ACE2-IgM-Fc fusion protein and uses thereof

Abstract

The present disclosure provides ACE2 fusion proteins comprising a multimerization moiety linked to an ACE2 moiety that specifically binds to the RBD region of SARS-CoV and / or SARS-CoV-2. The disclosure further relates to methods of producing the ACE2 fusion proteins, pharmaceutical compositions comprising the ACE2 fusion proteins, and methods of using the ACE2 fusion proteins to treat conditions associated with SARS-CoV and SARS-CoV-2 infection, such as COVID-19.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 487,399 (filed February 28, 2023), the contents of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML Sequence Listing was created on February 22, 2024, is named RGN-027WO_SL.xml, and is 67,033 bytes in size. [Background technology]

[0003] 3.Background technology Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, positive-sense, single-stranded RNA virus of the Betacoronavirus genus, which also includes SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus (HCoV)-OC43, and HCoV-HKU1 (Non-Patent Document 1). SARS-CoV-2 causes COVID-19, a potentially life-threatening disease that was first characterized in late 2019 and spread into a global pandemic in early 2020.

[0004] SARS-CoV-2 shares approximately 80% identity with SARS-CoV, and both viruses depend on interaction with angiotensin-converting enzyme 2 (ACE2) for cell entry. ACE2 is an enzyme expressed on the extracellular surface of many cell types, particularly respiratory epithelia, cardiac myocytes, endothelial cells, and arterial smooth muscle cells (Non-Patent Document 2). ACE2 is primarily involved in vascular tone regulation by catalyzing the cleavage of angiotensin precursors Ang I and / or Ang II, which are essential for angiotensin maturation (Non-Patent Document 3). SARS-CoV and SARS-CoV-2 compete with these precursors for ACE2 binding and use this interaction to enter host cells. Nevertheless, SARS-CoV-2 has been shown to have a higher affinity for human ACE2 (hACE2) and to bind to soluble hACE2 more strongly than SARS-CoV (Non-Patent Document 2). This enhanced affinity of SARS-CoV-2 to hACE2 may underlie its high infectivity.

[0005] Several vaccines against SARS-CoV-2 are currently available to prevent the development of severe disease. However, vaccination rates vary across populations, and even in areas with high vaccination rates, breakthrough infections causing COVID-19 have been observed in individuals immunized against SARS-CoV-2. Because some individuals have been diagnosed with COVID-19 multiple times, previous SARS-CoV-2 infection does not appear to confer complete immunity against any future infection. Furthermore, in some individuals, SARS-CoV-2 infection causes prolonged illness associated with the persistence of one or more symptoms of COVID-19 for weeks to months after clearance of the infection. These observations highlight the serious population health threat posed by COVID-19 and the need to combat SARS-CoV-2 infection with effective treatments.

[0006] Biologic therapies such as monoclonal antibodies may become obsolete due to the rapid emergence of new SARS-CoV-2 variants. Small molecule therapies such as Paxlovid (a combination of the oral antiviral drugs nilmatrervir and ritonavir) have been associated with a "Paxlovid rebound" effect, in which the virus re-emerges. 4 Therefore, there remains a need to develop novel therapies that are effective against different strains of SARS-CoV-2. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Jackson et al.,2022,Nat Rev Mol Cell Biol.23(1):3-20 [Non-patent document 2] Beyerstedt et al.,2021,Eur J Microbiol Infect Dis.40(5):905-919 [Non-patent document 3] Yan et al.,2020,Science.367:1444-1448 [Non-patent document 4] Callaway,Nature(News),11 August 2022 Summary of the Invention

[0008] 4. Summary of the Invention The present disclosure relates to ACE2 fusion proteins for inhibiting the interaction between coronaviruses and host cells. The ACE2 fusion proteins of the present disclosure do not have the drawbacks of vaccines and therapies specific to certain coronavirus strains.

[0009] ACE2 fusion proteins of the present disclosure generally comprise one or more polypeptide chains having the formula [A1]-[L1]-[MM]-[L2]-[A2], where [A1] represents a first ACE2 moiety, [L1] represents an optional first linker, [MM] represents a multimerization moiety, [L2] represents an optional second linker, and [A2] represents an optional second ACE2 moiety.

[0010] Exemplary configurations of ACE2 fusion proteins of the present disclosure are depicted in Figures 2A-2B and described in Section 6.2 and numbered embodiments 1-45. ACE2 moieties suitable for incorporation into ACE2 fusion proteins of the present disclosure are described in Section 6.3 and defined in numbered embodiments 2-24.

[0011] Linker moieties suitable for incorporation into the ACE2 fusion proteins of the present disclosure are described in Section 6.5 and defined in numbered embodiments 27 and 28. Multimerization moieties suitable for incorporation into ACE2 fusion proteins of the present disclosure are described in Section 6.4 and defined in numbered embodiments 29-32, 36, 37, 44, and 45.

[0012] Exemplary ACE2 fusion protein sequences are shown in embodiments 46-109. The present disclosure further provides nucleic acids encoding the ACE2 fusion proteins of the present disclosure, host cells engineered to express the ACE2 fusion proteins of the present disclosure, and recombinant methods for producing the ACE2 fusion proteins of the present disclosure. Such nucleic acids, host cells, and production methods are described in Section 6.6 and numbered embodiments 110-192.

[0013] The present disclosure further provides pharmaceutical compositions comprising the ACE2 fusion proteins of the present disclosure, as well as methods of using them in therapy. Pharmaceutical compositions are described in Section 6.7 and numbered embodiment 193. Methods of using the ACE2 fusion proteins and pharmaceutical compositions are described in Section 6.8 and numbered embodiments 194-202.

[0014] Other features and advantages of the fusion protein aspects of the present disclosure will become apparent from the following more detailed description, taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0015] 5. Brief description of the drawings [Figure 1A] The structure and domains of the ACE2 protein are shown. Figure 1A is a cartoon representation of the extracellular portion of human ACE2 shown in complex with the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein. The soluble extracellular portion of ACE2 contains the N-terminal peptidase domain (PD), corresponding to amino acids 18-615 of human ACE2, and the neck domain (ND) of the collectrin-like domain (CLD), corresponding to amino acids 616-740 of human ACE2. The peptide-binding cavity on the outer surface of ACE2-PD is directly involved in RBD binding, while ACE2-ND is involved in ACE2 dimerization. Figure 1B shows a diagram of the full-length ACE2 protein with the domains presented in an N- to C-terminal direction. [Figure 1B]The structure and domains of the ACE2 protein are shown. Figure 1A is a cartoon representation of the extracellular portion of human ACE2 shown in complex with the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein. The soluble extracellular portion of ACE2 contains the N-terminal peptidase domain (PD), corresponding to amino acids 18-615 of human ACE2, and the neck domain (ND) of the collectrin-like domain (CLD), corresponding to amino acids 616-740 of human ACE2. The peptide-binding cavity on the outer surface of ACE2-PD is directly involved in RBD binding, while ACE2-ND is involved in ACE2 dimerization. Figure 1B shows a diagram of the full-length ACE2 protein with the domains presented in an N- to C-terminal direction. [Figure 2A] 2A and 2B show exemplary ACE2 fusion protein constructs of the present disclosure, where (1) represents the ACE2 portion, (2) represents the IgM J chain domain, (3) represents the Cμ2 domain of IgM-Fc, (4) represents the Cμ3 domain of IgM-Fc, and (5) represents the Cμ4 domain of IgM-Fc. Figure 2A shows an exemplary decavalent ACE2 fusion protein having an IgM Fc domain containing the Cμ2, Cμ3, and Cμ4 domains (Fc2,3,4), and Figure 2B shows an exemplary ACE2 fusion protein having an IgM Fc domain containing the Cμ3 and Cμ4 domains (Fc3,4). [Figure 2B] 2A and 2B show exemplary ACE2 fusion protein constructs of the present disclosure, where (1) represents the ACE2 portion, (2) represents the IgM J chain domain, (3) represents the Cμ2 domain of IgM-Fc, (4) represents the Cμ3 domain of IgM-Fc, and (5) represents the Cμ4 domain of IgM-Fc. Figure 2A shows an exemplary decavalent ACE2 fusion protein having an IgM Fc domain containing the Cμ2, Cμ3, and Cμ4 domains (Fc2,3,4), and Figure 2B shows an exemplary ACE2 fusion protein having an IgM Fc domain containing the Cμ3 and Cμ4 domains (Fc3,4). [Figure 3]Representative 4-12% non-reducing SDS-PAGE of ACE2-Fc fusion constructs ("ACE2-Fc fusion protein" or "ACE2-Fc fusion construct" refers to an ACE2 fusion protein in which the multimerization moiety is an Fc domain). The gel shows different decavalent ACE2-Fc(IgM) fusion constructs (the term "ACE-Fc(IgM)" refers to an ACE2 fusion protein in which the multimerization moiety is an IgM-based Fc domain) in media samples collected from transfected Expi 293-F cells. Each well was filled with 20 μL of culture medium. NC: negative control. [Figure 4A] The SEC profiles of four ACE2-Fc(IgM) constructs are shown: ACE2(615)-Fc2,3,4(IgM) (Figure 4A), ACE2(615)-Fc3,4(IgM) (Figure 4B), ACE2(740)-Fc2,3,4(IgM) (Figure 4C), and ACE2(740)-Fc3,4(IgM) (Figure 4D). Molecular weight markers of 158 kDa and 670 kDa are labeled on each chromatogram. [Figure 4B] The SEC profiles of four ACE2-Fc(IgM) constructs are shown: ACE2(615)-Fc2,3,4(IgM) (Figure 4A), ACE2(615)-Fc3,4(IgM) (Figure 4B), ACE2(740)-Fc2,3,4(IgM) (Figure 4C), and ACE2(740)-Fc3,4(IgM) (Figure 4D). Molecular weight markers of 158 kDa and 670 kDa are labeled on each chromatogram. [Figure 4C] The SEC profiles of four ACE2-Fc(IgM) constructs are shown: ACE2(615)-Fc2,3,4(IgM) (Figure 4A), ACE2(615)-Fc3,4(IgM) (Figure 4B), ACE2(740)-Fc2,3,4(IgM) (Figure 4C), and ACE2(740)-Fc3,4(IgM) (Figure 4D). Molecular weight markers of 158 kDa and 670 kDa are labeled on each chromatogram. [Figure 4D]The SEC profiles of four ACE2-Fc(IgM) constructs are shown: ACE2(615)-Fc2,3,4(IgM) (Figure 4A), ACE2(615)-Fc3,4(IgM) (Figure 4B), ACE2(740)-Fc2,3,4(IgM) (Figure 4C), and ACE2(740)-Fc3,4(IgM) (Figure 4D). Molecular weight markers of 158 kDa and 670 kDa are labeled on each chromatogram. [Figure 5A] Figures 5A and 5D show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 pseudovirus D614G, respectively. Figures 5B and 5E show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 omicron BA.1, respectively. Finally, Figures 5C and 5F show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 omicron BA.2, respectively. [Figure 5B] Figures 5A and 5D show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 pseudovirus D614G, respectively. Figures 5B and 5E show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 omicron BA.1, respectively. Finally, Figures 5C and 5F show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 omicron BA.2, respectively. [Figure 5C]Figures 5A and 5D show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 pseudovirus D614G, respectively. Figures 5B and 5E show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 omicron BA.1, respectively. Finally, Figures 5C and 5F show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 omicron BA.2, respectively. [Figure 5D] Figures 5A and 5D show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 pseudovirus D614G, respectively. Figures 5B and 5E show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 omicron BA.1, respectively. Finally, Figures 5C and 5F show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 omicron BA.2, respectively. [Figure 5E]Figures 5A and 5D show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 pseudovirus D614G, respectively. Figures 5B and 5E show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 omicron BA.1, respectively. Finally, Figures 5C and 5F show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 omicron BA.2, respectively. [Figure 5F] Figures 5A and 5D show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 pseudovirus D614G, respectively. Figures 5B and 5E show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 omicron BA.1, respectively. Finally, Figures 5C and 5F show the neutralization efficacy of different ACE2-Fc2,3,4(IgM) and ACE2-Fc3,4(IgM) constructs against SARS-CoV2 omicron BA.2, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0016] 6. MODE FOR CARRYING OUT THE INVENTION 6.1.Definition As used herein, the following terms are intended to have the following meanings:

