complexes.
  • SARS-CoV-2 viral particles strain 2019-nCoV/USA_WAl/2020
  • the complexes were then added to Vero E6 cells expressing ACE2 and incubated at 37°C for Ih. Cells were overlaid with 1% (w/v) methylcellulose in MEM supplemented with 2% FBS. Plates were harvested 30 hours later by removing overlays and fixed with 4% PF A in PBS.
  • EC50 of ACE2(740)-IgM Fc mediated viral neutralization is 0.1 InM
  • EC50 of ACE2(740)-IgGl Fc mediated neutralization is 115nM.
  • the extracellular domain of ACE2 is heavily glycosylated.
  • ACE2 SEQ ID NO:3
  • glycosylation is predicted to be found at positions such as N53, N90, N103, N322, N432 and N546. Since glycosylation impacts metabolism (PK can be impacted and the multiple glycosylation sites may impact the manfacturability) of a therapeutic protein, ACE2-IgM polypeptide mutants with one or more glycosylation sites abolished were assayed for their cellular expression and binding capacity to coronavirus. 7 mutant ACE2(615)-IgM Fc(106) polypeptides were designed. Mutant 1 includes N53S in the ACE2(615).
  • Mutant 2 includes T92A in the ACE2(615).
  • Mutant 3 includes N103 S in the ACE2(615).
  • Mutant 4 includes T324A in the ACE2(615).
  • Mutant 5 includes N432S in the ACE2(615).
  • Mutant 6 includes N546S in the ACE2(615).
  • Mutant 7 includes N53S, T92A, N103S, T324A, N432S and N546S in the ACE2(615).
  • Polypeptides were expressed together with J-chain in Expi293FTM cells using standard transient transfection methods.
  • the polypeptides were purified by a variety of methods including affinity chromatography, size exclusion chromatography, and anion- exchange chromatography.
  • mutants except mutant 7 are expressed (between 125-150mg/L). Furthermore, these mutants are able to bind to the Spike protein of SARS-CoV-2 in ELISA assay (data not shown).
  • ACE2-IgM Fc fusion protein (ACE2 18-740 / IgM Fc 102-453) signal sequence underlined
  • ACE2-IgM Fc fusion protein (ACE2 18-740 / IgM Fc 102-453) signal sequence underlined
  • ACE2-IgM Fc fusion protein (ACE2 18-740 / IgM Fc 102-453) without signal sequence (SEQ ID NO: 17)
  • ACE2-IgM Fc fusion protein (ACE2 18-740 / IgM Fc 102-453-P311A/P313A) without signal sequence (SEQ ID NO: 18)
  • ACE2-IgM Fc fusion protein (ACE2 18-740 / IgM Fc 106-453) signal sequence underlined
  • ACE2-IgM Fc fusion protein (ACE2 18-740 / IgM Fc 106-453) signal sequence underlined
  • ACE2-IgM Fc fusion protein (ACE2 18-740 / IgM Fc 106-453) without signal sequence (SEQ ID NO:21)
  • ACE2-IgM Fc fusion protein (ACE2 18-740 / IgM Fc 106-453-P311A/P313A) without signal sequence (SEQ ID NO: 22)
  • ACE2-IgM Fc fusion protein (ACE2 18-615 / IgM Fc 102-453) signal sequence underlined
  • ACE2-IgM Fc fusion protein (ACE2 18-615 / IgM Fc 102-453-P311A/P313A) without signal sequence (SEQ ID NO: 26)
  • ACE2-IgM Fc fusion protein (ACE2 18-615 / IgM Fc 106-453) signal sequence underlined
  • ACE2-IgM Fc fusion protein (ACE2 18-615 / IgM Fc 106-453-P311A/P313A) without signal sequence (SEQ ID NO: 30)
  • SARS-CoV-2 SI Protein (aa 13-685) (SEQ ID NO:32)

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Abstract

La présente divulgation concerne de manière générale les agents se liant aux coronavirus, en particulier les coronavirus utilisant l'ACE2 comme récepteur. La présente divulgation concerne des agents, tels que des polypeptides et/ou des multimères de polypeptides qui se lient spécifiquement aux coronavirus, ainsi que des compositions comprenant les agents, et des procédés pour leur utilisation. La divulgation concerne également des polynucléotides et des vecteurs codant pour les agents; et des cellules comprenant ou produisant les agents.
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