WO2021189772A1 - Ace2-fc fusion proteins and use thereof - Google Patents

Ace2-fc fusion proteins and use thereof Download PDF

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WO2021189772A1
WO2021189772A1 PCT/CN2020/112364 CN2020112364W WO2021189772A1 WO 2021189772 A1 WO2021189772 A1 WO 2021189772A1 CN 2020112364 W CN2020112364 W CN 2020112364W WO 2021189772 A1 WO2021189772 A1 WO 2021189772A1
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ace2
seq
portions
terminus
fusion protein
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PCT/CN2020/112364
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French (fr)
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Renke LI
Yuan Liu
Cheng Liu
Junna JIANG
Yanling Xu
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Immed Therapeutics Co., Ltd.
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present disclosure relates to Fc fusion protein, especially fusion proteins of ACE2 and Fc and uses thereof.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • severe novel coronavirus pneumonia cases account for 13.8%, and critical cases account for 4.7%. Wherein, the mortality rate of the latter is up to 49%.
  • the clinical therapy is mainly symptomatic treatment supporting vital signs, so it is urgent to develop effective a medicament to inhibit virus infection.
  • SARS-CoV-2 genome shows a 70%sequence homology with SARS, and its S protein for infection shows a homology of up to 76.47%.
  • this virus infects the host cell by ACE2 protein (angiotensin-converting enzyme 2) , and its affinity with ACE2 reaches 15 nM, 10 to 20 times higher than the affinity between SARS and ACE2 protein.
  • ACE2 protein angiotensin-converting enzyme 2
  • ACE2 protein as an essential receptor for SARS-CoV-2 infection, is not affected by virus mutation and it can be applied to the clinical treatment of SARS and other viruses that also use ACE2 as the infection receptor.
  • Fc-fusion protein is a biopharmaceutical area that has developed rapidly in recent years. It uses the Fc segment of immunoglobulin as a molecular chaperone, and fuses the functional protein with it by molecular biology approach to greatly prolong the half-life of the functional protein. Fc fusion protein maintains the activity of the functional protein, while having the long half-life as immunoglobulin.
  • the first aspect of the present disclosure is to provide a fusion protein containing a ACE2 portion and a Fc segment; wherein the ACE2 portion is selected from the group consisting of ACE2 extracellular domain or a mutant thereof; a fragment of the extracellular domain containing ACE2 PD domain (ACE2-PD) or a mutant thereof; the Fc segment is selected from IgG1-Fc, IgG2-Fc and IgG4-Fc or mutants thereof, wherein the IgG1-Fc segment has no ADCC activity.
  • the ACE2 extracellular domain or the fragment containing ACE2-PD is a wild-type extracellular domain or a fragment thereof containing ACE2-PD; the mutant of the extracellular domain or fragment is an extracellular domain lacking enzyme activity or a fragment thereof containing ACE2-PD, or an extracellular domain with partial or complete mutations of glycosylation sites or a fragment thereof containing ACE2-PD, or an extracellular domain lacking enzyme activity and having partial or complete mutations of glycosylation sites or a fragment thereof containing ACE2-PD, or an extracellular domain with enzyme activity and having partial or complete mutations of glycosylation sites or a fragment thereof containing ACE2-PD.
  • the mutated glycosylation site includes one or more sites selected from the group consisting of N53, N90, N103, N322, N432, N546 and N690.
  • the mutated glycosylation site (s) is mutated to A.
  • the extracellular domain lacking enzyme activity or the fragment thereof is an extracellular domain or a fragment thereof mutated at site 374 and/or 378; preferably, an extracellular domain or a fragment thereof with mutation of H374N and/or H378N.
  • the sequence identity of the mutant of the extracellular domain is over 80%to the wild-type ACE2 extracellular domain, preferably over 85%, and more preferably over 90%, over 95%, over 97%, and over 99%; the sequence identity of the mutant of the fragment is over 80%to wild-type ACE2 PD, preferably over 85%, and more preferably over 90%, over 95%, over 97%, and over 99%.
  • the fusion protein is a bivalent fusion protein containing two ACE2 portions or a tetravalent fusion protein containing four ACE2 portions.
  • the fusion protein has a reduced ACE2 enzyme activity.
  • one ACE2 portion in the bivalent fusion protein, is a wild-type ACE2 extracellular domain or a fragment thereof containing the PD, and the other portion is a mutant ACE2 extracellular domain lacking enzyme activity or the fragment thereof containing the ACE2-PD.
  • both ACE2 portions are wild-type ACE2 extracellular domains or fragments thereof containing the PD, or mutant ACE2 extracellular domains lacking enzyme activity or fragments thereof containing the ACE2-PD.
  • At least one ACE2 portion is an extracellular domain lacking enzyme activity and having partial or complete mutations of glycosylation sites or a fragment thereof containing ACE2-PD, or at least one ACE2 portion is an extracellular domain having enzyme activity and having partial or complete mutations of glycosylation sites or a fragment thereof containing ACE2-PD.
  • At least one ACE2 portion is a wild-type ACE2 extracellular domain or a fragment thereof containing the PD, or at least one ACE2 portion is an ACE2 extracellular domain lacking enzyme activity or a fragment thereof containing PD; optionally, at least one ACE2 portion has partial or complete mutation of glycosylation sites.
  • At least one ACE2 portion is a wild-type extracellular domain or a fragment thereof containing the ACE2-PD, or at least one ACE2 portion is an extracellular domain having enzyme activity and having partial or complete mutations of glycosylation sites or the fragment thereof containing ACE2-PD, or at least one ACE2 portion is an extracellular domain that has no mutation of glycosylation sites without enzyme activity or a fragment thereof containing ACE2-PD, or at least one ACE2 portion is an extracellular domain lacking enzyme activity and having partial or complete mutations of glycosylation sites or the fragment thereof containing ACE2-PD.
  • the ACE2 portion is linked to Fc through a linker peptide; preferably, the linker peptide contains G and S.
  • the IgG1 Fc contains the mutation of the N297A glycosylation site, and the IgG4 Fc has S228P mutation and optional N297A mutation.
  • the two chains of IgG Fc have mutations forming a Knob and a Hole respectively.
  • the mutation forming a Knob occurs at site T366, and the mutation forming a Hole occurs at one or more sites selected from the group consisting of T366, L368 and Y407. More preferably, the mutation forming a Knob is T366W, and the mutation forming a Hole is one or more mutations selected from the group consisting of T366S, L368A and Y407V.
  • the fusion protein is expressed by yeast.
  • the ACE2-Fc fusion protein is a bivalent fusion protein comprising two ACE2 portions and a Fc, with the two ACE2 portions are directly linked to the N-terminus of the Fc.
  • the ACE2 portion has a sequence of any of SEQ ID NO: 1-4, and Fc has a sequence of any of SEQ ID NO: 5-8;
  • the ACE2 portion has glycosylation mutation (s) selected from the group consisting of N53A, N90A, N103A, N322A, N432A, N546A and N690A;
  • the two ACE2 portions are identical;
  • the ACE2 portion has the sequence of SEQ ID NO: 1 or 2, and Fc has a sequence of any of SEQ ID NO: 5-7;
  • the ACE2 portion has the sequence of SEQ ID NO: 3 or 4, and Fc has the sequence of SEQ ID NO: 7;
  • the ACE2 portion has the sequence of SEQ ID NO: 1
  • Fc has the sequence of SEQ ID NO: 8.
  • all ACE2 portions have one or more glycosylation mutations selected from the group consisting of N53A, N90A, N103A, N322A, N432A and N546A.
  • the ACE2-Fc fusion protein is a tetravalent fusion protein comprising four ACE2 portions and a Fc, with two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two ACE2 portions are respectively linked to the C-terminus of Fc directly or through (GGGGS) n , wherein n is a positive integer of 1-8;
  • the two ACE2 portions linked to the N-terminus of Fc have a sequence of any of SEQ ID No: 1-4
  • the two ACE2 portions linked to the C-terminus of Fc have a sequence of any of SEQ ID No: 1-4;
  • Fc has a sequence of any of SEQ ID NO: 5-8;
  • n 3, 4, 7 or 8;
  • the two ACE2 portions linked to the N-terminus of Fc are identical, and the two linked to the C-terminus of Fc are identical;
  • the ACE2 portion has glycosylation mutations of N53A, N90A, N103A, N322A, N432A, N546A and N690A;
  • the two ACE2 portions linked to the N-terminus of Fc have the sequence of SEQ ID No: 1 or 2
  • the two ACE2 portions linked to the C-terminus of Fc have the sequence of SEQ ID No: 1 or 2
  • Fc has a sequence of any of SEQ ID No: 5-7;
  • the two ACE2 portions linked to the N-terminus of Fc have the sequence of SEQ ID No: 3 or 4
  • the two ACE2 portions linked to the C-terminus of Fc have the sequence of SEQ ID No: 3 or 4
  • Fc has the sequence of SEQ ID No: 7;
  • the two ACE2 portions linked to the N-terminus of Fc have the sequence of SEQ ID No: 1
  • the two ACE2 portions linked to the C-terminus of Fc have the sequence of SEQ ID No: 2
  • Fc has the sequence of SEQ ID No: 8.
  • all ACE2 portions have one or more glycosylation mutations selected from the group consisting of N53A, N90A, N103A, N322A, N432A and N546A.
  • the present disclosure also provides a nucleic acid molecule selected from: (1) the polynucleotide sequence encoding the fusion protein described herein; (2) the sequence complementary to the polynucleotide sequence in (1) .
  • the present disclosure also provides a nucleic acid construct containing the nucleic acid molecule described herein.
  • the nucleic acid construct is a clone vector or an expression vector.
  • the present disclosure also provides an engineered cell, wherein the engineered cell: (1) expresses the fusion protein described herein; and/or (2) contains the nucleic acid molecule or the nucleic acid construct described herein.
  • the present disclosure also provides a pharmaceutical composition containing the ACE2-Fc fusion protein, the nucleic acid molecule or the nucleic acid construct described herein, and optional pharmaceutically acceptable carriers or excipients.
  • the present disclosure also provides the use of the fusion protein, the nucleic acid molecule containing a nucleic acid sequence encoding the fusion protein or its complementary sequence, and the nucleic acid construct containing the nucleic acid molecule in the preparation of medicament for the treatment of coronavirus-induced diseases, wherein the fusion protein contains ACE2 portion and Fc; wherein the ACE2 portion is selected from ACE2 extracellular domain or a fragment thereof or a mutant of the extracellular domain or the fragment thereof, and the fragment of the extracellular domain contains ACE2 PD domain or a mutant thereof; the Fc segment is selected from IgG1-Fc, IgG2-Fc and IgG4-Fc or mutants thereof, and IgG1-Fc segment has no ADCC activity.
  • coronaviruses are selected from one or more of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, and 2019-nCoV.
  • the coronavirus-induced diseases include but are not limited to Middle East respiratory syndrome, severe acute respiratory syndrome, coronavirus induced pneumonia, pulmonary arterial hypertension, acute respiratory distress syndrome, heart failure, and novel coronavirus pneumonia.
  • the fusion protein is as described in the first aspect herein.
  • the present disclosure also provides the use of the ACE2-Fc fusion protein, the nucleic acid molecule containing a nucleic acid sequence encoding the fusion protein or its complementary sequence, and the nucleic acid construct containing the nucleic acid molecule in the preparation of a medicament for the treatment of acute respiratory distress syndrome (ARDS) , wherein the ACE2-Fc fusion protein contains ACE2 portion and Fc; wherein the ACE2 is selected from ACE2 extracellular domain or a fragment thereof or a mutant of the extracellular domain or the fragment thereof, and the fragment of the extracellular domain contains ACE2 PD domain or a mutant thereof; the Fc segment is selected from IgG1-Fc, IgG2-Fc and IgG4-Fc or mutants thereof, and IgG1-Fc segment has no ADCC activity.
  • ARDS acute respiratory distress syndrome
  • the ACE2-Fc fusion protein is as described in the first aspect herein.
  • a method for producing ACE2-Fc fusion protein comprising a step of introducing the nucleic acid construct described herein into cells capable of expressing the ACE2-Fc fusion protein, and incubating the cells under a condition capable of expressing the ACE2-Fc fusion protein.
  • the cell is a mammalian cell CHO or a Pichia yeast cell.
  • the ACE2-Fc fusion protein is as described in the first aspect herein, and the nucleic acid construct expresses the ACE2-Fc fusion protein.
  • the ACE2-Fc fusion protein is a bivalent fusion protein comprising two ACE2 portions and a Fc, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and have the sequence of SEQ ID NO: 1, and the Fc has the sequence of SEQ ID No: 8.
  • all ACE2 portions have one or more glycosylation mutations selected from the group consisting of N53A, N90A, N103A, N322A, N432A and N546A.
  • the ACE2-Fc fusion protein is a tetravalent fusion protein comprising four ACE2 portions and a Fc.
  • the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two ACE2 portions are respectively linked to the C-terminus of Fc directly or through (GGGGS) n , wherein n is a positive integer 1-8; wherein, the two ACE2 portions linked to the N-terminus of Fc have the sequence of SEQ ID No: 1, and the two ACE2 portions linked to the C-terminus of Fc have the sequence of SEQ ID No: 2, and the Fc has the sequence of SEQ ID No: 6, 7 or 8.
  • all ACE2 portions have one or more glycosylation mutations selected from the group consisting of N53A, N90A, N103A, N322A, N432A and N546A.
  • Figure 1 Schematic diagram of neutralizing virus infection of the fusion protein in the present disclosure.
  • Figure 2 Schematic diagram of the fusion protein of the disclosure.
  • Figure 3 Schematic diagram of the mechanism of treating the acute respiratory distress syndrome with the fusion protein of the disclosure.