[0017] About, Approximately: The terms "about," "approximately," and the like are used throughout the specification before numerical values ​​to indicate that the numerical value is not necessarily exact (e.g., to account for fractions, variations in measurement precision and / or accuracy, timing, etc.). A disclosure of "about X" or "approximately X," where X is a numerical value, should be understood to also disclose "X." Thus, for example, disclosure of embodiments in which a sequence has "about X% sequence identity" to another sequence is also a disclosure of embodiments in which the sequence has "X% sequence identity" to the other sequence.

[0018] ACE2 portion: The term "ACE2 portion" refers to a portion comprising an amino acid sequence having at least 70% sequence identity to the extracellular portion of human ACE2 that is capable of binding to the RBD of SARS-CoV or SARS-CoV-2 RBD, e.g., an amino acid sequence having at least 70% sequence identity to the peptidase domain (PD) of human ACE2. In some embodiments, the ACE2 portion comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the peptidase domain of human ACE2. In further embodiments, the ACE2 portion comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the peptidase and neck domains of human ACE2. Typically, the ACE2 portion lacks the transmembrane domain. Exemplary ACE2 portions are provided in Section 6.3.

[0019] And / Or: Unless otherwise indicated, the conjunction "or" is intended to be used in its proper sense as a Boolean logic operator, encompassing both the selection of features in an alternative (A or B, where the selection of A is mutually exclusive of B) and the selection of conjoint features (A or B, where both A and B are selected). In several places in the text, the term "and / or" is used interchangeably and should not be construed to mean that "or" is used in reference to mutually exclusive alternatives.

[0020] Associated: The term "associated" in the context of an ACE2 fusion protein refers to a functional relationship between two or more polypeptide chains. In particular, the term "associated" means that two or more polypeptides are associated with one another, e.g., non-covalently through molecular interactions, or covalently through one or more disulfide bridges or chemical interconnections, to generate a functional ACE2 fusion protein. Examples of associations that may be present in ACE2 fusion proteins of the present disclosure include, but are not limited to, association between Fc domains to form an Fc region (e.g., a homopentameric or homohexameric IgM Fc region, as described in Section 6.4.1).

[0021] Bivalent: As used herein, the term "bivalent" refers to an ACE2 fusion protein that contains two ACE2 moieties, whether in the same polypeptide chain or on different polypeptide chains. The two ACE2 moieties can be the same or different.

[0022] COVID-19: The term "COVID-19" stands for "Coronavirus disease 2019" and refers to the infectious disease caused by SARS-CoV-2 infection. Patients with COVID-19 can experience a wide range of symptoms, ranging from mild to severe. Symptoms may include, but are not limited to, fever, chills, cough, shortness of breath, difficulty breathing, fatigue, muscle aches, body aches, headache, loss of smell, loss of taste, sore throat, congestion, runny nose, nausea, and diarrhea.

[0023] Decavalent: As used herein with respect to an ACE2 fusion protein, the term "decavalent" refers to an ACE2 fusion protein that contains ten ACE2 moieties. The ten ACE2 moieties can be the same or different. In some embodiments, the decavalent ACE2 fusion protein is a pentameric assembly of five IgM Fc dimers, each containing an ACE2 moiety at its N-terminus and connected via a J chain.

[0024] Dodecavalent: As used herein with respect to an ACE2 fusion protein, the term "dodecavalent" refers to an ACE2 fusion protein that contains 12 ACE2 moieties. The 12 ACE2 moieties can be the same or different. In some embodiments, the dodecavalent ACE2 fusion protein is a hexameric assembly of six IgM Fc dimers, each containing an ACE2 moiety at its N-terminus and no J-chain attachment.

[0025] EC50: The term "EC50" refers to the half maximal effective concentration of a molecule (e.g., an ACE2 fusion protein) that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an ACE2 fusion protein at which 50% of its maximal effect is observed. In certain embodiments, the EC50 value is equal to the concentration of an ACE2 fusion protein that confers half-maximal virus or pseudovirus neutralization in the assay described in Section 8.1.3.

[0026] Fc domain and Fc region: The term "Fc domain" refers to the portion of a heavy chain that pairs with the corresponding portion of another heavy chain. In some embodiments, an Fc domain comprises a Cμ2 domain followed by a Cμ3 domain, with or without a hinge region N-terminal to the Cμ2 domain. The term "Fc region" refers to the region formed by the association of two heavy chain Fc domains. The two Fc domains within an Fc region may be identical to one another or different. In natural antibodies, the Fc domains are typically identical, but one or both Fc domains may be modified to allow heterodimerization, for example, via knob-in-hole interactions.

[0027] Host cell: As used herein, the term "host cell" refers to a cell into which a nucleic acid of the present disclosure has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer to the particular subject cell, and also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but still fall within the scope of the term as used herein. Typical host cells are eukaryotic host cells, such as mammalian host cells. Exemplary eukaryotic host cells include yeast and mammalian cells, e.g., vertebrate cells such as mouse, rat, monkey, or human cell lines, e.g., HKB11 cells, PER.C6 cells, HEK cells, or CHO cells.

[0028] Linker: As used herein, the term "linker" refers to a connecting peptide between two moieties. For example, a linker can connect an ACE2 moiety to a multimerization moiety.

[0029] Multivalent: As used herein, the term "multivalent" refers to an ACE2 fusion protein that contains two or more ACE2 moieties on one, two, or more polypeptide chains. The two or more ACE2 moieties can be the same or different.

[0030] Operably linked: The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of a fusion protein or other polypeptide, the term "operably linked" means that two or more amino acid segments are linked to produce a functional polypeptide. For example, in the context of an ACE2 fusion protein of the present disclosure, the separate components (e.g., an ACE2 portion and a multimerization portion) can be operably linked directly or via a peptide linker sequence. In the context of a nucleic acid encoding a fusion protein, such as an ACE2 fusion protein of the present disclosure, "operably linked" means that the two nucleic acids are connected such that the amino acid sequences encoded by the two nucleic acids remain in frame.

[0031] Polypeptide, Peptide, and Protein: The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0032] Subject: The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" and "subject" are used interchangeably herein.

[0033] Tetravalent: As used herein, the term "tetravalent" refers to an ACE2 fusion protein that contains four ACE2 moieties, whether in the same polypeptide chain or on two or more polypeptide chains. The four ACE2 moieties can be the same or different.

[0034] Treat, Treatment, Treating: As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or condition, and / or the alleviation of one or more symptoms (preferably one or more discernible symptoms) of a disease or condition, resulting from the administration of one or more ACE2 fusion proteins of the present disclosure.

[0035] In some embodiments, the disease or condition is caused by a coronavirus infection, e.g., SARS-CoV or SARS-CoV-2 (e.g., COVID-19). In some embodiments, the disease or condition is a SARS-CoV or SARS-CoV-2 infection, or any other disease associated with a similar infection. With reference to these diseases and conditions, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of disease progression, severity, and / or duration, or alleviation of one or more symptoms (preferably one or more discernible symptoms) of the disease, resulting from administration of one or more ACE2 fusion proteins of the present disclosure. In certain embodiments, the terms "treat," "treatment," and "treating" refer to a reduction in at least one measurable (not necessarily discernible by the patient) physical parameter of COVID-19, such as blood oxygen saturation level. In other embodiments, the terms "treat," "treatment," and "treating" refer to an inhibition of COVID-19 progression, either physically (e.g., by stabilization of discernible symptoms), physiologically (e.g., by stabilization of physical parameters), or both. In other embodiments, the terms "treat", "treatment" and "treating" refer to the reduction or elimination of an infection.

[0036] ACE2 fusion proteins The present disclosure relates to an ACE2 fusion protein comprising one or more polypeptide chains having the formula [A1]-[L1]-[MM]-[L2]-[A2], where [A1] represents a first ACE2 moiety, [L1] represents an optional first linker, [MM] represents a multimerization moiety, [L2] represents an optional second linker, and [A2] represents an optional second ACE2 moiety.

[0037] Exemplary ACE2 moieties that can be incorporated into an ACE2 fusion protein as component(s) [A1] and / or [A2] are disclosed in Section 6.3. Exemplary linkers that can be incorporated into ACE2 fusion proteins as component(s) [L1] and / or [L2] are disclosed in Section 6.5.

[0038] Exemplary multimerization moieties that can be incorporated into ACE2 fusion proteins as building blocks [MM] are disclosed in Section 6.4. In some embodiments, the ACE2 fusion proteins of the present disclosure are pentameric or hexamers. Pentameric ACE2 fusion proteins typically contain five [A1]-[L1]-[MM] dimers, where [MM] contains a dimerized IgM-based Fc domain as disclosed in Section 6.4.1, and the five dimers are closed in a ring structure with a J chain polypeptide, as also disclosed in Section 6.4.1. Exemplary pentameric ACE2 fusion proteins are shown in Figures 2A and 2B. Hexameric ACE2 fusion proteins typically contain six [A1]-[L1]-[MM] dimers, where [MM] contains a dimerized IgM-based Fc domain as disclosed in Section 6.4.1, and the six dimers are closed in a ring structure without a J chain polypeptide.

[0039] 6.3.ACE2 part SARS-CoV-2 docks to the extracellular surface of host cells by binding to ACE2, an enzyme expressed in a variety of cells, including respiratory epithelia.

[0040] The amino acid sequence of human ACE2 has been assigned NCBI reference sequence NP_001358344.1 and UniProtKB accession number Q9BYF1, with the signal peptide reproduced below in underline.

[0041] [ka]

[0042] Under normal circumstances, ACE2 contributes to the regulation of vascular tone and blood pressure by cleaving the angiotensin precursor, which is achieved via its peptidase domain (PD). ACE2-PD is the largest domain of ACE2, corresponding to amino acids 18 to 615, the sequence of which is reproduced below.