  • Figure 4 Comparison of the affinity of M05 and M25 to 2019-nCoV RBD by SPR experiment.
  • Figure 5 Results of competitive blocking ELISA experiment with bivalent ACE2-Fc as the solid phase and 2019-nCoV RBD as the mobile phase.
  • Figure 7 Schematic diagram of the mechanism of tetravalent ACE2-Fc in blocking virus infection.
  • Figure 8 Effect of ACE2-Fc molecules on blood pressure in mice.
  • the present disclosure is intended to synthesize an antibody-like molecule binding to the coronavirus itself, thereby neutralizing the infection of the virus, instead of protecting the cells from infection.
  • the present disclosure discloses an ACE2-Fc fusion protein as a blocker to inhibit the invasion of novel coronavirus pneumonia.
  • the Fc fusion protein can bind to the FcRn receptor through the Fc segment thereof in the endosome, in order to be recycled outside the cell, thereby avoiding the degradation of the protein by the lysosomal pathway, prolonging the half-life of the drug, cutting down the frequency of administration, increasing the drug efficacy and reducing the treatment costs.
  • ACE2 is a transmembrane protein comprising a peptidase domain (PD) at the N-terminus and a Collectrin-like domain (CLD) at the C-terminus.
  • the end of CLD contains a transmembrane helix and an intracellular segment.
  • the peptidase domain (PD) is in the extracellular domain and is in charge of cleaving angiotensin II and angiotensin I.
  • the crystal structure of ACE2-PD also provides a direct binding site for coronavirus S protein. For the binding of coronavirus cells, PD is essential in ACE2.
  • the ACE2 portion in the ACE2-Fc fusion protein of the present disclosure may be an extracellular domain of ACE2 or a fragment thereof containing the peptidase domain of ACE2, or a mutant of the extracellular domain or the fragment thereof.
  • the ACE2 portion may be an extracellular domain of ACE2 or the fragment thereof containing a wild-type ACE2 peptidase domain, or the mutant thereof that retains the activity to bind to a ligand (e.g., virus S protein) .
  • a ligand e.g., virus S protein
  • Said mutant may be an extracellular domain of ACE2 losing the peptidase activity of cleaving angiotensin II and angiotensin I by mutation or a fragment thereof containing the peptidase domain, for example, the histidine at the active center site of ACE2 is mutated into other amino acid residues, specifically, the histidine (His) at sites 374 and/or 378 is mutated into a mutant after pretreatment with asparagine (Asn) .
  • the mutant may also be one that retains peptidase activity.
  • ACE2 peptidase of ACE2-FC fusion protein can be retained to supplement the above symptoms caused by the absence of ACE2.
  • the mutant also contains an ACE2 extracellular domain that has partial or complete mutation of glycosylation sites and with or without peptidase activity, or a fragment thereof containing ACE2 peptidase domain.
  • the glycosylation sites contain one or more sites selected from the group consisting of 53, 90, 103, 322, 432, 546 and 690 of asparagine residues, as described elsewhere herein.
  • the active ACE2-PD sequence is as shown in SEQ ID NO: 1
  • the inactivated ACE2-PD sequence is as shown in SEQ ID NO: 2
  • the active ACE2-extracellular domain sequence is as shown in SEQ ID NO: 3
  • the inactivated ACE2-extracellular domain sequence is as shown in SEQ ID NO: 4.
  • the mutation in the mutant of the ACE2 extracellular domain occurs outside PD.
  • ACE2 Fc fusion protein refers to a protein produced by fusing a Fc segment of a immunoglobulin with a ACE2 portion.
  • Fc fusion protein has the biological activity of the ACE2 portion, and also has the properties of antibody, e.g. antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP) .
  • the Fc of the disclosure can be selected from the Fc segment of different subtypes of human IgG1, IgG2, IgG4 and the like. IgG1 often has a strong effect function.
  • ADCC NK cells or macrophages with Fc ⁇ receptor to kill (ADCC) or phagocytize (ADCP) target cells or pathogens
  • FC ⁇ receptor Due to the weak binding affinity for FC ⁇ receptor, IgG2 and IgG4 molecules have a weak effect of ADCC or ADCP.
  • ADCC/ADCP has the potential to promote the removal of the virus by the body's immune system, thereby promoting the patient's recovery.
  • ADE antibody dependent enhancement
  • the Fc segment of the fusion protein of the disclosure may be IgG1-Fc, IgG2-Fc and IgG4-Fc or mutants thereof. These Fc segments can eliminate the activity of ADCC and ADCP by introducing the mutation at N297 site (e.g., mutation A) . And the IgG4 Fc segment can eliminate chain exchange by introducing the mutation at S228 site (e.g., S228P) . Therefore, the Fc segment of the fusion protein of the disclosure is selected from IgG1-Fc with or without mutation at N297 site, IgG2-Fc with or without mutation at N297 site, and IgG4-Fc with or without mutation at sites S228 and/or N297.
  • the Fc segment of the fusion protein of the disclosure is selected from IgG1-Fc with mutation at N297 site, Fc segment of wild-type IgG2, and IgG4-Fc with mutation at S228 site and optional N297 site.
  • IgG1 containing the N297A mutation is shown in SEQ ID NO: 5
  • wild-type IgG2 is shown in SEQ ID NO: 6
  • IgG4 containing the S228P mutation is shown in SEQ ID NO: 7
  • IgG4 containing N297A and S228P mutations is shown in SEQ ID NO: 8.
  • the ACE2-Fc fusion protein of the disclosure can have different valences. With the multivalent binding of multiple ACE2 portions to the spike protein of 2019-nCoV, it can simultaneously bind to multiple S proteins on a single spike, or multiple spikes of a virus, or even multiple spikes of multiple viruses to enhance its neutralization activity. Multivalent binding can significantly increase intermolecular forces through the affinity effect, thereby increasing the neutralization effect of blocking virus infection.
  • the bivalent ACE2-Fc fusion protein herein contains two ACE2 portions at the N-terminus of the Fc segment. In the bivalent fusion protein, both ACE2 portions may have or have no peptidase activity, or one of them has peptidase activity while the other does not.
  • one ACE2 portion is the full-length extracellular domain of the wild type ACE2 or a fragment containing peptidase domain or the mutant thereof with enzyme activity, while the other ACE2 portion is a extracellular domain without enzyme activity or a fragment containing peptidase domain or their mutants without enzyme activity.
  • the tetravalent ACE2-Fc fusion protein herein contains two ACE2 portions at the N-terminus of the Fc segment and two ACE2 portions at the C-terminus of the Fc segment. In the tetravalent ACE2-Fc fusion protein, all or part of the enzyme activity of each ACE2 portion can be retained.
  • At least one ACE2 portion in the tetravalent ACE2-Fc fusion protein may be a wild-type ACE2 extracellular domain or the fragment thereof containing PD or the mutant thereof with enzyme activity, or at least one ACE2 portion may be an ACE2 extracellular domain without enzyme activity or the fragment thereof containing PD or their mutants without enzyme activity. This ensures that the tetravalent ACE2-Fc fusion protein can effectively block virus infection, while preventing excessive ACE2 peptidase activity from causing blood pressure drop and other risks.
  • the two ACE2 portions at the N-terminus of the ACE2-Fc fusion protein are wild-type ACE2 extracellular domains or PDs thereof, with peptidase activity, while the two ACE2 portions at the C-terminus are ACE2 extracellular domains without peptidase activity or PDs thereof due to H374N and/or H378N mutations.
  • the two ACE2 portions at the N-terminus can have no peptidase activity, while the two ACE2 portions at the C-terminus have peptidase activity.
  • the linker peptide of the disclosure is a linker containing G and S, including but not limited to (GS) n, (GGS) n, (GGGS) n, (GGGGS) n, wherein G refers to glycine, S refers to serine, and n is a positive integer of 1-15, preferably 1-10, and more preferably 1-8.
  • the linker is (GGGGS) n, wherein n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • Other common linker peptides are well known in the art.
  • the ACE2 portion when the ACE2 portion is preferably linked to the C-terminus of Fc through the linker, the ACE2 portion can be directly linked to the N-terminus of Fc.
  • the fusion protein of the disclosure also contains signal peptide, and signal peptide sequence suitable for expression of cellular or subcellular structures is well known in the art.
  • the ACE2-Fc fusion protein contains the natural signal peptide of ACE2 at the N-terminus: MSSSSWLLLSLVAVTAA (SEQ ID NO: 9) .
  • the natural signal peptide is included in the amino acids numbering of ACE2 described herein only for the sake of convenience, not to limit whether the fusion protein contains signal peptides or limit the type and sequence of the signal peptides contained.
  • ACE2 Fc and the fusion protein of the disclosure also include mutants with at least 70%sequence identity that retain the respective required activity thereof.
  • the activity is the capability to bind to viral proteins e.g. S protein; for Fc segment, the activity is the capability to bind to FcRn receptor, thus avoiding degradation of protein by lysosomal pathway; for the fusion protein, the activity is the capability to bind to viral proteins e.g. S protein and FcRn receptor.
  • the mutant includes an amino acid sequence having at least 70%, at least 80%, preferably at least 85%, more preferably at least 90%, at least 95%, at least 97%and at least 99%of sequence identity compared to the reference and retaining the required activity of the reference (e.g., capability to bind to viral proteins e.g. S protein, and/or capability to bind to FcRn receptor) .
  • the sequence identity between two aligned sequences can be calculated by BLASTp of NCBI.
  • the mutant also includes an amino acid sequence that has one or more mutations (insertions, deletions or substitutions) in the amino acid sequence, while still retaining the required activity of the reference sequence.
  • the number of said mutations usually is in a range of 1-50, e.g.
  • amino acids with close or similar properties include, for example, families of amino acid residues with similar side chains, which include amino acids with basic side chains (e.g. lysine, arginine, and histidine) , amino acids with acidic side chains (e.g. aspartic acid, and glutamine) , and amino acids with non-charged polar side chains (e.g.
  • glycine asparagine, glutamine, serine, threonine, tyrosine, and cysteine
  • amino acids with non-polar side chains e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan
  • amino acids with ⁇ -branched side chains e.g. threonine, valine, and isoleucine
  • amino acids with aromatic side chains e.g. tyrosine, phenylalanine, tryptophan, and histidine
  • Exemplary ACE2-Fc fusion proteins of the disclosure are as follows:
  • M01 a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
  • M02 a bivalent fusion protein consisting of two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
  • M03 a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
  • M04 a bivalent fusion protein consisting of two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
  • M05 a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
  • M06 a bivalent fusion protein consisting of two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
  • M05F a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 3 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively;
  • M06F a bivalent fusion protein consisting of two inactivated ACE2 portions shown in SEQ ID NO: 4 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
  • M07 a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M08 a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M09 a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M10 a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two inactivated ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two active ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M11 a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M12 a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M13 a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M14 a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two inactivated ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two active ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M15 a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS) 3 ;
  • M16 a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M17 a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M18 a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M15F a tetravalent fusion protein comprising four active ACE2 portions shown in SEQ ID NO: 3 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M16F a tetravalent fusion protein comprising four inactivated ACE2 portions shown in SEQ ID NO: 4 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M17F a tetravalent fusion protein comprising two active ACE2 portions shown in SEQ ID NO: 3, two inactivated ACE2 portions shown in SEQ ID NO: 4 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M18F a tetravalent fusion protein comprising two active ACE2 portions shown in SEQ ID NO: 3, two inactivated ACE2 portions shown in SEQ ID NO: 4 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two inactivated ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two active ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS) 3 ;
  • M19 a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is directly linked to Fc;
  • M20 a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker GS;
  • M21 a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker GGS;
  • M22 a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker GGGS;
  • M23 a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker GGGGS;
  • M24 a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker (GGGGS) 2 ;
  • M25 a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker (GGGGS) 4 ;
  • M26 a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker (GGGGS) 5 ;
  • M27 a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker (GGGGS) 6 ;
  • M28 a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker (GGGGS) 7 ;
  • M29 a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker (GGGGS) 8 ;
  • M30 a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS) 4 ;
  • M31 a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS) 4 ;
  • M32 a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS) 4 ;
  • M33 a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS) 4 ;
  • M34 a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS) 4 ;
  • M35 a tetravalent fusion protein comprising four activated ACE2 portions shown in SEQ ID NO: 1 and IgG1 Fc shown in SEQ ID NO: 5, wherein two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS) 4 ;
  • M36 a tetravalent fusion protein comprising four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS) 4 ;
  • M37 a tetravalent fusion protein comprising two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS) 4 ;
  • Y01 a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 8, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
  • Y03 a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 8, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS) 4 .
  • the ACE2 portion in the fusion protein of the disclosure can also be the ACE2 portion with partial or complete mutation of glycosylation sites.
  • These glycosylation sites contain asparagine residues at sites 53, 90, 103, 322, 432, 546 or 690, and threonine or serine at sites 55, 92, 105, 324, 434, 548 or 692.
  • Their mutation into non-glycosylation patterns e.g. alanine
  • the ACE2 portion with partial or complete mutation of glycosylation sites may have or have no peptidase activity, for example, the peptidase activity is reduced or lost by the mutation described elsewhere herein.
  • the present disclosure also includes the ACE2-Fc fusion protein as described below:
  • Y02 a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 8, wherein all ACE2 portions have glycosylation mutations: N53A, N90A, N103A, N322A, N432A, N546A, and two ACE2 portions are directly linked to the N-terminus of Fc respectively;
  • Y04 a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 8, wherein the two active ACE2 portions shown in SEQ ID NO: 1 are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions shown in SEQ ID NO: 2 are respectively linked to the C-terminus of Fc through the linker (GGGGS) 4 . All ACE2 portions have glycosylation mutations: N53A, N90A, N103A, N322A, N432A, N546A.