[0043] (SEQ ID NO: 2) The other domain of ACE2 is its collectrin-like domain (CLD, aa 616-770), which contains an extracellular neck domain (ND, aa 616-740) that promotes dimerization and a single transmembrane domain (TM, aa 741-761) (Figures 1A and 1B).

[0044] The extracellular part of ACE2 consists of PD and ND (PD+ND, aa 18-740), the amino acid sequence of which is reproduced below. (SEQ ID NO: 3) The interaction of SARS-CoV or SARS-CoV-2 with ACE2 involves a large viral protrusion called the spike (S) protein. The SARS-CoV or SARS-CoV-2 S protein consists of two subunits (S1 and S2). The receptor-binding domain (RBD) of S1 is involved in binding ACE2-PD through polar interactions (Figure 1A). More specifically, the extended loop of the RBD spans the α1 helix of ACE2-PD like a bridge, but also interacts with the α2 helix and loop connecting the β3 and β4 strands of ACE2-PD (Yan et al., 2020, Science. 367:1444-1448). An exemplary SARS-CoV RBD sequence is reproduced below.

[0045] TNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPK (SEQ ID NO: 4) An exemplary SARS-CoV-2 RBD sequence is reproduced below.

[0046] TNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPK (SEQ ID NO: 5) The ACE2 fusion proteins of the present disclosure include an ACE2 portion having an amino acid sequence with at least 70% sequence identity to the extracellular portion of human ACE2 that can bind the RBD of SARS-CoV or SARS-CoV-2 RBD, e.g., an amino acid sequence with at least 70% sequence identity to the peptidase domain (PD) of human ACE2.

[0047] The binding affinity of the ACE2 portion to the RBD peptide can be assessed using various binding assays. For example, biolayer interferometry (BLI) can be used to measure the binding of free RBD to immobilized ACE2 or free ACE2 to immobilized RBD by analyzing the light reflection pattern from a sensor surface. BLI and other binding affinity assays can be used to determine the effect of ACE2 mutations on its affinity for RBD.

[0048] Several affinity-enhancing mutations of ACE2 have been reported. For example, a hydrophobic substitution at T27 of ACE2 increases hydrophobic packing with aromatic residues of the S protein, while a D30E mutation enables interaction with K417 of the S protein (Yan et al., 2020, Science. 367:1444-1448). Furthermore, a series of single amino acid substitutions that enhance ACE2 binding affinity to the RBD have been characterized (Chan et al., 2020, Science. 369:1261-1265; Laurini et al., 2021, ACS Nano 15(4):6929-6948), which may be used to generate ACE2 moieties with enhanced binding affinity to the RBD, e.g., the SARS-CoV-2 RBD.

[0049] In certain aspects, the ACE2 portion has an amino acid sequence at least 70%, at least 80%, or at least 90% identical to the PD of human ACE2 corresponding to amino acids 18 to 615 (SEQ ID NO: 2), and in various embodiments has an amino acid sequence at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the PD of human ACE2.

[0050] In further aspects, the ACE2 portion has an amino acid sequence that is at least 70%, at least 80%, or at least 90% identical to the PD of human ACE2 corresponding to amino acids 18-740 (SEQ ID NO: 3), and in various embodiments, has an amino acid sequence that is about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the PD+ND of human ACE2.

[0051] In some embodiments, the PD portion of the ACE2 portion can include one or more amino acid substitutions that increase binding affinity to the RBD, e.g., the SARS-COV2 RBD. These substitutions can include one or more of amino acids 19, 23, 24, 25, 26, 27, 29, 30, 31, 33, 34, 35, 39, 40, 41, 42, 65, 69, 72, 75, 76, 79, 82, 89, 90, 91, 92, 324, 325, 330, 357, 386, 393, or 519 of human ACE2, e.g., one or more of the amino acid substitutions listed in Table 1.

[0052] [Table 1]

[0053] In some embodiments, the PD portion of the ACE2 portion comprises a combination of two or more amino acid substitutions that enhance its affinity to the RBD, e.g., the SARS-COV-2 RBD, compared to the corresponding wild-type sequence. In certain specific embodiments, the PD portion of the ACE2 portion comprises two, three, four, five, six, or more of the substitutions listed in Table 1.

[0054] In some embodiments, the ACE2 portion comprises one or more amino acid substitutions associated with a high level of enhanced binding to the RBD, e.g., the SARS-CoV-2 RBD. For example, these combinations of amino acid substitutions can include one or more substitutions at amino acids 25, 27, 31, 34, 42, 79, 90, 92, 324, 325, 330, and 386 of human ACE2, e.g., one or more of the substitutions shown in Table 1 that are associated with the highest increases in binding affinity to the SARS-CoV-2 RBD.

[0055] In certain embodiments, the ACE2 portion can include a combination of amino acid substitutions that have been shown to be associated with increased RBD affinity. For example, one such example is ACE2v2.4, which combines the amino acid substitutions T27Y, L79T, and N330Y (Chan et al., 2020, Science. 369:1261-1265). Thus, in certain embodiments, the combination of amino acid substitutions in the ACE2 portion can include the amino acid substitutions T27Y, L79T, and N330Y, optionally with one or more additional substitutions. In some embodiments, the ACE2 portion includes an ACE2 PD with the amino acid substitutions T27Y, L79T, and N330Y (e.g., an amino acid sequence having the sequence of SEQ ID NO: 2). In further embodiments, the ACE2 portion includes an ACE2 PD+ND with the amino acid substitutions T27Y, L79T, and N330Y (e.g., an amino acid sequence having the sequence of SEQ ID NO: 3).

[0056] In certain specific embodiments, the ACE2 portion is (a) comprises an amino acid sequence having at least 90%, 95%, or 98% sequence identity to the ACE2 PD (SEQ ID NO: 2) and / or at least 90%, 95%, or 98% sequence identity to the ACE2 PD+ND (SEQ ID NO: 3); (b) comprises at least one amino acid substitution that increases affinity for a coronavirus RBD, e.g., the RBD of SEQ ID NO: 4 and / or SEQ ID NO: 5; (c) contains at least one amino acid substitution at position 25, 27, 31, 34, 42, 79, 90, 92, 324, 325, 330, or 386 of ACE2; (d) contains at least one amino acid substitution shown in Table 1; (e) containing the amino acid substitutions T27Y, L79T, and N330Y (referred to as v2.4); (f) has increased affinity for a coronavirus RBD, e.g., the RBD of SEQ ID NO: 4 and / or SEQ ID NO: 5, optionally wherein the increased affinity is at least 25%, at least 50%, at least 100%, at least 200%, or at least 300% compared to the corresponding sequence of wild-type ACE2 (SEQ ID NO: 1); or (g) Any combination of two, three, or more of (a) through (f).

[0057] 6.4. Multimerization moiety In some embodiments, the ACE2 fusion proteins of the present disclosure include one or more multimerization moieties, e.g., one or more multimerization moieties that are or include an Fc domain. In certain embodiments, the ACE2 fusion proteins of the present disclosure include a single multimerization moiety (e.g., a single Fc domain), and / or the ACE2 fusion proteins of the present disclosure include two or more multimerization moieties (e.g., two or more Fc domains that can associate to form an Fc region). In some embodiments, the ACE2 fusion protein is a pentamer or hexamer of five or six dimeric Fc regions from IgM, e.g., as described in Section 6.4.1.

[0058] Fc Domain The ACE2 fusion protein of the present disclosure can include an Fc domain, or a pair of Fc domains that associate to form an Fc region, from any suitable species operably linked to the ACE2 moiety. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the ACE2 moiety is fused to an IgM Fc domain.

[0059] The Fc domain that can be incorporated into the ACE2 fusion protein can be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgM.

[0060] In natural antibodies, the heavy chain Fc domain of IgA, IgD, and IgG consists of two heavy chain constant domains (Cμ2 and Cμ3), while the domain of IgE and IgM consists of three heavy chain constant domains (Cμ2, Cμ3, and Cμ4), which dimerize to create the Fc region.

[0061] In the ACE2 fusion proteins of the present disclosure, the Fc region, and / or the Fc domain therein, can comprise heavy chain constant domains from one or more different classes of antibodies, e.g., one, two, or three different classes.

[0062] In some embodiments, the ACE2 fusion protein of the present disclosure comprises an Fc domain derived from IgM. IgM naturally exists in humans as a covalent multimer of heavy (H) and light (L) chains that form a common H2L2 antibody unit. In addition to heavy and light chains, IgM also has a third chain known as the connecting (J) chain (Keyt et al., 2020, Antibodies. 9(4):53). IgM exists as a pentamer when the J chain is incorporated and as a hexamer when the J chain is absent.

[0063] The J chain is a small 137-residue polypeptide that associates with IgM by forming a disulfide bond with the Cμ4 tail. Incorporation of the J chain into pentameric IgM closes the ring structure by bridging the first and fifth monomer units, thereby precluding the addition of a sixth IgM monomer.

[0064] An exemplary amino acid sequence of a human mature wild-type J chain is reproduced below.

[0065] [ka]

[0066] In some embodiments, the engineered J chain is incorporated into an IgM pentamer. An exemplary amino acid sequence of an engineered human mature J chain is reproduced below.

[0067] [ka]

[0068] The heavy chain of IgM has an 18-amino acid extension to the C-terminal constant domain known as the tail. The tail contains cysteine ​​residues that form disulfide bonds between heavy chains within the polymer, and is thought to play an important role in polymerization. The tail also contains glycosylation sites. In certain embodiments, the ACE2 fusion protein of the present disclosure comprises a tail.

[0069] IgM assembly typically begins with the association of heavy (H) and light (L) chains in an HL configuration, which then dimerizes to form the H2L2 subunit. A key site for this intrasubunit assembly is Cys337, which forms a disulfide bond between the two Cμ2 domains and stabilizes the H2L2 domain. These subunits then join together through disulfide bridges to form multimers. A residue involved in this multimerization is Cys575 on the tail domain of Cμ4, which forms a disulfide bond and enables noncovalent Cμ4 interactions. Another key residue is Cys414 on Cμ3, which further connects the two Cμ3 domains of adjacent H2L2 subunits in tandem with the disulfide bond between the Cys337 residues of Cμ2. In the presence of J chain, IgM assembly results in a pentamer in which the Cys337 disulfide bond is in tandem with both the Cys414 and Cys575 disulfide bonds (Pasalic et al., 2017, Proc. Nat'l Acad. Sci USA 114(41)E8575-E8584; Keyt et al., 2020, Antibodies. 9(4):53; Casali, 1998. Encyclopedia of Immunology (2nd Ed), p1212-1217). For IgM assembly to include a J chain, the J chain polypeptide must be coexpressed with a polypeptide encoding the H2L2 subunit domain.

[0070] In certain embodiments, the multimerizing moieties provided by the present disclosure are pentameric or hexameric binding molecules comprising a dimeric IgM heavy chain constant region or a multimerizing fragment thereof. An exemplary sequence of a full-length human IgM heavy chain constant domain is reproduced below.

[0071] [ka]

[0072] Without wishing to be bound by theory, assembly of dimeric IgM Fc regions into pentameric or hexameric structures is thought to involve at least the Cμ4 and / or tail (TP) domains (Braathen, R., et al., 2002. J. Biol. Chem. 277:42755-42762). Thus, multimerization moieties based on IgM Fc domains typically contain at least the Cμ4 and / or TP domain sequences.

[0073] An IgM heavy chain constant domain can further comprise a Cμ3 domain or a fragment thereof, a Cμ2 domain or a fragment thereof, and / or other IgM or other immunoglobulin heavy chain domains.

[0074] Exemplary sequences of human IgM heavy chain constant domains are reproduced in Table 2 below.

[0075] [Table 2]

[0076] In some embodiments, the Fc domain comprises the amino acid sequence of the Cμ4 domain of IgM, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9.

[0077] In some embodiments, the Fc domain comprises the amino acid sequence of the Cμ4 and tail domain of IgM, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:10.

[0078] In some embodiments, the Fc domain comprises the amino acid sequence of the Cμ3 and Cμ4 domains of IgM, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:11.

[0079] In some embodiments, the Fc domain comprises the amino acid sequence of the Cμ3 and Cμ4 and tail domain of IgM, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:12.

[0080] In further embodiments, the Fc domain comprises the amino acid sequence of the Cμ2, Cμ3, and Cμ4 domains of IgM, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0081] In still further embodiments, the Fc domain comprises the amino acid sequence of the Cμ2, Cμ3, Cμ4 and tail domain of IgM, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14.

[0082] An ACE2 fusion protein (e.g., a pentameric ACE2 fusion protein) of the present disclosure can further comprise a J chain polypeptide associated with the CH4 tail. In various embodiments, the J chain polypeptide comprises the amino acid sequence of a naturally occurring mature J chain polypeptide or an engineered J chain polypeptide, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the J chain polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, or at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.

[0083] The heavy chain constant domains used in generating the IgM Fc region for the ACE2 fusion proteins of the present disclosure may comprise variants of the above-mentioned naturally occurring constant domains. In one example, the Fc region of the present disclosure comprises at least one constant domain whose sequence differs from that of the wild-type constant domain. It will be understood that the variant constant domains may be longer or shorter than their corresponding wild-type constant domains.

[0084] Linker In certain embodiments, the present disclosure provides ACE2 fusion proteins in which two or more components are connected to each other by a peptide linker. By way of example and not limitation, a linker can be used to connect an ACE2 moiety to a multimerization moiety.

[0085] Peptide linkers can range from 2 to 60 or more amino acids, and in certain embodiments, peptide linkers range from 3 to 50 amino acids in length, 4 to 30 amino acids in length, 5 to 25 amino acids in length, 10 to 25 amino acids in length, 10 to 60 amino acids in length, 12 to 20 amino acids in length, 20 to 50 amino acids in length, or 25 to 35 amino acids in length.

[0086] In certain embodiments, the peptide linker is at least 5, at least 6, or at least 7 amino acids in length, and optionally up to 30, up to 40, up to 50, or up to 60 amino acids in length.

[0087] In some of the aforementioned embodiments, the linker is between 5 and 50 amino acids in length, e.g., between 5 and 50, 5 and 45, 5 and 40, 5 and 35, 5 and 30, 5 and 25, or 5 and 20 amino acids in length. In other of the aforementioned embodiments, the linker is between 6 and 50 amino acids in length, e.g., between 6 and 50, 6 and 45, 6 and 40, 6 and 35, 6 and 30, 6 and 25, or 6 and 20 amino acids in length. In still other of the aforementioned embodiments, the linker is between 7 and 50 amino acids in length, e.g., between 7 and 50, 7 and 45, 7 and 40, 7 and 35, 7 and 30, 7 and 25, or 7 and 20 amino acids in length.

[0088] In some embodiments, the linker is a G4S linker (SEQ ID NO: 31). In some embodiments, the linker comprises two consecutive G4S sequences (SEQ ID NO: 32), three consecutive G4S sequences (SEQ ID NO: 33), four consecutive G4S sequences (SEQ ID NO: 34), five consecutive G4S sequences (SEQ ID NO: 35), or six consecutive G4S sequences (SEQ ID NO: 36).

[0089] 6.6. Nucleic Acids and Host Cells In another aspect, the present disclosure provides a nucleic acid encoding the ACE2 fusion protein of the present disclosure. In some embodiments, the ACE2 fusion protein is encoded by a single nucleic acid. In other embodiments, the ACE2 fusion protein can be encoded by multiple (e.g., 2, 3, 4, or more) nucleic acids.

[0090] A single nucleic acid can encode an ACE2 fusion protein antibody comprising a single polypeptide chain, an ACE2 fusion protein comprising two or more polypeptide chains, or a portion of an ACE2 fusion protein comprising more than two polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an ACE2 fusion protein comprising three, four, or more polypeptide chains, or three polypeptide chains of an ACE2 fusion protein comprising four or more polypeptide chains). To control their expression separately, open reading frames encoding two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). Open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory element and separated by an internal ribosome entry site (IRES) sequence, allowing translation into separate polypeptides.

[0091] In some embodiments, an ACE2 fusion protein comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the ACE2 fusion protein can be equal to or less than the number of polypeptide chains in the ACE2 fusion protein (e.g., when more than one polypeptide chain is encoded by a single nucleic acid).

[0092] The nucleic acids of the present disclosure can be DNA or RNA (eg, mRNA). In another aspect, the present disclosure provides host cells and vectors comprising the nucleic acids of the present disclosure. The nucleic acids may be present in a single vector or may be present in separate vectors that are present in the same host cell or in separate host cells, as described in more detail herein below.

[0093] Vectors The present disclosure provides a vector comprising a nucleotide sequence encoding an ACE2 fusion protein or its components described herein, such as one or two of the polypeptide chains of the ACE2 fusion protein. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).

[0094] Numerous vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, eastern equine encephalitis virus, and flaviviruses.

[0095] Additionally, cells that have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers may provide, for example, prototropy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be either directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0096] After the construct containing expression vector or DNA sequence is prepared for expression, the expression vector can be transfected or introduced into suitable host cells.To achieve this, various techniques can be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques.The method and conditions for culturing the obtained transfected cells and recovering the expressed polypeptide are known to those skilled in the art, and can be modified or optimized according to the specific expression vector and mammalian host cell used based on this specification.

[0097] 6.6.2.Cells The present disclosure also provides a host cell comprising a nucleic acid of the present disclosure. In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids described herein.

[0098] In one embodiment, the host cell is genetically engineered using an expression cassette. The term "expression cassette" refers to a nucleotide sequence capable of affecting the expression of a gene in a host compatible with such a sequence. Such a cassette may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in effecting expression, such as an inducible promoter, may also be used.

[0099] The present disclosure also provides host cells comprising the vectors described herein. The cell can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0100] Pharmaceutical Compositions The ACE2 fusion protein of the present disclosure may be in the form of a composition comprising the ACE2 fusion protein and one or more carriers, excipients, and / or diluents. The composition may be formulated for a specific use, such as veterinary use or pharmaceutical use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers used will depend on the intended use of the ACE2 fusion protein and, in the case of therapeutic use, the mode of administration.

[0101] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. This composition can be in any suitable form (depending on the desired method of administration to a patient). Pharmaceutical compositions can be administered to a patient by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local, or topical. The most suitable route for administration in any given case will depend on the particular antibody, the subject, the nature and severity of the disease, and the physical condition of the subject. Typically, pharmaceutical compositions will be administered intravenously or subcutaneously.

[0102] Pharmaceutical compositions can be conveniently presented in unit dosage forms containing a predetermined amount of the ACE2 fusion protein of the present disclosure per dose. The amount of ACE2 fusion protein contained in a unit dose will depend on the disease being treated, as well as other factors well known in the art. Such unit dosages can be in the form of a lyophilized dry powder containing an amount of ACE2 fusion protein suitable for a single administration, or in liquid form. The dry powder unit dosage form can be packaged in a kit with a syringe, a suitable amount of diluent, and / or other components useful for administration. The liquid unit dosage can be conveniently provided in the form of a syringe pre-filled with an amount of ACE2 fusion protein suitable for a single administration.

[0103] Pharmaceutical compositions may also be supplied in bulk, containing an amount of ACE2 fusion protein suitable for multiple administrations. Pharmaceutical compositions may be prepared for storage as lyophilized formulations or aqueous solutions by mixing ACE2 fusion proteins of the desired purity with any pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as "carriers") typically used in the art, i.e., buffers, stabilizers, preservatives, tonicity agents, non-ionic detergents, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to recipients at the dosages and concentrations used.

[0104] Buffering agents help maintain pH in a range close to physiological conditions. They may be present in a wide variety of concentrations, but will typically be present at concentrations ranging from about 2 mM to about 50 mM. Suitable buffering agents for use in the present disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconate buffers (e.g., gluconate-sodium glyconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium glyconate mixtures, etc.), Examples of buffers include oxalic acid buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (e.g., Tris) can be used.

[0105] Preservatives may be added to retard microbial growth and can be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, alkylparabens (e.g., methyl or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Tonicity adjusting agents, sometimes known as "stabilizers," can be added to ensure the isotonicity of the liquid compositions of the present disclosure and include trihydric alcohols, such as polyhydric sugar alcohols (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol). Stabilizers refer to a broad category of excipients that can range in function from bulking agents to additives, and serve to solubilize the therapeutic agent or prevent it from denaturing or adhering to the container wall.Typical stabilizers include polyhydric sugar alcohols (as listed above), amino acids (e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.), organic sugars or sugar alcohols (e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclitols such as inositol), polyethylene glycol, amino acid polymers, sulfur-containing reducing agents (e.g., urea, glutathione, thioctic acid, etc.), and the like. Stabilizers can be selected from the group consisting of: cellulose, cellulose acetate, cellulose acetate copolymer ...

[0106] Non-ionic surfactants or detergents (also known as "wetting agents") may be added to aid in solubilizing the glycoprotein and to protect it from agitation-induced aggregation, allowing the formulation to be exposed to stressful shear surfaces without denaturing the protein. Suitable non-ionic surfactants include polysorbates (e.g., 20, 80), poloxamers (e.g., 184, 188), and Pluronic® polyols. The non-ionic surfactant may be present in a range of about 0.05 mg / mL to about 1.0 mg / mL (e.g., about 0.07 mg / mL to about 0.2 mg / mL).

[0107] Additional miscellaneous excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

[0108] The ACE2 fusion protein of the present disclosure can be formulated as a pharmaceutical composition containing the ACE2 fusion protein, for example, containing one or more pharmaceutically acceptable excipients or carriers.To prepare a pharmaceutical composition or a sterile composition containing the ACE2 fusion protein of the present disclosure, the ACE2 fusion protein preparation can be combined with one or more pharmaceutically acceptable excipients or carriers.

[0109] For example, a formulation of an ACE2 fusion protein can be prepared by mixing the ACE2 fusion protein with a physiologically acceptable carrier, excipient, or stabilizer, e.g., in the form of a lyophilized powder, a slurry, an aqueous solution, a lotion, or a suspension (see, e.g., Hardman et al., 2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.), 1993, Pharmaceutical Dosage Forms: General Medications, Marcel Dekker, NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. al. (eds.), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0110] 6.8. Treatment Indications and Methods The present disclosure provides methods for using and applying the ACE2 fusion proteins of the present disclosure.