  • the fusion protein of the disclosure may also be heterodimer ACE2-Fc fusion protein at the N (or C) -terminus of Fc with different ACE2 activities.
  • the heterodimer ACE2-Fc fusion protein can also have Knob and Hole mutations in Fc. For example, by introducing a "Knob" mutation in one IgG Fc (e.g., mutation at site T366) and a "Hole” mutation (e.g. mutation at site T366 and/or L368 and/or Y407) into another IgG Fc, a stable heterodimer structure based on the Knob-Hole complementation is created. Therefore, the present disclosure also includes the ACE2-Fc fusion protein as described below:
  • KIH01 a bivalent fusion protein comprising one active ACE2 portion shown in SEQ ID NO: 1, one inactivated ACE2 portion shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, one chain of IgG4 Fc has T366W mutation, and the other chain of IgG4 Fc has one or more mutations selected from the group consisting of T366S, L368A and Y407V.
  • a pharmaceutical composition containing the ACE2 Fc fusion protein described herein or its encoded nucleic acid as an active substance and an optional pharmaceutically acceptable carrier or excipient.
  • Pharmaceutically acceptable carriers, excipients, or stabilizers are non-toxic to the recipient of the protein or nucleic acid at the dosage and concentration required, and may include various types of carriers or excipients commonly used in the delivery of protein or nucleic acid in treatment well known in the art.
  • the protein or nucleic acid in the pharmaceutical composition is in a therapeutically effective amount.
  • the effective dosage and dosage form of the protein or nucleic acid can be determined according to the type of active substance, the age, gender and the severity of the disease of the patient receiving the protein or nucleic acid.
  • the protein or nucleic acid described herein may be given by conventional administration, for example, orally or by injection. The administration methods for different dosage forms are well known in the art.
  • the fusion protein or pharmaceutical composition of the disclosure may be used for the treatment of coronavirus-induced diseases.
  • the fusion protein of the disclosure may bind to the coronavirus to neutralize its infection, and has an extended half-life.
  • the coronaviruses described herein are enveloped Coronavirus viruses with a single-strand positive-sense RNA, including but not limited to HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, and 2019-nCoV (COVID-19) .
  • Coronavirus S protein (spike, spike protein S) is a key protein for coronaviruses to infect cells, and it plays an important role in the tissue or host tropism and infection of the virus.
  • the fusion protein of the disclosure binds to the coronavirus S protein through ACE portion to block or reduce the binding of the coronavirus protein to the host cell to neutralize the infection. Therefore, the fusion protein of the disclosure may be used for the treatment of coronavirus-induced diseases. These diseases include but are not limited to Middle East respiratory syndrome, severe acute respiratory syndrome, acute respiratory distress syndrome, coronavirus induced pneumonia, pulmonary arterial hypertension, acute respiratory distress syndrome, heart failure, and novel coronavirus pneumonia.
  • a method for the treatment of coronavirus-induced diseases including administering a therapeutically effective amount of the fusion protein or pharmaceutical composition of the disclosure to subjects in need.
  • “Individuals” , “subjects” or “patients” herein refer to mammals, especially humans.
  • the present disclosure also includes the coding sequences and complementary sequences of various amino acid sequences, polypeptides, proteins or mutants thereof described herein, and nucleic acid constructs containing the coding sequences or complementary sequences.
  • the coding sequence described herein includes a sequence changed by codon optimization, provided the encoded amino acid sequence remains unchanged.
  • the codon-optimized sequence shows more suitable expression for specific species.
  • the method of codon optimization for coding sequences is well known in the art.
  • the nucleic acid constructs herein are artificially constructed segments of nucleic acids that can be introduced into target cells or tissues.
  • the nucleic acid construct contains the coding sequences described herein or complementary sequences thereof, and one or more regulatory sequences operatively linked to these sequences.
  • the regulatory sequence can be an appropriate promoter sequence.
  • the promoter sequence is usually operationally linked to coding sequence of amino acid sequence to be expressed.
  • a promoter including mutant, truncated and hybrid forms, may be any nucleotide sequence showing transcriptional activity in a selected host cell. It may be obtained from the gene encoding an extracellular or intracellular peptide homologous or heterologous to the host cell.
  • the regulatory sequence can also be a suitable transcriptional terminator sequence, identified by host cells to terminate transcription. The terminator sequence is linked to the 3' terminus of the nucleotide sequence encoding the peptide, and any terminator functional in the selected host cell can be used herein.
  • the nucleic acid construct is a vector.
  • the coding sequence described herein can be cloned into many types of vectors, including but not limited to plasmids, phages, phage derivatives, viruses, and mucous particles.
  • the vector can be an expression vector or a clone vector.
  • an appropriate vector contains the replication starting point, the promoter sequence, the convenient limiting enzyme site, and one or more optional markers that work in at least one organism.
  • the representative examples of promoters are lac or trp promoter of E. coli; PL promoter of ⁇ phage; eukaryotic promoter including CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, methanol oxidase promoter of Pichia yeast, and some other known promoters which control the gene expression in the prokaryotic cells, eukaryotic cells or virus.
  • Marker genes can be used to provide phenotypic traits for selection of the transformed host cells, including but not limited to dihydrofolate reductase, neomycin resistance gene and GFP (green florescent protein) for eukaryotic cells, as well as tetracycline or ampicillin resistance gene for E. coli.
  • Transcription of the polynucleotide of the disclosure in higher eukaryotes is increased by inserting an enhancer sequence into the vector.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase the gene transcription.
  • An expression vector containing the sequence of polynucleotide and appropriate transcription/translation control signals can be constructed by methods known by the skilled in the art. These methods include in vitro recombinant DNA technique, DNA synthesis technique, in vivo recombinant technique and the like.
  • the fusion protein of the disclosure can be prepared by chemical synthesis or biosynthesis.
  • the process and conditions for conventional chemical synthesis of proteins are well known in the art.
  • the biosynthesis method described herein is based on cell expression, including steps of introducing a vector expressing the ACE2-Fc fusion protein described herein into cells capable of expressing the fusion protein and incubating the cells under conditions capable of expressing the fusion protein.
  • the cells include, but are not limited to, mammalian cells CHO or Pichia yeast cells. Components e.g. vectors, promoters and the like required for expression in Pichia yeast cells are known in the art.
  • the ACE2 portion in the fusion protein of the disclosure can also be the ACE2 portion with partial or complete mutation of glycosylation sites.
  • Mutated ACE2-Fc fusion proteins at the exemplary glycosylation sites of the disclosure are as described in Y02 and Y04 above.
  • Host cells or engineered cells expressing the protein described herein, comprising the polynucleotide sequence described herein or a nucleic acid construct thereof are also included herein.
  • the host cells or engineered cells include prokaryote, e.g. bacteria; primary eukaryote, e.g. yeast cell; filamentous fungal cells or advanced eukaryotic, e.g. mammalian cells.
  • the representative examples are E. coli, Streptomyces, bacterial cells of salmonella typhimurium; fungal cells e.g. yeast, filamentous fungi and plant cells; insect cells e.g. drosophila S2 or Sf9; animal cells e.g. CHO, COS, 293 or Bowes melanoma cell, etc.
  • the vectors herein can be introduced into host cells by conventional methods, including microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation and the like.
  • coronavirus-induced diseases include but are not limited to Middle East respiratory syndrome, severe acute respiratory syndrome, coronavirus induced pneumonia, pulmonary arterial hypertension, acute respiratory distress syndrome, heart failure, and novel coronavirus pneumonia.
  • the causes of acute respiratory distress syndrome include but are not limited to pneumonia, aspiration, pulmonary contusion, drowning, inhalation of toxic substances; severe systemic infection (bacteria, viruses, fungi and atypical pathogens) , malignant tumors, severe multiple injuries (multiple fractures, flail chest, severe brain trauma and burns) , shock, high-risk surgery (cardiac surgery, arterial surgery, etc. ) , massive blood transfusion, drug poisoning, pancreatitis and cardiopulmonary bypass.
  • the Chronic obstructive pulmonary disease includes but not limited Chronic bronchitis, emphysema, pulmonary heart disease, Respiratory failure. Based on this, a method for the treatment of coronavirus-induced diseases is also provided, including administering a therapeutically effective amount of the fusion protein or pharmaceutical composition of the disclosure to subjects in need thereof.
  • Example 1 Expression of different forms of ACE2-Fc fusion proteins in HEK293 cells
  • ACE2 was selected from the peptidase domain (PD, labeled M + number, where M represents mammalian cell expression) or the full-length extracellular domain (labeled M + number + F, where F represents full length) .
  • ACE2 was active or inactivated.
  • His histidine
  • Asparagine Asparagine
  • the Fc segment was selected from human IgG1 Fc, IgG2 Fc, and IgG4 Fc.
  • IgG1 Fc contained N297A glycosylation site mutations to eliminate binding to Fc ⁇ receptors and thereby eliminate the effect functions.
  • IgG4 Fc introduced S228P mutation to eliminate generation of chain exchange.
  • the amino acid sequences of the above 24 molecules were delivered to Suzhou Genewiz for codon optimization of mammalian cells, synthesis and construction into pTT5 vector, and the signal peptide sequence was selected from the ACE2 natural signal peptide (MSSSSWLLLSLVAVTAA) .
  • the plasmids of said 24 molecules were prepared and 200 ⁇ g of plasmid was transfected into 200 ml of logarithmic growth phase HEK293 cells with density of 1 E6/ml by polyethyleneimine (PEI) reagent. After 24 hours of transfection, 5/1000 of peptone was added to promote cell growth.
  • PEI polyethyleneimine
  • the harvested cells were centrifuged at 4000 rpm for 30 min, the supernatant was purified with Protein A resin (Bestchrom) after filtration with 0.22 ⁇ m membrane and dialyzed into PBS; then it was filtered with 0.22 ⁇ m membrane again for storage.
  • Protein A resin Protein A resin
  • BCA protein concentration quantitative kit (Yeasen Biotech) was used to quantify twenty-four ACE2 fusion proteins according to the operation procedures, and the polymer and purity were analyzed by SEC-HPLC (G3000SWXL, TOSOH) . Results were as follows:
  • Example 2 ELISA experiment for binding of ACE2 Fc fusion proteins with RBD domain of 2019-nCoV S protein
  • Enzyme-linked immunosorbent assay was conducted on the above 24 molecules to characterize the binding activity of different molecules to 2019-nCoV S protein.
  • 96-well ELISA plate (Maxisorp, NUNC) was incubated with 100 ⁇ L of 1 ⁇ g/mL 2019-nCoV RBD (COV-VM4BD, Kactus Biosystems) at 4°C overnight, then was blocked with 1%BSA (bovine serum albumin) solution at room temperature for 2 hours and washed three times with PBST.
  • BSA bovine serum albumin
  • Example 3 ACE2-Fc fusion proteins competitively blocked the binding of 2019-nCoV S protein RBD to ACE2 protein
  • ACE2-Fc fusion proteins 50 ⁇ l of human ACE2-Fc fusion proteins of different concentrations were respectively added into 96 well plate, and 50 ⁇ l of ACE2 His-Biotin with a final concentration of 0.3 nM (ACE-HM401 B, Kactus Biosystems) was also added into each well, so that their mixture could bind to the coated 2019-nCoV RBD at room temperature for 1.5 hours, then the plate was washed three times with PBST. Then High Sensitivity Streptavidin-HRP (21130, 1: 15000, Thermo Fisher Scientific) was added for binding at room temperature for 1 hour, and then the plate was washed three times with PBST.
  • High Sensitivity Streptavidin-HRP 21130, 1: 15000, Thermo Fisher Scientific
  • Peptidase activity determination of the above 24 molecules was carried out. Use 75 mM Tris, 1 M NaCl, 10 ⁇ M ZnCl 2 , pH 7.5 Assay Buffer to prepare a 0.2 ng/ ⁇ L solution of human ACE2-Fc fusion proteins, and dilute the enzyme reaction substrate Mca-Y-V-A-D-A-P-K (Dnp) -OH Fluorogenic Peptide Substrate VI (ES007, R&D) to 40 ⁇ M with the same Assay Buffer.
  • Linker peptides of ACE2 and C-terminus of Fc were screened on the basis of the M17 molecule, and a series of linker peptides of different lengths were designed as follows:
  • Example 7 Investigation of the neutralization effect of ACE2-Fc fusion proteins on 2019-nCoV infection by pseudovirus infection experiment
  • 2019-nCoV S pseudovirus neutralization experiment was conducted on thirteen ACE2-Fc fusion protein molecules selected in example 1-7 to characterize the neutralization and blocking activity of different molecules to 2019-nCoV S protein infection.
  • pCMVR8.2 lentiviral packaging plasmid, pHRCMV-Luc reporter gene plasmid and CMVR-2019-nCoV-Svirus eukaryotic expression plasmid were transiently co-transfected with 293T cells; the supernatant was collected 48 hours after transfection, centrifuged and filtered with a 0.45 ⁇ m filter to get the packaged 2019-nCoV S pseudovirus. After subpackaging, some of the pseudoviruses were preserved at -4°C, and some frozen at -80°C for later use.
  • DNA3-ACE2 eukaryotic expression plasmid was transiently transfected with 293T cells to get 293T-ACE2 cells expressing ACE2 transiently. 1x10 4 293T-ACE2 cells were arranged into each well of 96-well cell culture plate, 100 ⁇ L per well, and cultured overnight. Different concentrations of human ACE2-Fc fusion proteins were mixed with 2019-nCoV S pseudovirus solution standardized by p24 content in a volume ratio of 1: 1 for interaction at room temperature for 10 minutes.