[0111] In certain embodiments, the present disclosure provides methods for preventing or treating diseases or conditions involving the interaction between the RBD of a coronavirus and cellular ACE2. The ACE2 fusion proteins and pharmaceutical compositions of the present disclosure can be used to inhibit the interaction between the RBD of a coronavirus and cellular ACE2. In some embodiments, the present disclosure provides methods for inhibiting the interaction between the RBDs of SARS-CoV. In other embodiments, the present disclosure provides methods for inhibiting the interaction between the RBDs of SARS-CoV-2. Accordingly, in some embodiments, the present disclosure provides a method for inhibiting the interaction between the RBD of a coronavirus and cellular ACE2, comprising administering to a subject in need thereof an ACE2 fusion protein pharmaceutical composition described herein.

[0112] In some embodiments, the present disclosure provides methods of administering an ACE2 fusion protein pharmaceutical composition described herein to a subject who has been exposed to a coronavirus but has not been diagnosed with an infection. In other embodiments, the subject is coronavirus-positive but asymptomatic. In yet other embodiments, the subject is coronavirus-positive and pre-symptomatic. In further embodiments, the subject is coronavirus-positive and symptomatic. In other embodiments, the subject has developed COVID-19 or another coronavirus-mediated disease or condition.

[0113] In some embodiments, the present disclosure provides a method of reducing the severity of a coronavirus infection, comprising administering to a subject in need thereof an ACE2 fusion protein pharmaceutical composition described herein.

[0114] In some other embodiments, the present disclosure provides a method of reducing coronavirus viral load, comprising administering to a subject in need thereof an ACE2 fusion protein pharmaceutical composition described herein.

[0115] In a further embodiment, the present disclosure provides a method of preventing disease progression in a subject having a coronavirus infection, comprising administering to a subject in need thereof an ACE2 fusion protein pharmaceutical composition described herein.

[0116] In some embodiments, the present disclosure provides a method of reducing the duration of a coronavirus infection, comprising administering to a subject in need thereof an ACE2 fusion protein pharmaceutical composition described herein.

[0117] In other embodiments, the present disclosure provides a method of reducing the risk of severe disease or death in a subject having a coronavirus infection, comprising administering to a subject in need thereof an ACE2 fusion protein.

[0118] 7. Numbered Embodiments While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure(s). The present disclosure is exemplified by the numbered embodiments set forth below. Unless otherwise specified, any concept, aspect, and / or feature of any of the embodiments described in the above detailed description is applicable mutatis mutandis to any of the numbered embodiments below.

[0119] 1.Formula: 1. An ACE2 fusion protein comprising one or more polypeptide chains having the formula: [A1]-[L1]-[MM]-[L2]-[A2], wherein: (a) [A1] represents the first ACE2 portion; (b) [L1] represents a first linker; (c) [MM] represents a multimerization moiety; (d) [L2] represents a second linker; (e) [A2] represents a second ACE2 portion; An ACE2 fusion protein, wherein [L1] and [L2] are optional, and [A2] is optional.

[0120] 2.[A1] is (a) comprises an amino acid sequence having at least 90%, 95%, or 98% sequence identity to the ACE2 PD (SEQ ID NO: 2) and / or at least 90%, 95%, or 98% sequence identity to the ACE2 PD+ND (SEQ ID NO: 3); (b) comprises at least one amino acid substitution that increases affinity for a coronavirus RBD, e.g., the RBD of SEQ ID NO: 4 and / or SEQ ID NO: 5; (c) contains at least one amino acid substitution at position 25, 27, 31, 34, 42, 79, 90, 92, 324, 325, 330, or 386 of ACE2; (d) contains at least one amino acid substitution shown in Table 1; (e) containing the amino acid substitutions T27Y, L79T, and N330Y; (f) has increased affinity for a coronavirus RBD, e.g., the RBD of SEQ ID NO: 4 and / or SEQ ID NO: 5, optionally wherein the increased affinity is at least 25%, at least 50%, at least 100%, at least 200%, or at least 300% compared to the corresponding sequence of wild-type ACE2 (SEQ ID NO: 1); or (g) An ACE2 fusion protein according to embodiment 1, which is any combination of two, three or more of (a) to (f).

[0121] 3. An ACE2 fusion protein described in embodiment 2, wherein [A1] comprises an amino acid sequence having at least 90%, 95%, or 98% sequence identity to ACE2 PD (SEQ ID NO: 2).

[0122] 4. An ACE2 fusion protein described in embodiment 3, wherein [A1] comprises an amino acid sequence having at least 90% sequence identity to ACE2 PD (sequence number 2). 5. An ACE2 fusion protein described in embodiment 3, wherein [A1] comprises an amino acid sequence having at least 95% sequence identity to ACE2 PD (sequence number 2).

[0123] 6. An ACE2 fusion protein described in embodiment 3, wherein [A1] comprises an amino acid sequence having at least 98% sequence identity to ACE2 PD (sequence number 2). 7. An ACE2 fusion protein described in embodiment 3, wherein [A1] comprises an amino acid sequence having 100% sequence identity to ACE2 PD (sequence number 2).

[0124] 8. An ACE2 fusion protein according to any one of embodiments 3 to 7, wherein [A1] lacks ND. 9. An ACE2 fusion protein according to any one of embodiments 3 to 7, wherein [A1] comprises ND.

[0125] 10. An ACE2 fusion protein as described in embodiment 9, wherein [A1] comprises an amino acid sequence having at least 90%, 95%, or 98% sequence identity to ACE2 PD+ND (SEQ ID NO: 3).

[0126] 11. An ACE2 fusion protein described in embodiment 10, wherein [A1] comprises an amino acid sequence having at least 90% sequence identity to ACE2 PD+ND (sequence number 3).

[0127] 12. An ACE2 fusion protein described in embodiment 10, wherein [A1] comprises an amino acid sequence having at least 95% sequence identity to ACE2 PD+ND (sequence number 3).

[0128] 13. An ACE2 fusion protein according to embodiment 10, wherein [A1] comprises an amino acid sequence having at least 98% sequence identity to ACE2 PD+ND (SEQ ID NO: 3).

[0129] 14. An ACE2 fusion protein described in embodiment 10, wherein [A1] comprises an amino acid sequence having 100% sequence identity to ACE2 PD+ND (sequence number 3). 15. An ACE2 fusion protein according to any one of embodiments 2 to 14, wherein [A1] comprises at least one amino acid substitution that increases affinity for coronavirus RBD, e.g., the RBD of SEQ ID NO: 4 and / or SEQ ID NO: 5.

[0130] 16. An ACE2 fusion protein according to any one of embodiments 2 to 15, wherein [A1] comprises at least one amino acid substitution at position 25, 27, 31, 34, 42, 79, 90, 92, 324, 325, 330, or 386 of ACE2.

[0131] 17. An ACE2 fusion protein according to any one of embodiments 2 to 16, wherein [A1] comprises at least one amino acid substitution as shown in Table 1. 18. An ACE2 fusion protein according to any one of embodiments 2 to 17, wherein [A1] comprises the amino acid substitutions T27Y, L79T, and N330Y.

[0132] 19. An ACE2 fusion protein according to any one of embodiments 2 to 18, wherein [A1] has increased affinity for a coronavirus RBD, e.g., the RBD of SEQ ID NO: 4 and / or SEQ ID NO: 5, and optionally the increased affinity is at least 25%, at least 50%, at least 100%, at least 200%, or at least 300%, compared to the corresponding sequence of wild-type ACE2 (SEQ ID NO: 1).

[0133] 20. An ACE2 fusion protein according to any one of embodiments 2 to 18, wherein the increase in affinity is at least 25% compared to the corresponding sequence of wild-type ACE2 (SEQ ID NO: 1).

[0134] 21. An ACE2 fusion protein according to any one of embodiments 2 to 18, wherein the increase in affinity is at least 50% compared to the corresponding sequence of wild-type ACE2 (SEQ ID NO: 1).

[0135] 22. An ACE2 fusion protein according to any one of embodiments 2 to 18, wherein the increase in affinity is at least 100% compared to the corresponding sequence of wild-type ACE2 (SEQ ID NO: 1).

[0136] 23. An ACE2 fusion protein according to any one of embodiments 2 to 18, wherein the increase in affinity is at least 200% compared to the corresponding sequence of wild-type ACE2 (SEQ ID NO: 1).

[0137] 24. An ACE2 fusion protein according to any one of embodiments 2 to 18, wherein the increase in affinity is at least 300% compared to the corresponding sequence of wild-type ACE2 (SEQ ID NO: 1).

[0138] 25. An ACE2 fusion protein according to any one of embodiments 1 to 24, in which [A2] is absent. 26. An ACE2 fusion protein according to any one of embodiments 1 to 25, wherein [L1] is absent.

[0139] 27. An ACE2 fusion protein according to any one of embodiments 1 to 25, wherein [L1] is present. 28. An ACE2 fusion protein according to embodiment 27, wherein [L1] is 5 to 35 amino acids in length.

[0140] 29. An ACE2 fusion protein according to any one of embodiments 1 to 28, wherein [MM] comprises an Fc domain. 30. The ACE2 fusion protein of embodiment 29, wherein the Fc domain is an IgM Fc domain.

[0141] 31. An ACE2 fusion protein according to embodiment 30, wherein the Fc domain comprises a Cμ3 domain and a Cμ4 domain. 32. An ACE2 fusion protein according to embodiment 30 or embodiment 31, wherein the Fc domain comprises a Cμ2 domain.

[0142] 33. An ACE2 fusion protein according to any one of embodiments 30 to 32, which is a pentamer. 34. An ACE2 fusion protein according to embodiment 33, which is a pentamer of five dimers, each dimer comprising two polypeptides, each polypeptide comprising an ACE2 domain (as [A1]), an optional linker (as [L1]), and an IgM Fc domain (as [MM]).

[0143] 35. The ACE2 fusion protein of embodiment 33 or embodiment 34, which is a homopentamer. 36. An ACE2 fusion protein according to any one of embodiments 33 to 35, wherein some or all of the Cμ3 and / or Cμ4 domains are disulfide-linked.

[0144] 37. An ACE2 fusion protein according to any one of embodiments 33 to 36, comprising a J chain. 38. An ACE2 fusion protein according to any one of embodiments 30 to 37, which is decavalent for ACE2.

[0145] 39. An ACE2 fusion protein, optionally an ACE2 fusion protein according to any one of embodiments 1 to 38, having the configuration depicted in FIG. 2A. 40. An ACE2 fusion protein, optionally an ACE2 fusion protein according to any one of embodiments 1 to 38, having the configuration depicted in Figure 2B.

[0146] 41. An ACE2 fusion protein according to any one of embodiments 30 to 32, which is a hexamer. 42. The ACE2 fusion protein of embodiment 41, which is a hexamer of six dimers, each dimer comprising two polypeptides, each polypeptide comprising an ACE2 domain (as [A1]), an optional linker (as [L1]), and an IgM Fc domain (as [MM]).