  • SPR experiment was carried based on Biacore T200 to determine the affinity of M05 and M25 to 2019-nCoV RBD.
  • the anti-His antibody was covalently coupled to the channels 1, 2, 3, and 4 of the CM5 chip by the amino coupling method, at a coupling level of about 9000 RU.
  • His-RBD (COV-VM4BD, Kactus Biosystems) as the ligand was diluted to 2 ⁇ g/mL with pH7.4, 1 ⁇ HBS-EP buffer, and captured in 2 channels for 30 s, at a flow rate of 10 ⁇ l/min.
  • Chip was regenerated for 90s with 10 mM pH1.5 glycine as the regeneration buffer, at a flow rate of 30 ⁇ l/min.
  • Channel 1 is the reference channel
  • channel 2 is the active channel.
  • Example 10 Competitive blocking ELISA with bivalent ACE2-Fc as solid phase and 2019-nCoV RBD as mobile phase
  • ACE2 protein existed as a dimer on the cell surface.
  • the bivalent ACE2-Fc protein M05 was coated at 2 ⁇ g/ml overnight into a 96-well ELISA plate (Maxisorp, NUNC) , then was blocked with 1%BSA (bovine serum albumin) solution for 2 hours at room temperature, and the plate was washed three times with PBST.
  • BSA bovine serum albumin
  • TMB substrate Solarbio, PR1200
  • stop buffer Solarbio, C1058
  • Absorbance at 450 nm was measured with a microplate reader (BioTek Elx800) .
  • Data processing was carried out with GraphPad Prism software, and median inhibitory concentration (IC 50 ) was calculated with a four-parameter regression model.
  • 2019-nCoV S protein was packed into a lentiviral particle carrying a luciferase reporter gene to simulate the infection of Hela cells overexpressing ACE2 by the 2019-nCoV virus.
  • the lentivirus was incubated with different concentrations of the test samples at room temperature for 1 hour, then the mixture was incubated with 20,000 Hela cells overexpressing ACE2 for 48 hours in 5%carbon dioxide at 37°C; the supernatant was removed, the cells were rinsed with DPBS and lysed with the lysis buffer, and Bio-Glo TM fluorescent substrate was added for reaction to read the fluorescence value with EnVision; the inhibition curve was drawn and IC 50 was calculated with GraphPad, with the results as shown in Figure 6.
  • Multivalent binding effect that is, tetravalent ACE2-Fc binds up to four 2019-nCoV spikes at the same time. In this case, only when four ACE2 molecules dissociate at the same time, the whole molecule can dissociate from the virus particle, leading to a significant decrease in its dissociation rate (Kd) .
  • Figure 7 shows the schematic diagram of the mechanism of tetravalent ACE2-Fc blocking virus infection.
  • Example 12 Use of (GGGGS) 4 linker peptide in IgG1, IgG2, and IgG4 tetravalent molecules
  • mice Normal healthy female Balb/c mice were intravenously administrated with M05 (10 mg/kg) and M25 (17.4 mg/kg) , and a PBS control group was set up, with 3 mice for each group. Blood pressures of each group of mice were monitored with the blood pressure monitoring system (Visitech Systems, BP-2000 SERIES II) 1 day before administration, 4 hours, 1 day, 2 days, 5 days, and 9 days after administration, and the data was recorded (the instrument automatically recorded data for 10 consecutive times for each mouse, and eliminated the zero values caused by detection failure) . Data processing and graphing was carried out with Excel, and the results were shown in Figure 8.
  • the blood pressure monitoring system Visitech Systems, BP-2000 SERIES II

Abstract

Provided are a fusion protein and its use in the treatment of coronavirus-induced diseases. The fusion protein contains ACE2 portion and Fc segment. The ACE2 portion is selected from ACE2 protein extracellular domain or a mutant thereof, and the extracellular domain contains a fragment of ACE2 PD domain or a mutant thereof. The Fc segment is selected from IgG1-Fc, IgG2-Fc and IgG4-Fc segment or mutants thereof, and IgG1-Fc segment has no ADCC activity.

Description

ACE2-FC FUSION PROTEINS AND USE THEREOF
Field of the Disclosure
The present disclosure relates to Fc fusion protein, especially fusion proteins of ACE2 and Fc and uses thereof.
Background
Due to emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , severe novel coronavirus pneumonia cases account for 13.8%, and critical cases account for 4.7%. Wherein, the mortality rate of the latter is up to 49%. At present, there is no specific antiviral drug for novel coronavirus pneumonia and the clinical therapy is mainly symptomatic treatment supporting vital signs, so it is urgent to develop effective a medicament to inhibit virus infection.
According to the studies, SARS-CoV-2 genome shows a 70%sequence homology with SARS, and its S protein for infection shows a homology of up to 76.47%. Like SARS, this virus infects the host cell by ACE2 protein (angiotensin-converting enzyme 2) , and its affinity with ACE2 reaches 15 nM, 10 to 20 times higher than the affinity between SARS and ACE2 protein. This high affinity makes SARS-CoV-2 highly infectious, and suggests that ACE2 protein can be used as a candidate target for blocking SARS-CoV-2 infection in vivo.
Traditional neutralizing antibodies against viruses often have a single epitope, which may have the disadvantages of low blocking efficiency and the neutralization result affected by virus mutations. However, the ACE2 protein, as an essential receptor for SARS-CoV-2 infection, is not affected by virus mutation and it can be applied to the clinical treatment of SARS and other viruses that also use ACE2 as the infection receptor.
Fc-fusion protein is a biopharmaceutical area that has developed rapidly in recent years. It uses the Fc segment of immunoglobulin as a molecular chaperone, and fuses the functional protein with it by molecular biology approach to greatly prolong the half-life of the functional protein. Fc fusion protein maintains the activity of the functional protein, while having the long half-life as immunoglobulin.
Summary of the Disclosure
The first aspect of the present disclosure is to provide a fusion protein containing a ACE2 portion and a Fc segment; wherein the ACE2 portion is selected from the group consisting of ACE2 extracellular domain or a mutant thereof; a fragment of the  extracellular domain containing ACE2 PD domain (ACE2-PD) or a mutant thereof; the Fc segment is selected from IgG1-Fc, IgG2-Fc and IgG4-Fc or mutants thereof, wherein the IgG1-Fc segment has no ADCC activity.
In one or more embodiments, the ACE2 extracellular domain or the fragment containing ACE2-PD is a wild-type extracellular domain or a fragment thereof containing ACE2-PD; the mutant of the extracellular domain or fragment is an extracellular domain lacking enzyme activity or a fragment thereof containing ACE2-PD, or an extracellular domain with partial or complete mutations of glycosylation sites or a fragment thereof containing ACE2-PD, or an extracellular domain lacking enzyme activity and having partial or complete mutations of glycosylation sites or a fragment thereof containing ACE2-PD, or an extracellular domain with enzyme activity and having partial or complete mutations of glycosylation sites or a fragment thereof containing ACE2-PD.
In one or more embodiments, the mutated glycosylation site includes one or more sites selected from the group consisting of N53, N90, N103, N322, N432, N546 and N690. Preferably, the mutated glycosylation site (s) is mutated to A.
In one or more embodiments, the extracellular domain lacking enzyme activity or the fragment thereof is an extracellular domain or a fragment thereof mutated at site 374 and/or 378; preferably, an extracellular domain or a fragment thereof with mutation of H374N and/or H378N.
In one or more embodiments, the sequence identity of the mutant of the extracellular domain is over 80%to the wild-type ACE2 extracellular domain, preferably over 85%, and more preferably over 90%, over 95%, over 97%, and over 99%; the sequence identity of the mutant of the fragment is over 80%to wild-type ACE2 PD, preferably over 85%, and more preferably over 90%, over 95%, over 97%, and over 99%.
In one or more embodiments, the fusion protein is a bivalent fusion protein containing two ACE2 portions or a tetravalent fusion protein containing four ACE2 portions.
In one or more embodiments, the fusion protein has a reduced ACE2 enzyme activity.
In one or more embodiments, in the bivalent fusion protein, one ACE2 portion is a wild-type ACE2 extracellular domain or a fragment thereof containing the PD, and the other portion is a mutant ACE2 extracellular domain lacking enzyme activity or the fragment thereof containing the ACE2-PD.
In one or more embodiments, in the bivalent fusion protein, both ACE2 portions are  wild-type ACE2 extracellular domains or fragments thereof containing the PD, or mutant ACE2 extracellular domains lacking enzyme activity or fragments thereof containing the ACE2-PD.
In one or more embodiments, in the bivalent fusion protein, at least one ACE2 portion is an extracellular domain lacking enzyme activity and having partial or complete mutations of glycosylation sites or a fragment thereof containing ACE2-PD, or at least one ACE2 portion is an extracellular domain having enzyme activity and having partial or complete mutations of glycosylation sites or a fragment thereof containing ACE2-PD.
In one or more embodiments, in the tetravalent fusion protein, at least one ACE2 portion is a wild-type ACE2 extracellular domain or a fragment thereof containing the PD, or at least one ACE2 portion is an ACE2 extracellular domain lacking enzyme activity or a fragment thereof containing PD; optionally, at least one ACE2 portion has partial or complete mutation of glycosylation sites.
In one or more embodiments, in the tetravalent fusion protein, at least one ACE2 portion is a wild-type extracellular domain or a fragment thereof containing the ACE2-PD, or at least one ACE2 portion is an extracellular domain having enzyme activity and having partial or complete mutations of glycosylation sites or the fragment thereof containing ACE2-PD, or at least one ACE2 portion is an extracellular domain that has no mutation of glycosylation sites without enzyme activity or a fragment thereof containing ACE2-PD, or at least one ACE2 portion is an extracellular domain lacking enzyme activity and having partial or complete mutations of glycosylation sites or the fragment thereof containing ACE2-PD.
In one or more embodiments, the ACE2 portion is linked to Fc through a linker peptide; preferably, the linker peptide contains G and S.
In one or more embodiments, the IgG1 Fc contains the mutation of the N297A glycosylation site, and the IgG4 Fc has S228P mutation and optional N297A mutation.
In one or more embodiments, the two chains of IgG Fc have mutations forming a Knob and a Hole respectively. Preferably, the mutation forming a Knob occurs at site T366, and the mutation forming a Hole occurs at one or more sites selected from the group consisting of T366, L368 and Y407. More preferably, the mutation forming a Knob is T366W, and the mutation forming a Hole is one or more mutations selected from the group consisting of T366S, L368A and Y407V.
In one or more embodiments, the fusion protein is expressed by yeast.
In one or more embodiments, the ACE2-Fc fusion protein is a bivalent fusion protein comprising two ACE2 portions and a Fc, with the two ACE2 portions are directly  linked to the N-terminus of the Fc.
Preferably, the ACE2 portion has a sequence of any of SEQ ID NO: 1-4, and Fc has a sequence of any of SEQ ID NO: 5-8;
Preferably, the ACE2 portion has glycosylation mutation (s) selected from the group consisting of N53A, N90A, N103A, N322A, N432A, N546A and N690A;
Preferably, the two ACE2 portions are identical;
Preferably, the ACE2 portion has the sequence of SEQ ID NO: 1 or 2, and Fc has a sequence of any of SEQ ID NO: 5-7;
Preferably, the ACE2 portion has the sequence of SEQ ID NO: 3 or 4, and Fc has the sequence of SEQ ID NO: 7;
Preferably, the ACE2 portion has the sequence of SEQ ID NO: 1, and Fc has the sequence of SEQ ID NO: 8. Optionally, all ACE2 portions have one or more glycosylation mutations selected from the group consisting of N53A, N90A, N103A, N322A, N432A and N546A.
In one or more embodiments, the ACE2-Fc fusion protein is a tetravalent fusion protein comprising four ACE2 portions and a Fc, with two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two ACE2 portions are respectively linked to the C-terminus of Fc directly or through (GGGGS)  n, wherein n is a positive integer of 1-8;
Preferably, the two ACE2 portions linked to the N-terminus of Fc have a sequence of any of SEQ ID No: 1-4, and the two ACE2 portions linked to the C-terminus of Fc have a sequence of any of SEQ ID No: 1-4;
Preferably, Fc has a sequence of any of SEQ ID NO: 5-8;
Preferably, n is 3, 4, 7 or 8;
Preferably, the two ACE2 portions linked to the N-terminus of Fc are identical, and the two linked to the C-terminus of Fc are identical;
Preferably, the ACE2 portion has glycosylation mutations of N53A, N90A, N103A, N322A, N432A, N546A and N690A;
Preferably, the two ACE2 portions linked to the N-terminus of Fc have the sequence of SEQ ID No: 1 or 2, and the two ACE2 portions linked to the C-terminus of Fc have the sequence of SEQ ID No: 1 or 2, and Fc has a sequence of any of SEQ ID No: 5-7;
Preferably, the two ACE2 portions linked to the N-terminus of Fc have the sequence of SEQ ID No: 3 or 4, and the two ACE2 portions linked to the C-terminus of Fc have the sequence of SEQ ID No: 3 or 4, and Fc has the sequence of SEQ ID No: 7;
Preferably, the two ACE2 portions linked to the N-terminus of Fc have the  sequence of SEQ ID No: 1, the two ACE2 portions linked to the C-terminus of Fc have the sequence of SEQ ID No: 2, and Fc has the sequence of SEQ ID No: 8. Optionally, all ACE2 portions have one or more glycosylation mutations selected from the group consisting of N53A, N90A, N103A, N322A, N432A and N546A.
The present disclosure also provides a nucleic acid molecule selected from: (1) the polynucleotide sequence encoding the fusion protein described herein; (2) the sequence complementary to the polynucleotide sequence in (1) .
The present disclosure also provides a nucleic acid construct containing the nucleic acid molecule described herein.
In one or more embodiments, the nucleic acid construct is a clone vector or an expression vector.
The present disclosure also provides an engineered cell, wherein the engineered cell: (1) expresses the fusion protein described herein; and/or (2) contains the nucleic acid molecule or the nucleic acid construct described herein.
The present disclosure also provides a pharmaceutical composition containing the ACE2-Fc fusion protein, the nucleic acid molecule or the nucleic acid construct described herein, and optional pharmaceutically acceptable carriers or excipients.
The present disclosure also provides the use of the fusion protein, the nucleic acid molecule containing a nucleic acid sequence encoding the fusion protein or its complementary sequence, and the nucleic acid construct containing the nucleic acid molecule in the preparation of medicament for the treatment of coronavirus-induced diseases, wherein the fusion protein contains ACE2 portion and Fc; wherein the ACE2 portion is selected from ACE2 extracellular domain or a fragment thereof or a mutant of the extracellular domain or the fragment thereof, and the fragment of the extracellular domain contains ACE2 PD domain or a mutant thereof; the Fc segment is selected from IgG1-Fc, IgG2-Fc and IgG4-Fc or mutants thereof, and IgG1-Fc segment has no ADCC activity.
In one or more embodiments, coronaviruses are selected from one or more of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, and 2019-nCoV.
In one or more embodiments, the coronavirus-induced diseases include but are not limited to Middle East respiratory syndrome, severe acute respiratory syndrome, coronavirus induced pneumonia, pulmonary arterial hypertension, acute respiratory distress syndrome, heart failure, and novel coronavirus pneumonia.
In one or more embodiments, the fusion protein is as described in the first aspect herein.
The present disclosure also provides the use of the ACE2-Fc fusion protein, the nucleic acid molecule containing a nucleic acid sequence encoding the fusion protein or its complementary sequence, and the nucleic acid construct containing the nucleic acid molecule in the preparation of a medicament for the treatment of acute respiratory distress syndrome (ARDS) , wherein the ACE2-Fc fusion protein contains ACE2 portion and Fc; wherein the ACE2 is selected from ACE2 extracellular domain or a fragment thereof or a mutant of the extracellular domain or the fragment thereof, and the fragment of the extracellular domain contains ACE2 PD domain or a mutant thereof; the Fc segment is selected from IgG1-Fc, IgG2-Fc and IgG4-Fc or mutants thereof, and IgG1-Fc segment has no ADCC activity.
In one or more embodiments, the ACE2-Fc fusion protein is as described in the first aspect herein.
A method for producing ACE2-Fc fusion protein, comprising a step of introducing the nucleic acid construct described herein into cells capable of expressing the ACE2-Fc fusion protein, and incubating the cells under a condition capable of expressing the ACE2-Fc fusion protein.
In one or more embodiments, the cell is a mammalian cell CHO or a Pichia yeast cell.
In one or more embodiments, the ACE2-Fc fusion protein is as described in the first aspect herein, and the nucleic acid construct expresses the ACE2-Fc fusion protein.
Preferably, the ACE2-Fc fusion protein is a bivalent fusion protein comprising two ACE2 portions and a Fc, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and have the sequence of SEQ ID NO: 1, and the Fc has the sequence of SEQ ID No: 8. Optionally, all ACE2 portions have one or more glycosylation mutations selected from the group consisting of N53A, N90A, N103A, N322A, N432A and N546A.
Preferably, the ACE2-Fc fusion protein is a tetravalent fusion protein comprising four ACE2 portions and a Fc. The two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two ACE2 portions are respectively linked to the C-terminus of Fc directly or through (GGGGS)  n, wherein n is a positive integer 1-8; wherein, the two ACE2 portions linked to the N-terminus of Fc have the sequence of SEQ ID No: 1, and the two ACE2 portions linked to the C-terminus of Fc have the sequence of SEQ ID No: 2, and the Fc has the sequence of SEQ ID No: 6, 7 or 8. Optionally, all ACE2 portions have one or more glycosylation mutations selected from the group consisting of N53A, N90A, N103A, N322A, N432A and N546A.
Description of Figures
Figure 1: Schematic diagram of neutralizing virus infection of the fusion protein in the present disclosure.
Figure 2: Schematic diagram of the fusion protein of the disclosure.
Figure 3: Schematic diagram of the mechanism of treating the acute respiratory distress syndrome with the fusion protein of the disclosure.
Figure 4: Comparison of the affinity of M05 and M25 to 2019-nCoV RBD by SPR experiment.
Figure 5: Results of competitive blocking ELISA experiment with bivalent ACE2-Fc as the solid phase and 2019-nCoV RBD as the mobile phase.
Figure 6: Results of pseudovirus neutralization experiment of Hela cells overexpressing ACE2.
Figure 7: Schematic diagram of the mechanism of tetravalent ACE2-Fc in blocking virus infection.
Figure 8: Effect of ACE2-Fc molecules on blood pressure in mice.
Description of the Disclosure
It should be understood that in the scope of the present disclosure, the above technical features of the present disclosure and the technical features described in the following part (e.g. embodiments) can be combined with each other to form a preferred technical solution.
The present disclosure is intended to synthesize an antibody-like molecule binding to the coronavirus itself, thereby neutralizing the infection of the virus, instead of protecting the cells from infection. Specifically, the present disclosure discloses an ACE2-Fc fusion protein as a blocker to inhibit the invasion of novel coronavirus pneumonia. The Fc fusion protein can bind to the FcRn receptor through the Fc segment thereof in the endosome, in order to be recycled outside the cell, thereby avoiding the degradation of the protein by the lysosomal pathway, prolonging the half-life of the drug, cutting down the frequency of administration, increasing the drug efficacy and reducing the treatment costs.
Full-length ACE2 is a transmembrane protein comprising a peptidase domain (PD) at the N-terminus and a Collectrin-like domain (CLD) at the C-terminus. The end of CLD contains a transmembrane helix and an intracellular segment. The peptidase domain (PD) is in the extracellular domain and is in charge of cleaving angiotensin II and angiotensin I. The crystal structure of ACE2-PD also provides a direct binding site for  coronavirus S protein. For the binding of coronavirus cells, PD is essential in ACE2. The ACE2 portion in the ACE2-Fc fusion protein of the present disclosure may be an extracellular domain of ACE2 or a fragment thereof containing the peptidase domain of ACE2, or a mutant of the extracellular domain or the fragment thereof. Specifically, the ACE2 portion may be an extracellular domain of ACE2 or the fragment thereof containing a wild-type ACE2 peptidase domain, or the mutant thereof that retains the activity to bind to a ligand (e.g., virus S protein) . Said mutant may be an extracellular domain of ACE2 losing the peptidase activity of cleaving angiotensin II and angiotensin I by mutation or a fragment thereof containing the peptidase domain, for example, the histidine at the active center site of ACE2 is mutated into other amino acid residues, specifically, the histidine (His) at sites 374 and/or 378 is mutated into a mutant after pretreatment with asparagine (Asn) . The mutant may also be one that retains peptidase activity. In patients infected with 2019-nCoV or SARS, due to the internalization of ACE2 with virus, its expression level is down-regulated, resulting in impairment of its function of regulating renin-angiotensin, thereby leading to pulmonary arterial hypertension, acute respiratory distress syndrome, heart failure and many other symptoms. Therefore, in clinical use, the activity of ACE2 peptidase of ACE2-FC fusion protein can be retained to supplement the above symptoms caused by the absence of ACE2. The mutant also contains an ACE2 extracellular domain that has partial or complete mutation of glycosylation sites and with or without peptidase activity, or a fragment thereof containing ACE2 peptidase domain. The glycosylation sites contain one or more sites selected from the group consisting of 53, 90, 103, 322, 432, 546 and 690 of asparagine residues, as described elsewhere herein. Exemplarily, the active ACE2-PD sequence is as shown in SEQ ID NO: 1, the inactivated ACE2-PD sequence is as shown in SEQ ID NO: 2, the active ACE2-extracellular domain sequence is as shown in SEQ ID NO: 3, and the inactivated ACE2-extracellular domain sequence is as shown in SEQ ID NO: 4. In some embodiments, the mutation in the mutant of the ACE2 extracellular domain occurs outside PD.
ACE2 Fc fusion protein refers to a protein produced by fusing a Fc segment of a immunoglobulin with a ACE2 portion. Fc fusion protein has the biological activity of the ACE2 portion, and also has the properties of antibody, e.g. antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP) . The Fc of the disclosure can be selected from the Fc segment of different subtypes of human IgG1, IgG2, IgG4 and the like. IgG1 often has a strong effect function. By recruiting NK cells or macrophages with Fc γ receptor to kill (ADCC) or phagocytize (ADCP) target cells or pathogens, it plays a clearance effect. Due to the  weak binding affinity for FC γ receptor, IgG2 and IgG4 molecules have a weak effect of ADCC or ADCP. Studies have shown that the binding of Fc segment to Fc γ receptor depends on the glycosylation of aspartic acid at site 297, and the glycosylation site mutation will cause the loss of its activity to bind to Fc γ receptor, thus losing the activity of ADCC and ADCP. For antiviral therapy, ADCC/ADCP has the potential to promote the removal of the virus by the body's immune system, thereby promoting the patient's recovery. However, on the other hand, studies have shown that neutralizing antibodies or fusion proteins may mediate the binding of viruses to macrophages, thus promoting virus infection in macrophages, and then virus amplification and replication in macrophages, resulting in the increase of viral load and negative impact on the course of disease. This effect is called antibody dependent enhancement (ADE) . By using IgG2 or IgG4 subtypes, or adopting Fc engineering strategies, including modification of mutation at the N297 glycosylation site (e.g., N297A) , it reduces the risk of ADE and improves the safety of the drug in patients. Therefore, the Fc segment of the fusion protein of the disclosure may be IgG1-Fc, IgG2-Fc and IgG4-Fc or mutants thereof. These Fc segments can eliminate the activity of ADCC and ADCP by introducing the mutation at N297 site (e.g., mutation A) . And the IgG4 Fc segment can eliminate chain exchange by introducing the mutation at S228 site (e.g., S228P) . Therefore, the Fc segment of the fusion protein of the disclosure is selected from IgG1-Fc with or without mutation at N297 site, IgG2-Fc with or without mutation at N297 site, and IgG4-Fc with or without mutation at sites S228 and/or N297. In specific embodiments, the Fc segment of the fusion protein of the disclosure is selected from IgG1-Fc with mutation at N297 site, Fc segment of wild-type IgG2, and IgG4-Fc with mutation at S228 site and optional N297 site. Exemplarily, IgG1 containing the N297A mutation is shown in SEQ ID NO: 5, wild-type IgG2 is shown in SEQ ID NO: 6, IgG4 containing the S228P mutation is shown in SEQ ID NO: 7, and IgG4 containing N297A and S228P mutations is shown in SEQ ID NO: 8.
The ACE2-Fc fusion protein of the disclosure can have different valences. With the multivalent binding of multiple ACE2 portions to the spike protein of 2019-nCoV, it can simultaneously bind to multiple S proteins on a single spike, or multiple spikes of a virus, or even multiple spikes of multiple viruses to enhance its neutralization activity. Multivalent binding can significantly increase intermolecular forces through the affinity effect, thereby increasing the neutralization effect of blocking virus infection. The bivalent ACE2-Fc fusion protein herein contains two ACE2 portions at the N-terminus of the Fc segment. In the bivalent fusion protein, both ACE2 portions may have or have no peptidase activity, or one of them has peptidase activity while the other does not. For  example, one ACE2 portion is the full-length extracellular domain of the wild type ACE2 or a fragment containing peptidase domain or the mutant thereof with enzyme activity, while the other ACE2 portion is a extracellular domain without enzyme activity or a fragment containing peptidase domain or their mutants without enzyme activity. The tetravalent ACE2-Fc fusion protein herein contains two ACE2 portions at the N-terminus of the Fc segment and two ACE2 portions at the C-terminus of the Fc segment. In the tetravalent ACE2-Fc fusion protein, all or part of the enzyme activity of each ACE2 portion can be retained. That is, only one, two, three or four ACE2 portions in the tetravalent ACE2-Fc fusion protein have peptidase activity. Therefore, at least one ACE2 portion in the tetravalent ACE2-Fc fusion protein may be a wild-type ACE2 extracellular domain or the fragment thereof containing PD or the mutant thereof with enzyme activity, or at least one ACE2 portion may be an ACE2 extracellular domain without enzyme activity or the fragment thereof containing PD or their mutants without enzyme activity. This ensures that the tetravalent ACE2-Fc fusion protein can effectively block virus infection, while preventing excessive ACE2 peptidase activity from causing blood pressure drop and other risks. For example, the two ACE2 portions at the N-terminus of the ACE2-Fc fusion protein are wild-type ACE2 extracellular domains or PDs thereof, with peptidase activity, while the two ACE2 portions at the C-terminus are ACE2 extracellular domains without peptidase activity or PDs thereof due to H374N and/or H378N mutations. Of course, in the ACE2-Fc fusion protein of the disclosure, the two ACE2 portions at the N-terminus can have no peptidase activity, while the two ACE2 portions at the C-terminus have peptidase activity.
ACE2 portions in the fusion protein of the disclosure can be linked to Fc directly or through the common linker peptide. Exemplarily, the linker peptide of the disclosure is a linker containing G and S, including but not limited to (GS) n, (GGS) n, (GGGS) n, (GGGGS) n, wherein G refers to glycine, S refers to serine, and n is a positive integer of 1-15, preferably 1-10, and more preferably 1-8. In specific embodiments, the linker is (GGGGS) n, wherein n is 1, 2, 3, 4, 5, 6, 7, or 8. Other common linker peptides are well known in the art. Generally, when the ACE2 portion is preferably linked to the C-terminus of Fc through the linker, the ACE2 portion can be directly linked to the N-terminus of Fc.