[0147] 43. The ACE2 fusion protein of embodiment 41 or embodiment 42, which is a homohexamer. 44. An ACE2 fusion protein according to any one of embodiments 41 to 43, wherein some or all of the Cμ3 and / or Cμ4 domains are disulfide-linked.

[0148] 45. An ACE2 fusion protein according to any one of embodiments 41 to 44, which lacks a J chain. 46. ​​The ACE2 fusion protein of any one of embodiments 1 to 45, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 23, optionally wherein the amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23.

[0149] 47. The ACE2 fusion protein of embodiment 46, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 23. 48. The ACE2 fusion protein of embodiment 46, comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 23.

[0150] 49. The ACE2 fusion protein of embodiment 46, comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 23. 50. The ACE2 fusion protein of embodiment 46, comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 23.

[0151] 51. The ACE2 fusion protein of embodiment 46, comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 23. 52. The ACE2 fusion protein of embodiment 46, comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 23.

[0152] 53. The ACE2 fusion protein of embodiment 46, comprising an amino acid sequence having 100% sequence identity to SEQ ID NO: 23. 54. The ACE2 fusion protein of any one of embodiments 1 to 45, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 24, optionally wherein the amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24.

[0153] 55. The ACE2 fusion protein of embodiment 54, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24. 56. The ACE2 fusion protein of embodiment 54, comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 24.

[0154] 57. The ACE2 fusion protein of embodiment 54, comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 24. 58. The ACE2 fusion protein of embodiment 54, comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 24.

[0155] 59. The ACE2 fusion protein of embodiment 54, comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 24. 60. The ACE2 fusion protein of embodiment 54, comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 24.

[0156] 61. The ACE2 fusion protein of embodiment 54, comprising an amino acid sequence having 100% sequence identity to SEQ ID NO: 24. 62. The ACE2 fusion protein of any one of embodiments 1 to 45, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 25, optionally wherein the amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25.

[0157] 63. The ACE2 fusion protein of embodiment 62, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 25. 64. The ACE2 fusion protein of embodiment 62, comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 25.

[0158] 65. The ACE2 fusion protein of embodiment 62, comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 25. 66. The ACE2 fusion protein of embodiment 62, comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 25.

[0159] 67. The ACE2 fusion protein of embodiment 62, comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 25. 68. The ACE2 fusion protein of embodiment 62, comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 25.

[0160] 69. The ACE2 fusion protein of embodiment 62, comprising an amino acid sequence having 100% sequence identity to SEQ ID NO: 25. 70. The ACE2 fusion protein of any one of embodiments 1 to 45, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 26, optionally wherein the amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26.

[0161] 71. The ACE2 fusion protein of embodiment 70, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26. 72. The ACE2 fusion protein of embodiment 70, comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 26.

[0162] 73. The ACE2 fusion protein of embodiment 70, comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 26. 74. The ACE2 fusion protein of embodiment 70, comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 26.

[0163] 75. The ACE2 fusion protein of embodiment 70, comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 26. 76. The ACE2 fusion protein of embodiment 70, comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 26.

[0164] 77. The ACE2 fusion protein of embodiment 70, comprising an amino acid sequence having 100% sequence identity to SEQ ID NO: 26. 78. The ACE2 fusion protein of any one of embodiments 1 to 45, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 27, optionally wherein the amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27.

[0165] 79. The ACE2 fusion protein of embodiment 78, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 27. 80. The ACE2 fusion protein of embodiment 78, comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 27.

[0166] 81. The ACE2 fusion protein of embodiment 78, comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 27. 82. The ACE2 fusion protein of embodiment 78, comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 27.

[0167] 83. The ACE2 fusion protein of embodiment 78, comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 27. 84. The ACE2 fusion protein of embodiment 78, comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 27.

[0168] 85. The ACE2 fusion protein of embodiment 78, comprising an amino acid sequence having 100% sequence identity to SEQ ID NO: 27. 86. The ACE2 fusion protein of any one of embodiments 1 to 45, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 28, optionally wherein the amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0169] 87. The ACE2 fusion protein of embodiment 86, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28. 88. The ACE2 fusion protein of embodiment 86, comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 28.

[0170] 89. The ACE2 fusion protein of embodiment 86, comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 28. 90. The ACE2 fusion protein of embodiment 86, comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 28.

[0171] 91. The ACE2 fusion protein of embodiment 86, comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 28. 92. The ACE2 fusion protein of embodiment 86, comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 28.

[0172] 93. The ACE2 fusion protein of embodiment 86, comprising an amino acid sequence having 100% sequence identity to SEQ ID NO: 28. 94. The ACE2 fusion protein of any one of embodiments 1 to 45, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 29, optionally wherein the amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29.

[0173] 95. The ACE2 fusion protein of embodiment 94, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29. 96. The ACE2 fusion protein of embodiment 94, comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 29.

[0174] 97. The ACE2 fusion protein of embodiment 94, comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 29. 98. The ACE2 fusion protein of embodiment 94, comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 29.

[0175] 99. The ACE2 fusion protein of embodiment 94, comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 29. 100. The ACE2 fusion protein of embodiment 94, comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 29.

[0176] 101. The ACE2 fusion protein of embodiment 94, comprising an amino acid sequence having 100% sequence identity to SEQ ID NO: 29. 102. The ACE2 fusion protein of any one of embodiments 1 to 45, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 30, optionally wherein the amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30.

[0177] 103. The ACE2 fusion protein of embodiment 102, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 30. 104. The ACE2 fusion protein of embodiment 102, comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 30.

[0178] 105. The ACE2 fusion protein of embodiment 102, comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 30. 106. The ACE2 fusion protein of embodiment 102, comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 30.

[0179] 107. The ACE2 fusion protein of embodiment 102, comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 30. 108. The ACE2 fusion protein of embodiment 102, comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 30.

[0180] 109. The ACE2 fusion protein of embodiment 102, comprising an amino acid sequence having 100% sequence identity to SEQ ID NO: 30. 110. A nucleic acid or nucleic acids encoding an ACE2 fusion protein according to any one of embodiments 1 to 109.

[0181] 111. The nucleic acid of embodiment 110, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 15, and optionally, the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15.

[0182] 112. The nucleic acid of embodiment 111, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 15. 113. The nucleic acid of embodiment 111, comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 15.

[0183] 114. The nucleic acid of embodiment 111, comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15. 115. The nucleic acid of embodiment 111, comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 15.

[0184] 116. The nucleic acid of embodiment 111, comprising a nucleotide sequence having at least 96% sequence identity to SEQ ID NO: 15. 117. The nucleic acid of embodiment 111, comprising a nucleotide sequence having at least 97% sequence identity to SEQ ID NO: 15.

[0185] 118. The nucleic acid of embodiment 111, comprising a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 15. 119. The nucleic acid of embodiment 111, comprising a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 15.

[0186] 120. The nucleic acid of embodiment 111, comprising a nucleotide sequence having 100% sequence identity to SEQ ID NO: 15. 121. The nucleic acid of embodiment 110, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 16, and optionally, the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16.

[0187] 122. The nucleic acid of embodiment 121, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 16. 123. The nucleic acid of embodiment 121, comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 16.

[0188] 124. The nucleic acid of embodiment 121, comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 16. 125. The nucleic acid of embodiment 121, comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 16.

[0189] 126. The nucleic acid of embodiment 121, comprising a nucleotide sequence having at least 96% sequence identity to SEQ ID NO: 16. 127. The nucleic acid of embodiment 121, comprising a nucleotide sequence having at least 97% sequence identity to SEQ ID NO: 16.

[0190] 128. The nucleic acid of embodiment 121, comprising a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 16. 129. The nucleic acid of embodiment 121, comprising a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 16.

[0191] 130. The nucleic acid of embodiment 121, comprising a nucleotide sequence having 100% sequence identity to SEQ ID NO: 16. 131. The nucleic acid of embodiment 110, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 17, and optionally, the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17.

[0192] 132. The nucleic acid of embodiment 131, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 17. 133. The nucleic acid of embodiment 131, comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 17.

[0193] 134. The nucleic acid of embodiment 131, comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 17. 135. The nucleic acid of embodiment 131, comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 17.

[0194] 136. The nucleic acid of embodiment 131, comprising a nucleotide sequence having at least 96% sequence identity to SEQ ID NO: 17. 137. The nucleic acid of embodiment 131, comprising a nucleotide sequence having at least 97% sequence identity to SEQ ID NO: 17.

[0195] 138. The nucleic acid of embodiment 131, comprising a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 17. 139. The nucleic acid of embodiment 131, comprising a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 17.

[0196] 140. The nucleic acid of embodiment 131, comprising a nucleotide sequence having 100% sequence identity to SEQ ID NO: 17. 141. The nucleic acid of embodiment 110, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 18, and optionally, the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18.

[0197] 142. The nucleic acid of embodiment 141, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 18. 143. The nucleic acid of embodiment 141, comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 18.

[0198] 144. The nucleic acid of embodiment 141, comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 18. 145. The nucleic acid of embodiment 141, comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 18.

[0199] 146. The nucleic acid of embodiment 141, comprising a nucleotide sequence having at least 96% sequence identity to SEQ ID NO: 18. 147. The nucleic acid of embodiment 141, comprising a nucleotide sequence having at least 97% sequence identity to SEQ ID NO: 18.

[0200] 148. The nucleic acid of embodiment 141, comprising a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 18. 149. The nucleic acid of embodiment 141, comprising a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 18.

[0201] 150. The nucleic acid of embodiment 141, comprising a nucleotide sequence having 100% sequence identity to SEQ ID NO: 18. 151. The nucleic acid of embodiment 110, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 19, and optionally, the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.

[0202] 152. The nucleic acid of embodiment 151, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 19. 153. The nucleic acid of embodiment 151, comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 19.

[0203] 154. The nucleic acid of embodiment 151, comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 19. 155. The nucleic acid of embodiment 151, comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 19.

[0204] 156. The nucleic acid of embodiment 151, comprising a nucleotide sequence having at least 96% sequence identity to SEQ ID NO: 19. 157. The nucleic acid of embodiment 151, comprising a nucleotide sequence having at least 97% sequence identity to SEQ ID NO: 19.

[0205] 158. The nucleic acid of embodiment 151, comprising a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 19. 159. The nucleic acid of embodiment 151, comprising a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 19.

[0206] 160. The nucleic acid of embodiment 151, comprising a nucleotide sequence having 100% sequence identity to SEQ ID NO: 19. 161. The nucleic acid of embodiment 110, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:20, and optionally, the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:20.

[0207] 162. The nucleic acid of embodiment 161, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 20. 163. The nucleic acid of embodiment 161, comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 20.

[0208] 164. The nucleic acid of embodiment 161, comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 20. 165. The nucleic acid of embodiment 161, comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 20.

[0209] 166. The nucleic acid of embodiment 161, comprising a nucleotide sequence having at least 96% sequence identity to SEQ ID NO: 20. 167. The nucleic acid of embodiment 161, comprising a nucleotide sequence having at least 97% sequence identity to SEQ ID NO: 20.

[0210] 168. The nucleic acid of embodiment 161, comprising a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 20. 169. The nucleic acid of embodiment 161, comprising a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 20.