The fusion protein of the disclosure also contains signal peptide, and signal peptide sequence suitable for expression of cellular or subcellular structures is well known in the art. Exemplarily, the ACE2-Fc fusion protein contains the natural signal peptide of ACE2 at the N-terminus: MSSSSWLLLSLVAVTAA (SEQ ID NO: 9) . The natural signal peptide is included in the amino acids numbering of ACE2 described herein only for the  sake of convenience, not to limit whether the fusion protein contains signal peptides or limit the type and sequence of the signal peptides contained.
ACE2, Fc and the fusion protein of the disclosure also include mutants with at least 70%sequence identity that retain the respective required activity thereof. For ACE2, the activity is the capability to bind to viral proteins e.g. S protein; for Fc segment, the activity is the capability to bind to FcRn receptor, thus avoiding degradation of protein by lysosomal pathway; for the fusion protein, the activity is the capability to bind to viral proteins e.g. S protein and FcRn receptor. The mutant includes an amino acid sequence having at least 70%, at least 80%, preferably at least 85%, more preferably at least 90%, at least 95%, at least 97%and at least 99%of sequence identity compared to the reference and retaining the required activity of the reference (e.g., capability to bind to viral proteins e.g. S protein, and/or capability to bind to FcRn receptor) . For example, the sequence identity between two aligned sequences can be calculated by BLASTp of NCBI. The mutant also includes an amino acid sequence that has one or more mutations (insertions, deletions or substitutions) in the amino acid sequence, while still retaining the required activity of the reference sequence. The number of said mutations usually is in a range of 1-50, e.g. 1-20, 1-10, 1-8, 1-5 or 1-3. The substitution is preferably in a conservative manner. For example, when conservative substitutions are made with amino acids of close or similar properties in the art, the function of the protein or polypeptide is usually not changed. "Amino acids with close or similar properties" include, for example, families of amino acid residues with similar side chains, which include amino acids with basic side chains (e.g. lysine, arginine, and histidine) , amino acids with acidic side chains (e.g. aspartic acid, and glutamine) , and amino acids with non-charged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine) , amino acids with non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan) , amino acids with β -branched side chains (e.g. threonine, valine, and isoleucine) and amino acids with aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, and histidine) . Therefore, substitution of one or more sites with another amino acid residue from the same side chain in the polypeptide or protein of the disclosure will not substantially affect its activity.
Exemplary ACE2-Fc fusion proteins of the disclosure are as follows:
M01: a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
M02: a bivalent fusion protein consisting of two inactivated ACE2 portions shown in  SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
M03: a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
M04: a bivalent fusion protein consisting of two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
M05: a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
M06: a bivalent fusion protein consisting of two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
M05F: a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 3 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively;
M06F: a bivalent fusion protein consisting of two inactivated ACE2 portions shown in SEQ ID NO: 4 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
M07: a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M08: a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M09: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M10: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two inactivated ACE2 portions are directly linked  to the N-terminus of Fc, respectively, and the two active ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M11: a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M12: a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M13: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M14: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two inactivated ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two active ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M15: a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  3;
M16: a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M17: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M18: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the  N-terminus of Fc, respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M15F: a tetravalent fusion protein comprising four active ACE2 portions shown in SEQ ID NO: 3 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M16F: a tetravalent fusion protein comprising four inactivated ACE2 portions shown in SEQ ID NO: 4 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M17F: a tetravalent fusion protein comprising two active ACE2 portions shown in SEQ ID NO: 3, two inactivated ACE2 portions shown in SEQ ID NO: 4 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M18F: a tetravalent fusion protein comprising two active ACE2 portions shown in SEQ ID NO: 3, two inactivated ACE2 portions shown in SEQ ID NO: 4 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two inactivated ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two active ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
M19: a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is directly linked to Fc;
M20: a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker GS;
M21: a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker GGS;
M22: a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker GGGS;
M23: a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker GGGGS;
M24: a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker (GGGGS)  2;
M25: a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker (GGGGS)  4;
M26: a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker (GGGGS)  5;
M27: a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker (GGGGS)  6;
M28: a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker (GGGGS)  7;
M29: a protein having the same structure with M17, but the ACE2 portion at the C-terminus of Fc is linked to Fc through the linker (GGGGS)  8;
M30: a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
M31: a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
M32: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  4;
M33: a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
M34: a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
M35: a tetravalent fusion protein comprising four activated ACE2 portions shown in SEQ ID NO: 1 and IgG1 Fc shown in SEQ ID NO: 5, wherein two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
M36: a tetravalent fusion protein comprising four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein two ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
M37: a tetravalent fusion protein comprising two active ACE2 portions shown in  SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
Y01: a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 8, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively;
Y03: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 8, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  4.
To better express in the protein expression system (e.g. Pichia yeast system) , the ACE2 portion in the fusion protein of the disclosure can also be the ACE2 portion with partial or complete mutation of glycosylation sites. These glycosylation sites contain asparagine residues at  sites  53, 90, 103, 322, 432, 546 or 690, and threonine or serine at sites 55, 92, 105, 324, 434, 548 or 692. Their mutation into non-glycosylation patterns (e.g. alanine) can significantly improve the yield, purity and enzyme activity of the fusion protein of the disclosure produced by the Pichia yeast system. In the present disclosure, the ACE2 portion with partial or complete mutation of glycosylation sites may have or have no peptidase activity, for example, the peptidase activity is reduced or lost by the mutation described elsewhere herein.
In multivalent fusion proteins, at least one, two, three, or four ACE2 portions have partial or complete mutation of glycosylation sites. Therefore, the present disclosure also includes the ACE2-Fc fusion protein as described below:
Y02: a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 8, wherein all ACE2 portions have glycosylation mutations: N53A, N90A, N103A, N322A, N432A, N546A, and two ACE2 portions are directly linked to the N-terminus of Fc respectively;
Y04: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 8, wherein the two active ACE2 portions shown in SEQ ID NO: 1 are directly linked to the N-terminus of Fc, respectively, and the two inactivated ACE2 portions shown in SEQ ID NO: 2 are respectively linked to the C-terminus of Fc through the linker (GGGGS)  4. All ACE2 portions have glycosylation mutations: N53A, N90A, N103A, N322A, N432A, N546A.
As described above, the fusion protein of the disclosure may also be heterodimer ACE2-Fc fusion protein at the N (or C) -terminus of Fc with different ACE2 activities. Further, the heterodimer ACE2-Fc fusion protein can also have Knob and Hole mutations in Fc. For example, by introducing a "Knob" mutation in one IgG Fc (e.g., mutation at site T366) and a "Hole" mutation (e.g. mutation at site T366 and/or L368 and/or Y407) into another IgG Fc, a stable heterodimer structure based on the Knob-Hole complementation is created. Therefore, the present disclosure also includes the ACE2-Fc fusion protein as described below:
KIH01: a bivalent fusion protein comprising one active ACE2 portion shown in SEQ ID NO: 1, one inactivated ACE2 portion shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc, respectively, one chain of IgG4 Fc has T366W mutation, and the other chain of IgG4 Fc has one or more mutations selected from the group consisting of T366S, L368A and Y407V.
In some embodiments, a pharmaceutical composition is provided, containing the ACE2 Fc fusion protein described herein or its encoded nucleic acid as an active substance and an optional pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers, excipients, or stabilizers are non-toxic to the recipient of the protein or nucleic acid at the dosage and concentration required, and may include various types of carriers or excipients commonly used in the delivery of protein or nucleic acid in treatment well known in the art. The protein or nucleic acid in the pharmaceutical composition is in a therapeutically effective amount. The effective dosage and dosage form of the protein or nucleic acid can be determined according to the type of active substance, the age, gender and the severity of the disease of the patient receiving the protein or nucleic acid. The protein or nucleic acid described herein may be given by conventional administration, for example, orally or by injection. The administration methods for different dosage forms are well known in the art.
The fusion protein or pharmaceutical composition of the disclosure may be used for the treatment of coronavirus-induced diseases. As mentioned above, the fusion protein of the disclosure may bind to the coronavirus to neutralize its infection, and has an extended half-life. The coronaviruses described herein are enveloped Coronavirus viruses with a single-strand positive-sense RNA, including but not limited to HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, and 2019-nCoV (COVID-19) . Coronavirus S protein (spike, spike protein S) is a key protein for coronaviruses to infect cells, and it plays an important role in the tissue or host tropism and infection of the virus. As shown in Figure 1, the fusion protein of the  disclosure binds to the coronavirus S protein through ACE portion to block or reduce the binding of the coronavirus protein to the host cell to neutralize the infection. Therefore, the fusion protein of the disclosure may be used for the treatment of coronavirus-induced diseases. These diseases include but are not limited to Middle East respiratory syndrome, severe acute respiratory syndrome, acute respiratory distress syndrome, coronavirus induced pneumonia, pulmonary arterial hypertension, acute respiratory distress syndrome, heart failure, and novel coronavirus pneumonia. Based on this, a method for the treatment of coronavirus-induced diseases is also provided, including administering a therapeutically effective amount of the fusion protein or pharmaceutical composition of the disclosure to subjects in need. "Individuals" , "subjects" or "patients" herein refer to mammals, especially humans.
The present disclosure also includes the coding sequences and complementary sequences of various amino acid sequences, polypeptides, proteins or mutants thereof described herein, and nucleic acid constructs containing the coding sequences or complementary sequences. The coding sequence described herein includes a sequence changed by codon optimization, provided the encoded amino acid sequence remains unchanged. The codon-optimized sequence shows more suitable expression for specific species. The method of codon optimization for coding sequences is well known in the art. The nucleic acid constructs herein are artificially constructed segments of nucleic acids that can be introduced into target cells or tissues. The nucleic acid construct contains the coding sequences described herein or complementary sequences thereof, and one or more regulatory sequences operatively linked to these sequences. The regulatory sequence can be an appropriate promoter sequence. The promoter sequence is usually operationally linked to coding sequence of amino acid sequence to be expressed. A promoter, including mutant, truncated and hybrid forms, may be any nucleotide sequence showing transcriptional activity in a selected host cell. It may be obtained from the gene encoding an extracellular or intracellular peptide homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcriptional terminator sequence, identified by host cells to terminate transcription. The terminator sequence is linked to the 3' terminus of the nucleotide sequence encoding the peptide, and any terminator functional in the selected host cell can be used herein.
In some embodiments, the nucleic acid construct is a vector. Specifically, the coding sequence described herein can be cloned into many types of vectors, including but not limited to plasmids, phages, phage derivatives, viruses, and mucous particles. The vector can be an expression vector or a clone vector.
Typically, an appropriate vector contains the replication starting point, the promoter sequence, the convenient limiting enzyme site, and one or more optional markers that work in at least one organism. The representative examples of promoters are lac or trp promoter of E. coli; PL promoter of λ phage; eukaryotic promoter including CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, methanol oxidase promoter of Pichia yeast, and some other known promoters which control the gene expression in the prokaryotic cells, eukaryotic cells or virus. Marker genes can be used to provide phenotypic traits for selection of the transformed host cells, including but not limited to dihydrofolate reductase, neomycin resistance gene and GFP (green florescent protein) for eukaryotic cells, as well as tetracycline or ampicillin resistance gene for E. coli. Transcription of the polynucleotide of the disclosure in higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase the gene transcription.
The skilled in the art know how to select appropriate vectors, promoters, enhancers and host cells. An expression vector containing the sequence of polynucleotide and appropriate transcription/translation control signals can be constructed by methods known by the skilled in the art. These methods include in vitro recombinant DNA technique, DNA synthesis technique, in vivo recombinant technique and the like.
The fusion protein of the disclosure can be prepared by chemical synthesis or biosynthesis. The process and conditions for conventional chemical synthesis of proteins are well known in the art. The biosynthesis method described herein is based on cell expression, including steps of introducing a vector expressing the ACE2-Fc fusion protein described herein into cells capable of expressing the fusion protein and incubating the cells under conditions capable of expressing the fusion protein. The cells include, but are not limited to, mammalian cells CHO or Pichia yeast cells. Components e.g. vectors, promoters and the like required for expression in Pichia yeast cells are known in the art. To better express in the Pichia yeast system, the ACE2 portion in the fusion protein of the disclosure can also be the ACE2 portion with partial or complete mutation of glycosylation sites. Mutated ACE2-Fc fusion proteins at the exemplary glycosylation sites of the disclosure are as described in Y02 and Y04 above.
Host cells or engineered cells expressing the protein described herein, comprising the polynucleotide sequence described herein or a nucleic acid construct thereof are also included herein. The host cells or engineered cells include prokaryote, e.g. bacteria; primary eukaryote, e.g. yeast cell; filamentous fungal cells or advanced eukaryotic, e.g. mammalian cells. The representative examples are E. coli,  Streptomyces, bacterial cells of salmonella typhimurium; fungal cells e.g. yeast, filamentous fungi and plant cells; insect cells e.g. drosophila S2 or Sf9; animal cells e.g. CHO, COS, 293 or Bowes melanoma cell, etc.
The vectors herein can be introduced into host cells by conventional methods, including microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation and the like.
Further, the use of various products involved herein is also included herein, including pharmaceutical use. Specifically, the use of the fusion protein, the nucleic acid molecule, and the nucleic acid construct described herein in the preparation of a medicament for the treatment of coronavirus-induced diseases or acute respiratory distress syndrome is included herein. Coronavirus-induced diseases include but are not limited to Middle East respiratory syndrome, severe acute respiratory syndrome, coronavirus induced pneumonia, pulmonary arterial hypertension, acute respiratory distress syndrome, heart failure, and novel coronavirus pneumonia. The causes of acute respiratory distress syndrome (ARDS) include but are not limited to pneumonia, aspiration, pulmonary contusion, drowning, inhalation of toxic substances; severe systemic infection (bacteria, viruses, fungi and atypical pathogens) , malignant tumors, severe multiple injuries (multiple fractures, flail chest, severe brain trauma and burns) , shock, high-risk surgery (cardiac surgery, arterial surgery, etc. ) , massive blood transfusion, drug poisoning, pancreatitis and cardiopulmonary bypass. The Chronic obstructive pulmonary disease includes but not limited Chronic bronchitis, emphysema, pulmonary heart disease, Respiratory failure. Based on this, a method for the treatment of coronavirus-induced diseases is also provided, including administering a therapeutically effective amount of the fusion protein or pharmaceutical composition of the disclosure to subjects in need thereof.
The present disclosure will be explained herein in the form of specific examples. It should be understood that these examples are merely illustrative, not intended to limit the present disclosure. Unless otherwise specified, the materials and methods used in the examples are those conventional in the art.
The sequences of the disclosure are as follows:
Active ACE2-PD sequence, SEQ ID NO: 1
Figure PCTCN2020112364-appb-000001
Figure PCTCN2020112364-appb-000002
Inactivated ACE2-PD sequence, SEQ ID NO: 2
Figure PCTCN2020112364-appb-000003
Active ACE2-extracellular domain sequence, SEQ ID NO: 3
Figure PCTCN2020112364-appb-000004
Inactivated ACE2-extracellular domain sequence, SEQ ID NO: 4
Figure PCTCN2020112364-appb-000005
Figure PCTCN2020112364-appb-000006
IgG1 with N297A mutation, SEQ ID NO: 5
Figure PCTCN2020112364-appb-000007
Wild-type IgG2, SEQ ID NO: 6
Figure PCTCN2020112364-appb-000008
IgG4 with S228P mutation, SEQ ID NO: 7
Figure PCTCN2020112364-appb-000009
IgG4 with N297A and S228P mutation, SEQ ID NO: 8
Figure PCTCN2020112364-appb-000010
Example 1: Expression of different forms of ACE2-Fc fusion proteins in  HEK293 cells
A series of different forms of ACE2-Fc fusion proteins were designed and the changes were as follows:
1) ACE2 was selected from the peptidase domain (PD, labeled M + number, where M represents mammalian cell expression) or the full-length extracellular domain (labeled M + number + F, where F represents full length) .
2) ACE2 was active or inactivated. For the inactivated form, histidine (His) at sites 374 and 378 was mutated to asparagine (Asn) , and the mutation positions were shown as those underlined in SEQ ID NO: 2 and 4.
3) The Fc segment was selected from human IgG1 Fc, IgG2 Fc, and IgG4 Fc. IgG1 Fc contained N297A glycosylation site mutations to eliminate binding to Fcγreceptors and thereby eliminate the effect functions. IgG4 Fc introduced S228P mutation to eliminate generation of chain exchange.
4) Different valences: when ACE2 molecules were linked to the N-terminus and the C-terminus of Fc at the same time, (GGGGS)  3 linker peptide was added to the C-terminus to connect to ACE2 to form a tetrameric ACE2-Fc molecule. Total designed molecules were as shown in the table below:
Figure PCTCN2020112364-appb-000011
Figure PCTCN2020112364-appb-000012
The amino acid sequences of the above 24 molecules were delivered to Suzhou Genewiz for codon optimization of mammalian cells, synthesis and construction into pTT5 vector, and the signal peptide sequence was selected from the ACE2 natural signal peptide (MSSSSWLLLSLVAVTAA) . The plasmids of said 24 molecules were prepared and 200 μg of plasmid was transfected into 200 ml of logarithmic growth phase HEK293 cells with density of 1 E6/ml by polyethyleneimine (PEI) reagent. After  24 hours of transfection, 5/1000 of peptone was added to promote cell growth. After 7 days of transfection, the harvested cells were centrifuged at 4000 rpm for 30 min, the supernatant was purified with Protein A resin (Bestchrom) after filtration with 0.22 μm membrane and dialyzed into PBS; then it was filtered with 0.22 μm membrane again for storage.
BCA protein concentration quantitative kit (Yeasen Biotech) was used to quantify twenty-four ACE2 fusion proteins according to the operation procedures, and the polymer and purity were analyzed by SEC-HPLC (G3000SWXL, TOSOH) . Results were as follows:
Figure PCTCN2020112364-appb-000013
Figure PCTCN2020112364-appb-000014
Example 2: ELISA experiment for binding of ACE2 Fc fusion proteins with RBD domain of 2019-nCoV S protein
Enzyme-linked immunosorbent assay was conducted on the above 24 molecules to characterize the binding activity of different molecules to 2019-nCoV S protein. 96-well ELISA plate (Maxisorp, NUNC) was incubated with 100 μL of 1 μg/mL 2019-nCoV RBD (COV-VM4BD, Kactus Biosystems) at 4℃ overnight, then was blocked with 1%BSA (bovine serum albumin) solution at room temperature for 2 hours and washed three times with PBST. 100 μl of human ACE2-Fc fusion proteins of different concentrations were respectively added into the 96-well plate to bind to the coated 2019-nCoV RBD at room temperature for 1 hour, and then the plate was washed three times with PBST. Then the anti-Human IgG enzyme HRP (Goat anti-Human IgG Fc Highly Cross-Adsorbed Secondary Antibody, HRP, A18829, 1: 5000, Thermo Fisher Scientific) was added for binding for 0.5 hour at room temperature, and then the plate was washed three times with PBST. Finally, 100 μl of TMB substrate (Solarbio, PR1200) was added to each well for color development, and 50 μl of stop buffer (Solarbio, C1058) to stop the reaction. Absorbance at 450 nm was measured with a microplate reader (BioTek Elx800) . Data processing was carried out with GraphPad Prism software, and half effect concentration (EC50) was calculated with a four-parameter regression model. Results were summarized as follows:
Molecule ID. ELISA EC 50 (nM)
M01 0.263
M02 0.186
M03 0.365
M04 0.334
M05 0.194
M06 0.223
M05F 0.198
M06F 0.263
M07 0.033
M08 0.043
M09 0.037
M10 0.046
M11 0.028
M12 0.036
M13 0.039
M14 0.029
M15 0.018
M16 0.025
M17 0.015
M18 0.017
M15F 0.026
M16F 0.037
M17F 0.023
M18F 0.027
The above experimental results have shown that the binding affinity of the tetramer ACE2-Fc fusion protein is about 10 times higher than that of the dimer form, which may be due to the affinity addition effect (Avidity) between the tetramer and the 2019-nCoV S protein, suggesting that it has stronger activity against 2019-nCoV infection. 
Example 3: ACE2-Fc fusion proteins competitively blocked the binding of 2019-nCoV S protein RBD to ACE2 protein
Competitive enzyme-linked immunosorbent assay was conducted on the above 24 molecules to characterize the blocking activity of different molecules to 2019-nCoV S protein. 96-well ELISA plate (Maxisorp, NUNC) was incubated with 100 μL of 1 μg/mL 2019-nCoV RBD (COV-VM4BD, Kactus Biosystems) at 4℃ overnight, then was blocked with 1%BSA (bovine serum albumin) solution at room temperature for 2 hours and washed three times with PBST. 50 μl of human ACE2-Fc fusion proteins of different concentrations were respectively added into 96 well plate, and 50 μl of ACE2 His-Biotin with a final concentration of 0.3 nM (ACE-HM401 B, Kactus Biosystems) was also added into each well, so that their mixture could bind to the coated 2019-nCoV RBD at room temperature for 1.5 hours, then the plate was washed three times with PBST. Then
Figure PCTCN2020112364-appb-000015
High Sensitivity Streptavidin-HRP (21130, 1: 15000, Thermo Fisher Scientific) was added for binding at room temperature for 1 hour, and then the plate was washed three times with PBST. Finally, 100 μl of TMB substrate (Solarbio, PR1200) was added to each well for color development, and 50 μl of stop buffer (Solarbio, C1058) to stop the reaction. Absorbance at 450 nm was measured with a  microplate reader (BioTek Elx800) . Data processing was carried out with GraphPad Prism software, and median inhibitory concentration (IC 50) was calculated with a four-parameter regression model. Results were summarized as follows:
Molecule ID. ELISA IC 50 (nM)
M01 8.9
M02 7.2
M03 9.6
M04 7.8
M05 8.3
M06 6.5
M05F 9.6
M06F 7.3
M07 0.39
M08 0.62
M09 0.43
M10 0.38
M11 0.36
M12 0.42
M13 0.69
M14 0.81
M15 0.26
M16 0.36
M17 0.23
M18 0.34
M15F 0.27
M16F 0.39
M17F 0.33
M18F 0.42
Example 4: Determination of peptidase activity of ACE2-Fc fusion proteins
Peptidase activity determination of the above 24 molecules was carried out. Use 75 mM Tris, 1 M NaCl, 10 μM ZnCl 2, pH 7.5 Assay Buffer to prepare a 0.2 ng/μL solution of human ACE2-Fc fusion proteins, and dilute the enzyme reaction substrate Mca-Y-V-A-D-A-P-K (Dnp) -OH Fluorogenic Peptide Substrate VI (ES007, R&D) to 40  μM with the same Assay Buffer. Add 250 μL of 0.2 ng/μL human ACE2-Fc fusion protein solution to the cuvette, then add 250 μL of 40 μM reaction substrate solution for reaction, and set the control wells without protein but with Assay Buffer. Use F-180 fluorescence spectrophotometer (Tianjin Gangdong Scientific and Technical Development Co., Ltd. ) to read for 5 minutes in dynamic mode under the excitation wavelength and the emission wavelength of 320 nm and 405 nm, respectively. Calculate the specific activity of human ACE2-Fc fusion proteins by specific activity calculation formula.
Specific activity (pmol/min/μg) = corrected Vmax (RFU/min) x conversion factor (pmol/RFU) /enzyme per well (μg) , where the conversion factor is calculated by making a standard curve based on different concentrations of MCA-Pro-Leu-OH (Bachem, Catalog #M-1975) , and the enzyme amount refers to the human ACE2-Fc fusion proteins.
Results were summarized as follows:
Molecule ID. Peptidase activity (pmol/min/μg)
M01 1025
M02 3.2
M03 1056
M04 4.1
M05 1106
M06 1.8
M05F 1326
M06F 3.6
M07 1436
M08 3.8
M09 823
M10 756
M11 1268
M12 3.5
M13 669
M14 728
M15 1459
M16 2.7
M17 879
M18 754
M15F 1563
M16F 3.8
M17F 987
M18F 862
Example 5: Screening of linker peptides of tetramer ACE2-Fc fusion protein
Linker peptides of ACE2 and C-terminus of Fc were screened on the basis of the M17 molecule, and a series of linker peptides of different lengths were designed as follows:
Figure PCTCN2020112364-appb-000016
Figure PCTCN2020112364-appb-000017
The codons of the above different molecular sequences were optimized, genes were synthesized and cloned into the pTT5 vector; expression and purification, and SEC purity analysis, binding activity test, blocking activity test and enzyme activity test were conducted by the methods described in Examples 1-4. Results were summarized as follows:
Figure PCTCN2020112364-appb-000018
Figure PCTCN2020112364-appb-000019
Example 6: Expression of ACE2-Fc fusion proteins by the Pichia yeast system
Four molecules were selected for expression by the Pichia yeast system based on the above screening results. To avoid the influence of high mannose type glycosylation modification of Pichia yeast system on the half-life and immunogenicity of the drug, glycosylation site (s) in Fc segment of all yeast based proteins were mutated, with the glycosylation site (s) in ACE2 molecule retained or mutated. Four molecules were designed as follows:
Figure PCTCN2020112364-appb-000020
Figure PCTCN2020112364-appb-000021
At ACE2 glycosylation mutation sites, asparagine residues at sites 53, 90, 103, 322, 432 and 546 were mutated to alanine to produce a non-glycosylation pattern. The codons of these four molecules were optimized based on Pichia yeast, genes were synthesized and cloned into Invitrogen pPICZ α vector. According to the instructions of EasySelect Pichia Expression Kit provided by Invitrogen, linearized plasmid was transformed into GS115 competent cells by electroporation, and positive clones were screened by Zeocin resistance. 10 clones were selected from 3000 μg/ml Zeocin resistance plate, a small amount of expression was induced in 200mL of BMMY medium with 0.5%methanol for three days, the supernatant was extracted for Protein A purification, and the above SEC purity analysis, binding activity analysis, inhibitory activity analysis and enzyme activity analysis were carried out for the clone with the highest molecular yield for each protein. Results were summarized as follows:
Figure PCTCN2020112364-appb-000022
Example 7: Investigation of the neutralization effect of ACE2-Fc fusion proteins on 2019-nCoV infection by pseudovirus infection experiment
2019-nCoV S pseudovirus neutralization experiment was conducted on thirteen ACE2-Fc fusion protein molecules selected in example 1-7 to characterize the neutralization and blocking activity of different molecules to 2019-nCoV S protein infection. pCMVR8.2 lentiviral packaging plasmid, pHRCMV-Luc reporter gene plasmid and CMVR-2019-nCoV-Svirus eukaryotic expression plasmid were transiently co-transfected with 293T cells; the supernatant was collected 48 hours after transfection, centrifuged and filtered with a 0.45 μm filter to get the packaged 2019-nCoV S pseudovirus. After subpackaging, some of the pseudoviruses were preserved at -4℃, and some frozen at -80℃ for later use. p24 was tested with HIV-1  p24 test kit and its content was calculated to determine the dose of pseudovirus required for neutralization tests. DNA3-ACE2 eukaryotic expression plasmid was transiently transfected with 293T cells to get 293T-ACE2 cells expressing ACE2 transiently. 1x10 4 293T-ACE2 cells were arranged into each well of 96-well cell culture plate, 100 μL per well, and cultured overnight. Different concentrations of human ACE2-Fc fusion proteins were mixed with 2019-nCoV S pseudovirus solution standardized by p24 content in a volume ratio of 1: 1 for interaction at room temperature for 10 minutes. 100 μl of the mixture of fusion protein and virus was added to the 96-well cell culture plate above for interaction with each other for 16 hours. The culture medium was replaced with a new one (fresh culture medium without virus) , 200 μL per well, and culturing continued for 48 hours. The expression of firefly luciferase was determined with a firefly luciferase detection kit and a microplate reader (TACAN, 
Figure PCTCN2020112364-appb-000023
M1000 Pro multimode microplate reader) . Data processing was carried out with GraphPad Prism software, and median inhibitory concentration (IC50) was calculated with a four-parameter regression model. Results were summarized as follows:
Molecule ID. Inhibitory activity IC 50 (nM)
Y01 3.1
Y02 2.2
Y03 0.33
Y04 0.18
M01 4.2
M02 6.3
M03 3.6
M04 4.8
M05 2.6
M06 2.8
M05F 3.2
M06F 3.0
M25 0.13
Example 8: Production of heterodimer ACE2-Fc by “Knob into Hole” mutation
By introducing the T366W mutation ( "Knob" mutation) in one chain of IgG4 Fc  (containing S228P mutation) , and introducing the T366S/L368A/Y407V mutation ( "Hole" mutation) in the other chain of IgG4 Fc, a stable heterodimer structure based on Knob-Hole complementation was produced. The active and inactivated ACE2 PDs were fused into the above IgG4 Fc (containing S228P) segment containing “Knob” and “Hole” mutations respectively, and the heterodimer molecule is named KIH01. Co-transfection, expression and purification were conducted for plasmids by the method described in Example 1, and their binding activity, competitive inhibitory activity, peptidase activity and virus neutralization activity were tested. The results were summarized as follows:
Figure PCTCN2020112364-appb-000024
Example 9: Comparison of the affinity between M05 and M25 for 2019-nCoV RBD by SPR experiment
In this example, SPR experiment was carried based on Biacore T200 to determine the affinity of M05 and M25 to 2019-nCoV RBD. The anti-His antibody was covalently coupled to the  channels  1, 2, 3, and 4 of the CM5 chip by the amino coupling method, at a coupling level of about 9000 RU.