[0211] 170. The nucleic acid of embodiment 161, comprising a nucleotide sequence having 100% sequence identity to SEQ ID NO: 20. 171. The nucleic acid of embodiment 110, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:21, and optionally, the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:21.

[0212] 172. The nucleic acid of embodiment 171, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 21. 173. The nucleic acid of embodiment 171, comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 21.

[0213] 174. The nucleic acid of embodiment 171, comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 21. 175. The nucleic acid of embodiment 171, comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 21.

[0214] 176. The nucleic acid of embodiment 171, comprising a nucleotide sequence having at least 96% sequence identity to SEQ ID NO: 21. 177. The nucleic acid of embodiment 171, comprising a nucleotide sequence having at least 97% sequence identity to SEQ ID NO: 21.

[0215] 178. The nucleic acid of embodiment 171, comprising a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 21. 179. The nucleic acid of embodiment 171, comprising a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 21.

[0216] 180. The nucleic acid of embodiment 171, comprising a nucleotide sequence having 100% sequence identity to SEQ ID NO: 21. 181. The nucleic acid of embodiment 110, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 22, optionally wherein the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22.

[0217] 182. The nucleic acid of embodiment 181, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 22. 183. The nucleic acid of embodiment 181, comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 22.

[0218] 184. The nucleic acid of embodiment 181, comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 22. 185. The nucleic acid of embodiment 181, comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 22.

[0219] 186. The nucleic acid of embodiment 181, comprising a nucleotide sequence having at least 96% sequence identity to SEQ ID NO: 22. 187. The nucleic acid of embodiment 181, comprising a nucleotide sequence having at least 97% sequence identity to SEQ ID NO: 22.

[0220] 188. The nucleic acid of embodiment 181, comprising a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 22. 189. The nucleic acid of embodiment 181, comprising a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 22.

[0221] 190. The nucleic acid of embodiment 181, comprising a nucleotide sequence having 100% sequence identity to SEQ ID NO: 22. 191. A host cell engineered to express an ACE2 fusion protein according to any one of embodiments 1 to 109 or a nucleic acid(s) according to any one of embodiments 110 to 190.

[0222] 192. A method for producing an ACE2 fusion protein described in any one of embodiments 1 to 109, comprising culturing a host cell described in embodiment 191 and recovering the ACE2 fusion protein expressed thereby.

[0223] 193. A pharmaceutical composition comprising an ACE2 fusion protein according to any one of embodiments 1 to 109 and an excipient. 194. A method for treating a coronavirus disease, comprising administering to a subject in need thereof an ACE2 fusion protein described in any one of embodiments 1 to 109 or a pharmaceutical composition described in embodiment 193.

[0224] 195. A method for inhibiting the interaction between coronavirus RBD and cellular ACE2, comprising administering to a subject in need thereof an ACE2 fusion protein described in any one of embodiments 1 to 109 or a pharmaceutical composition described in embodiment 193.

[0225] 196. A method for reducing the severity of a coronavirus infection, comprising administering to a subject in need thereof an ACE2 fusion protein described in any one of embodiments 1 to 109 or a pharmaceutical composition described in embodiment 193.

[0226] 197. A method for reducing the viral load of coronavirus, comprising administering to a subject in need thereof an ACE2 fusion protein described in any one of embodiments 1 to 109 or a pharmaceutical composition described in embodiment 193.

[0227] 198. A method for preventing disease progression in a subject with a coronavirus infection, comprising administering to a subject in need thereof an ACE2 fusion protein described in any one of embodiments 1 to 109 or a pharmaceutical composition described in embodiment 193.

[0228] 199. A method for reducing the duration of a coronavirus infection, comprising administering to a subject in need thereof an ACE2 fusion protein described in any one of embodiments 1 to 109 or a pharmaceutical composition described in embodiment 193.

[0229] 200. A method for reducing the risk of severe disease or death in a subject with a coronavirus infection, comprising administering to a subject in need thereof an ACE2 fusion protein described in any one of embodiments 1 to 109 or a pharmaceutical composition described in embodiment 193.

[0230] 201. The method of any one of embodiments 194 to 200, wherein the coronavirus is SARS-CoV. 202. The method of any one of embodiments 194 to 200, wherein the coronavirus is SARS-CoV-2. [Example]

[0231] 8. Working Example 8.1. Materials and Methods 8.1.1. Design, Construction, and Production of ACE2 Fusion Proteins Exemplary decavalent ACE2 fusion proteins were designed as DNA fragments containing the following components from the 5' to 3' end: the ACE2 ectodomain (amino acids 1-615 or 1-740), an optional IgM Cμ2 domain, an IgM Cμ3 domain, an IgM Cμ4 domain, and a tail. The DNA fragments were then synthesized and cloned into the mammalian expression vector pcDNA3.4. For comparison, affinity-matured versions of the decavalent ACE2 fusion proteins (e.g., ACE2 v2.4(615)-Fc(IgM)) were created using the reported v2.4 mutations (T27Y, L79T, N330Y).

[0232] The DNA and amino acid sequences of exemplary ACE2 fusion proteins are shown in Tables 3 and 4, respectively.

[0233] [Table 3-1]

[0234] [Table 3-2]

[0235] [Table 3-3]

[0236] [Table 3-4]

[0237] [Table 3-5]

[0238] [Table 3-6]

[0239] [Table 3-7]

[0240] Table 3-8

[0241] Table 3-9

[0242] Table 3-10

[0243] Table 3-11

[0244] Table 3-12

[0245] Table 4-1

[0246] Table 4-2

[0247] Table 4-3

[0248] Table 4-4

[0249] Table 4-5

[0250] [Table 4-6]

[0251] Expression plasmids containing the constructs of interest were used to transiently transfect FreeStyle™ 293-F cells (ThermoFisher) according to the manufacturer's protocol. After 6 days, the culture supernatants were harvested, centrifuged at 3900 rpm for 15 minutes at 4°C, and filtered through a 0.2 μm filter for further purification. Further details about the decavalent constructs are shown in Table 5.

[0252] [Table 5]

[0253] 8.1.2. Purification and Quality Assessment Fusion protein isolation from the supernatant was performed using 1 mL of POROS CaptureSelect IgM Affinity Matrix (ThermoFisher). First, the column was equilibrated with 5 column volumes (CV) of PBS. The sterile-filtered supernatant containing the fusion protein was then loaded onto the pre-equilibrated column at a flow rate of approximately 2.0 mL / min. Any nonspecifically bound material was washed off the column using 50 mM Tris-HCl, 500 mM NaCl, pH 7.5 at a flow rate of 2.0 mL / min for 5 CV. The affinity-bound fusion protein was eluted from the column using Pierce™ IgG Elution Buffer (pH 2.8, ThermoFisher) at a flow rate of 0.5 mL / min for 5 CV. After elution, the protein was neutralized using 1 / 10 (v / v) of 1 M Tris-HCl (pH 8.0) and dialyzed into a final buffer of phosphate-buffered saline (PBS) containing 5% glycerol. Furthermore, in order to detect a lower percentage of J chain by mass for detection by Coomassie staining, samples were run on a 4-20% Tris-Glycine gel loaded with 10 μg of sample per well at a constant voltage of 200 V for 1 hour.

[0254] Protein samples were evaluated by UV-Vis and their protein concentrations were determined using a Labchip Dropsense instrument. The fractions were further analyzed by SE-UPLC to determine the presence of high- or low-molecular-weight species relative to the target species. The size-exclusion chromatography (SEC) column used was an Acquity BEH, 200 Å, 1.7 μm, 4.6 × 150 mm column (Waters) at a flow rate of 0.3 mL / min in 1 × DPBS, 0.5 M NaCl, pH 7.1.

[0255] Proteins isolated from each fraction pool were analyzed using SDS-PAGE under denaturing conditions. Samples were run on a 4-20% Tris-Glycine gel loaded with 2 μg of sample per well at a constant voltage of 200 V for 1 hour.

[0256] 8.1.3. SARS-CoV-2 Pseudovirus Neutralization Assay Vero cells were cultured in glutamine-free DMEM high-glucose medium containing sodium pyruvate, supplemented with 10% heat-inactivated FBS and penicillin / streptomycin / L-glutamine at 37°C in 5% CO2 (complete DMEM) and seeded at 20,000 cells / well in 96-well black / clear-bottom cell culture plates. On the day of the assay, test articles (antibodies and proteins) were diluted to 2x assay concentration and serially diluted 3x to obtain a total of 11 concentrations (e.g., 40 nM to 677.4 fM for all except ACE2-IgM-8, 9, and 10, which were diluted to 20 nM to 338.7 fM due to their low starting concentrations). All dilutions were performed using infection medium consisting of glutamine-free DMEM high-glucose medium containing sodium pyruvate supplemented with sodium pyruvate, 0.2% IgG-free BSA, and gentamicin.

[0257] The pVSV-Luc-SARS-CoV-2-S pseudovirus used herein is a non-replicating VSV-DG, expressing a dual GFP / firefly luciferase reporter in place of its native glycoprotein and pseudotyped with a SARS-CoV-2 spike. The pseudovirus was diluted 1:4 in infection medium and then combined 1:1 with the test article dilutions to achieve a final pseudovirus dilution of 1:8, resulting in final test article concentrations ranging from 20 nM to 338.7 fM (for all except ACE2-IgM-8, 9, and 10, where final concentrations ranged from 10 nM to 169.4 fM). Wells containing no test article (virus control) or no pseudovirus (media control) served as controls. The combined test article and pseudovirus were incubated at room temperature for 30 minutes. The culture medium was then removed from the cells, and the combined test article and pseudovirus were added to the wells in duplicate at 100 μL / well, followed by incubation at 37°C, 5% CO2 for 24 hours. At 24 hours post-infection, the medium was removed from the wells, and the cells were lysed using 100 μL / well of Glo-Lysis buffer (Promega). Immediately before reading luminescence on a Spectramax i3X plate reader, 100 μL of prepared Bright-Glo substrate (Promega) was added to the lysate. The results were exported to Microsoft Excel, and the % neutralization was calculated using the following equation: % neutralization = ((1 - (well value - medium control) / (virus control - medium control)) × 100. The % neutralization was then plotted in GraphPad Prism and analyzed using a nonlinear regression of response: log (inhibitor) - variable slope (4 parameters) to calculate IC50 values.

[0258] 8.2. Example 1: Generation of Multivalent Fusion Proteins The extracellular portion of the ACE2 protein consists of two major domains: a peptidase domain corresponding to amino acids 18–615 from the N-terminus, referred to herein as ACE2-PD or ACE2(615), and a portion of the collectrin-like domain (CLD), referred to herein as ACE2-ND, corresponding to amino acids 616–740 from the N-terminus (Figures 1A and 1B). The PD and ND domains, together corresponding to amino acids 18–740 from the N-terminus, are referred to herein as ACE2(740). The peptide-binding cavity on the outer surface of ACE2-PD is directly involved in RBD binding, while ACE2-ND is important for ACE2 dimerization. Different ACE2 ectodomain constructs were devised herein by containing either ACE2(615) or ACE2(740) connected to IgM Cμ2 (Figure 2A) or IgM Cμ3 (Figure 2B).

[0259] Multivalent fusion proteins were designed and prepared as described in Section 8.1.1. Transfected cells successfully expressed the multivalent fusion proteins contained in the transfected vectors. SDS-PAGE analysis of culture medium samples taken from transfected cells shows that expression levels of native ACE2-IgM-Fc or the affinity-enhanced version of the ACE2 v2.4-IgM-Fc construct were comparable (Figure 3).

[0260] 8.3. Example 2: SEC Profile of ACE2-Fc(IgM) Fusion Constructs Four representative Fc(IgM) fusion constructs: ACE2(615)-Fc 2,3,4 (IgM) (Fig. 4A), ACE2(615)-Fc 3,4 (IgM) (Fig. 4B), ACE2(740)-Fc 2,3,4 (IgM) (Fig. 4C), and ACE2(740)-Fc 3,4 The size-exclusion chromatography (SEC) profiles of ACE2-Fc(IgM) (Figure 4D) were determined as described in Section 8.1.2. All ACE2-Fc(IgM) constructs showed a major peak accompanied by several smaller peaks.

[0261] 8.4. Example 3: Neutralizing Activity of Multivalent ACE2-Fc Constructs Against SARS-CoV-2 Variants and Pseudoviruses Cell culture and virus neutralization assays were performed using Fc ligated to ACE2(615), ACE2 v2.4(615), ACE2(740), or ACE2 v2.4(740). 2,3,4 (IgM) (Figures 5A-5C) or Fc 3,4 (IgM) (Figures 5D-5F) were used as described in Section 8.1.3. 2,3,4 (IgM) and Fc 3,4 Both ACE2-Fc (IgM) constructs neutralized pseudovirus (Figures 5A and 5D), SARS-CoV2 variants BA.1 (Figures 5B and 5E), and BA.2 (Figures 5C and 5F). 3,4 (IgM) fusion constructs are generally 2,3,4 Among the tested SARS-CoV2 pseudovirus variants D614G, BA.1, and BA.2, most ACE2-IgM-Fc molecules exhibited neutralizing activity similar to that of bivalent ACE2(740)-IgG-Fc. However, in D614G, ACE2(615) and (740)-Fc were associated with better neutralization. 3,4 (IgM) fusions had 5- to 10-fold higher neutralizing potency than bivalent ACE2-IgG-Fc fusions ( Figure 5D ).

[0262] In summary, multivalent soluble ACE2-IgM-Fc fusions are a novel antiviral strategy that exploits alternative high-level multimerization of the soluble SARS-CoV-2 cell entry receptor ACE2 using a native human IgM scaffold. This approach is also widely utilized in areas where multivalency beyond 2–4 copies is desired for the development of other antiviral therapies, receptor clustering for activation, or target blockade with enhanced efficacy.

[0263] 9. Citation of References All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. In the event of a conflict between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present disclosure are intended.

Claims

1. 1. An ACE2 fusion protein that is a pentamer of five dimers and that includes a J chain, wherein each dimer has the formula: [A1]-[L1]-[MM], wherein: (a) [A1] represents a first ACE2 moiety; (b) [L1] represents an optional first linker; (c) ACE2 fusion protein, where [MM] represents a multimerization moiety containing an IgM Fc domain.

2. [A1] is (a) comprises an amino acid sequence having at least 90%, 95%, or 98% sequence identity to ACE2 PD (SEQ ID NO: 2) and / or at least 90%, 95%, or 98% sequence identity to ACE2 PD+ND (SEQ ID NO: 3); (b) comprises at least one amino acid substitution that increases affinity for a coronavirus RBD, e.g., the RBD of SEQ ID NO: 4 and / or SEQ ID NO: 5; (c) contains at least one amino acid substitution at position 25, 27, 31, 34, 42, 79, 90, 92, 324, 325, 330, or 386 of ACE2; (d) contains at least one amino acid substitution shown in Table 1; (e) containing the amino acid substitutions T27Y, L79T, and N330Y; (f) has increased affinity for a coronavirus RBD, e.g., the RBD of SEQ ID NO:4 and / or SEQ ID NO:5, optionally wherein the increased affinity is at least 25%, at least 50%, at least 100%, at least 200%, or at least 300% compared to the corresponding sequence of wild-type ACE2 (SEQ ID NO:1); or (g) The ACE2 fusion protein of claim 1, which is any combination of two, three, or more of (a) to (f).

3. 3. The ACE2 fusion protein of claim 2, wherein [A1] comprises an amino acid sequence having at least 90%, 95%, or 98% sequence identity to ACE2 PD (SEQ ID NO: 2).

4. The ACE2 fusion protein of claim 3, wherein [A1] lacks ND.

5. The ACE2 fusion protein of claim 3, wherein [A1] comprises ND.

6. The ACE2 fusion protein of claim 5, wherein [A1] comprises an amino acid sequence having at least 90%, 95%, or 98% sequence identity to ACE2 PD+ND (SEQ ID NO: 3).

7. ACE2 fusion protein according to any one of claims 2 to 6, wherein [A1] comprises at least one amino acid substitution that increases affinity for a coronavirus RBD, for example, the RBD of SEQ ID NO: 4 and / or SEQ ID NO:

5.

8. 8. The ACE2 fusion protein according to any one of claims 2 to 7, wherein [A1] comprises at least one amino acid substitution at position 25, 27, 31, 34, 42, 79, 90, 92, 324, 325, 330, or 386 of ACE2.

9. 9. The ACE2 fusion protein according to any one of claims 2 to 8, wherein [A1] contains at least one amino acid substitution shown in Table 1.

10. 10. The ACE2 fusion protein according to any one of claims 2 to 9, wherein [A1] comprises the amino acid substitutions T27Y, L79T, and N330Y.

11. 11. The ACE2 fusion protein of any one of claims 2 to 10, wherein [A1] has increased affinity for a coronavirus RBD, for example the RBD of SEQ ID NO: 4 and / or SEQ ID NO: 5, and optionally the increased affinity is at least 25%, at least 50%, at least 100%, at least 200%, or at least 300% compared to the corresponding sequence of wild-type ACE2 (SEQ ID NO: 1).

12. ACE2 fusion protein according to any one of claims 1 to 11, wherein [L1] is absent.

13. The ACE2 fusion protein according to any one of claims 1 to 11, wherein [L1] is present.

14. 14. The ACE2 fusion protein according to any one of claims 1 to 13, wherein [L1] is 5 to 35 amino acids in length.

15. ACE2 fusion protein according to any one of claims 1 to 14, wherein the IgM Fc domain comprises a Cμ3 domain and a Cμ4 domain.

16. 16. The ACE2 fusion protein of claim 15, wherein the IgM Fc domain comprises a Cμ2 domain.

17. 17. The ACE2 fusion protein of claim 15 or 16, which is a homopentamer.

18. ACE2 fusion protein according to any one of claims 15 to 17, wherein some or all of the Cμ3 and / or Cμ4 domains are disulfide-linked.

19. ACE2 fusion protein according to any one of claims 1 to 18, which is decavalent for ACE2.

20. 20. The ACE2 fusion protein according to any one of claims 1 to 19, which is decavalent with respect to the ACE2 moiety.

21. ACE2 fusion protein according to any one of claims 1 to 20, wherein the dimers are connected via the J chain.

22. 22. The ACE2 fusion protein of any one of claims 1 to 21, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:23, optionally wherein said amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

23.

23. 22. The ACE2 fusion protein of any one of claims 1 to 21, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:24, optionally wherein said amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

24.

24. 22. The ACE2 fusion protein of any one of claims 1 to 21, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:25, optionally wherein said amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

25.

25. 22. The ACE2 fusion protein of any one of claims 1 to 21, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:26, optionally wherein said amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

26.

26. 22. The ACE2 fusion protein of any one of claims 1 to 21, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:27, optionally wherein said amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

27.

27. 22. The ACE2 fusion protein of any one of claims 1 to 21, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:28, optionally wherein said amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

28.

28. 22. The ACE2 fusion protein of any one of claims 1 to 21, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO:29, optionally wherein said amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

29.

29. 22. The ACE2 fusion protein of any one of claims 1 to 21, comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 30, optionally wherein said amino acid sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

30.

30. A nucleic acid or nucleic acids encoding an ACE2 fusion protein according to any one of claims 1 to 29.

31. 31. The nucleic acid of claim 30, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 15, optionally wherein the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

15.

32. 31. The nucleic acid of claim 30, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 16, optionally wherein the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

16.

33. 31. The nucleic acid of claim 30, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 17, optionally wherein the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

17.

34. 31. The nucleic acid of claim 30, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 18, optionally wherein the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

18.

35. 31. The nucleic acid of claim 30, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 19, optionally wherein the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

19.

36. 31. The nucleic acid of claim 30, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:20, optionally wherein the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

20.

37. 31. The nucleic acid of claim 30, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:21, optionally wherein the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

21.

38. 31. The nucleic acid of claim 30, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:22, optionally wherein the nucleotide sequence has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

22.

39. A host cell engineered to express an ACE2 fusion protein according to any one of claims 1 to 29 or a nucleic acid(s) according to any one of claims 30 to 38.

40. 40. A method for producing an ACE2 fusion protein according to any one of claims 1 to 29, comprising culturing a host cell according to claim 39 and recovering the ACE2 fusion protein expressed thereby.

41. A pharmaceutical composition comprising an ACE2 fusion protein according to any one of claims 1 to 29 and an excipient.

42. 42. A method for treating a coronavirus disease, comprising administering to a subject in need thereof an ACE2 fusion protein according to any one of claims 1 to 29 or a pharmaceutical composition according to claim 41.

43. 42. A method for inhibiting the interaction between coronavirus RBD and cellular ACE2, comprising administering to a subject in need thereof an ACE2 fusion protein according to any one of claims 1 to 29 or a pharmaceutical composition according to claim 41.

44. 42. A method for reducing the severity of a coronavirus infection, comprising administering to a subject in need thereof an ACE2 fusion protein according to any one of claims 1 to 29 or a pharmaceutical composition according to claim 41.

45. 42. A method for reducing coronavirus viral load, comprising administering to a subject in need thereof an ACE2 fusion protein according to any one of claims 1 to 29 or a pharmaceutical composition according to claim 41.

46. 42. A method for preventing disease progression in a subject with a coronavirus infection, comprising administering to a subject in need thereof an ACE2 fusion protein according to any one of claims 1 to 29 or a pharmaceutical composition according to claim 41.

47. 42. A method for reducing the duration of a coronavirus infection, comprising administering to a subject in need thereof an ACE2 fusion protein according to any one of claims 1 to 29 or a pharmaceutical composition according to claim 41.

48. 42. A method for reducing the risk of severe disease or death in a subject with a coronavirus infection, comprising administering to a subject in need thereof an ACE2 fusion protein according to any one of claims 1 to 29 or a pharmaceutical composition according to claim 41.

49. The method of any one of claims 42 to 48, wherein the coronavirus is SARS-CoV.

50. The method of any one of claims 42 to 48, wherein the coronavirus is SARS-CoV-2.

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