His-RBD (COV-VM4BD, Kactus Biosystems) as the ligand was diluted to 2 μg/mL with pH7.4, 1×HBS-EP buffer, and captured in 2 channels for 30 s, at a flow rate of 10 μl/min. M05 or M25 as analyte was diluted with buffer to the gradient concentrations of 30 nM, 15 nM, 7.5 nM, 3.125 nM, 1.875 nM and 0.9375 nM, and then flowed through  channels  1 and 2 of the chip for 120 s at a flow rate of 30 μl/min, with the dissociation time of 2400 s. Chip was regenerated for 90s with 10 mM pH1.5 glycine as the regeneration buffer, at a flow rate of 30 μl/min. Channel 1 is the reference channel, and channel 2 is the active channel. After the data was processed by the analysis software and the reference signal was subtracted, the binding and dissociation curves were fit in the 1: 1 binding mode.
The results were shown in Figure 4 and the table below:
Figure PCTCN2020112364-appb-000025
Example 10: Competitive blocking ELISA with bivalent ACE2-Fc as solid phase and 2019-nCoV RBD as mobile phase
ACE2 protein existed as a dimer on the cell surface. To better simulate the role of ACE2-Fc molecules in antagonizing 2019-nCoV infection, the bivalent ACE2-Fc protein M05 was coated at 2 μg/ml overnight into a 96-well ELISA plate (Maxisorp, NUNC) , then was blocked with 1%BSA (bovine serum albumin) solution for 2 hours at room temperature, and the plate was washed three times with PBST. Different concentrations of human ACE2-Fc fusion proteins were pre-mixed with an equal volume of 2019-nCoV RBD His-Biotin (COV-VM4BDB, Kactus Biosystems) with a final concentration of 0.74 nM.100 μL of such mixed sample was added to the 96-well plate in order to bind to the coated ACE2-Fc protein for 1.5 hours at room temperature, and then the plate was washed three times with PBST. Then
Figure PCTCN2020112364-appb-000026
High Sensitivity Streptavidin-HRP (21130, 1: 15000, Thermo Fisher Scientific) was added for binding at room temperature for 1 hour, and then the plate was washed three times with PBST. Finally, 100 μl of TMB substrate (Solarbio, PR1200) was added to each well for color development, and 50 μl of stop buffer (Solarbio, C1058) to stop the reaction. Absorbance at 450 nm was measured with a microplate reader (BioTek Elx800) . Data processing was carried out with GraphPad Prism software, and median inhibitory concentration (IC 50) was calculated with a four-parameter regression model.
The results were shown in Figure 5 and the table below:
Molecule ID ELISA IC50 (nM)
M05 29.48nM
M25 0.1927nM
The results have shown that the competitive activity of the tetravalent ACE2-Fc fusion protein is better than that of the bivalent ACE2-Fc by 153-folds under such experimental condition.
Example 11: Pseudovirus neutralization experiment in Hela cells overexpressing ACE2
2019-nCoV S protein was packed into a lentiviral particle carrying a luciferase reporter gene to simulate the infection of Hela cells overexpressing ACE2 by the 2019-nCoV virus. First, the lentivirus was incubated with different concentrations of the test samples at room temperature for 1 hour, then the mixture was incubated with 20,000 Hela cells overexpressing ACE2 for 48 hours in 5%carbon dioxide at 37℃; the supernatant was removed, the cells were rinsed with DPBS and lysed with the lysis buffer, and Bio-Glo TM fluorescent substrate was added for reaction to read the fluorescence value with EnVision; the inhibition curve was drawn and IC 50 was calculated with GraphPad, with the results as shown in Figure 6.
The above experimental results have shown that the virus blocking activity of tetravalent ACE2-Fc is significantly better than that of bivalent ACE2-Fc. Without restriction by any theory, we believe the possible reasons for the increase in activity include:
1. Multivalent binding effect (Avidity effect) , that is, tetravalent ACE2-Fc binds up to four 2019-nCoV spikes at the same time. In this case, only when four ACE2 molecules dissociate at the same time, the whole molecule can dissociate from the virus particle, leading to a significant decrease in its dissociation rate (Kd) .
2. Longer spatial distance between two ACE2 portions at N-terminus and C-terminus of tetravalent ACE2-Fc than that between two ACE2 portions of bivalent ACE2-Fc molecule, which leads to the possibility that it may simultaneously bind to multiple spikes on virus particles. The cross-linking between the spikes may result in the deformation and inactivation of the virus.
3. Longer distance between two ACE2 portions at N-terminus and C-terminus of the tetravalent ACE2-Fc, providing more flexibility. As a result, different virus particles can be cross-linked through different ACE2 molecules to promote virus aggregation, which will cause the virus to become less mobile and become unable to infect, eventually being cleared by the human body.
Figure 7 shows the schematic diagram of the mechanism of tetravalent ACE2-Fc blocking virus infection.
Example 12: Use of (GGGGS)  4 linker peptide in IgG1, IgG2, and IgG4 tetravalent molecules
By use of (GGGGS)  4 linker peptide in IgG1, IgG2 and IgG4 tetravalent molecules, the following molecules were obtained:
Figure PCTCN2020112364-appb-000027
Figure PCTCN2020112364-appb-000028
Gene synthesis, vector construction, competitive ELISA (with ACE2-Fc used as a solid phase) , SEC, enzyme activity and other characterization were conducted by the method described in the examples. The results were as follows:
Figure PCTCN2020112364-appb-000029
Example 13: Investigation of the effect of ACE2-Fc molecules on blood pressure in mice
Normal healthy female Balb/c mice were intravenously administrated with M05 (10 mg/kg) and M25 (17.4 mg/kg) , and a PBS control group was set up, with 3 mice for each group. Blood pressures of each group of mice were monitored with the blood  pressure monitoring system (Visitech Systems, BP-2000 SERIES II) 1 day before administration, 4 hours, 1 day, 2 days, 5 days, and 9 days after administration, and the data was recorded (the instrument automatically recorded data for 10 consecutive times for each mouse, and eliminated the zero values caused by detection failure) . Data processing and graphing was carried out with Excel, and the results were shown in Figure 8.
From Figure 8, after the mice were treated with M05 and M25, both systolic and diastolic blood pressures remained at normal physiological levels.

Claims (20)

  1. A fusion protein, wherein the fusion protein contains a ACE2 portion and a Fc segment; the ACE2 portion is selected from the group consisting of ACE2 extracellular domain or a mutant thereof, a fragment of the extracellular domain containing ACE2 PD domain or a mutant thereof; the Fc segment is selected from IgG1-Fc, IgG2-Fc and IgG4-Fc segment or mutants thereof, and IgG1-Fc segment has no ADCC activity.
  2. The fusion protein according to Claim 1, wherein the ACE2 protein extracellular domain or the fragment thereof containing ACE2-PD is a wild-type extracellular domain or a fragment thereof containing ACE2-PD; the mutant of the extracellular domain or the fragment is an extracellular domain lacking enzyme activity or a fragment containing ACE2-PD, or an extracellular domain with partial or complete mutation of glycosylation sites or a fragment thereof containing ACE2-PD, or an extracellular domain lacking enzyme activity and having partial or complete mutation of glycosylation sites without enzyme activity or a fragment thereof containing ACE2-PD, or an extracellular domain with enzyme activity and having partial or complete mutations of glycosylation sites or a fragment thereof containing ACE2-PD; and/or
    the IgG1 Fc contains the mutation of the N297A glycosylation site, and the IgG4 Fc has S228P mutation and optional N297A mutation, or two chains of IgG Fc have mutations forming Knob and Hole, respectively, preferably, the mutation forming Knob occurs at site T366, and the mutation forming Hole occurs at one or more sites selected from the group consisting of T366, L368 and Y407.
  3. The fusion protein according to Claim 2, wherein the extracellular domain lacking enzyme activity or the fragment thereof is an extracellular domain or a fragment thereof mutated at site 374 and/or 378; preferably, an extracellular domain or the fragment thereof with mutation of H374N and/or H378N.
  4. The fusion protein according to Claim 1, wherein the sequence identity of the mutant of the extracellular domain to the wild-type ACE2 extracellular domain is over 80%, preferably over 85%, and more preferably over 90%, over 95%, over 97%, and over 99%; the sequence identity of the mutant of the fragment to wild-type ACE2 PD is over 80%, preferably over 85%, and more preferably over 90%, over 95%, over 97%, and over 99%; preferably, the amino acid sequence of the extracellular domain of the wild-type ACE2 protein is SEQ ID No: 3, and the amino acid sequence of the PD of the wild-type ACE2 protein is SEQ ID No: 1.
  5. The fusion protein according to Claim 1, wherein, the amino acid sequences of the extracellular domain of ACE2 protein and the mutant thereof are SEQ ID No: 3 and SEQ ID No: 4, respectively; the amino acid sequences of ACE2-PD and the mutant  thereof are SEQ ID No: 1 and SEQ ID No: 2, respectively; the amino acid sequence of the mutant of the IgG1 Fc segment is SEQ ID No: 5; the amino acid sequence of the IgG2 Fc segment is SEQ ID No: 6; the amino acid sequence of the mutant of the IgG4 Fc segment is SEQ ID No: 7 or 8; the amino acid sequence of the IgG4 Fc segment is SEQ ID No: 7, but the amino acid residue at site 228 is S.
  6. The fusion protein according to any term of Claim 1-5, wherein the fusion protein is a bivalent fusion protein containing two ACE2 portions or a tetravalent fusion protein containing four ACE2 portions.
  7. The fusion protein according to Claim 1, wherein the ACE2 enzyme activity of the fusion protein is reduced.
  8. The fusion protein according to Claim 6, wherein in the bivalent fusion protein, one ACE2 portion is a wild-type ACE2 extracellular domain or a fragment thereof containing the ACE2-PD, and the other ACE2 portion is a mutant ACE2 extracellular domain without enzyme activity or a fragment thereof containing the ACE2-PD, or
    in the bivalent fusion protein, both ACE2 portions are wild-type ACE2 extracellular domains or fragments thereof containing the ACE2-PD, or both ACE2 portions are mutant ACE2 extracellular domains without enzyme activity or fragments thereof containing the ACE2-PD, or
    in the bivalent fusion protein, at least one ACE2 portion is an extracellular domain lacking enzyme activity and having partial or complete mutation of glycosylation sites or a fragment thereof containing ACE2-PD, or at least one ACE2 portion is an extracellular domain with enzyme activity and having partial or complete mutation of glycosylation sites or a fragment thereof containing ACE2-PD.
  9. The fusion protein according to Claim 8, wherein in the tetravalent fusion protein, at least one ACE2 portion is a wild-type ACE2 extracellular domain or a fragment thereof containing the ACE2-PD, or at least one ACE2 portion is an ACE2 extracellular domain without enzyme activity or the fragment thereof containing ACE2-PD; optionally, at least one ACE2 portion has partial or complete mutation of glycosylation sites.
  10. The fusion protein according to Claim 1, wherein the ACE2 portion is linked to the Fc segment through a linker peptide; preferably, the linker contains G and S.
  11. A fusion protein, comprising an ACE2 portion and a Fc segment, wherein the fusion protein is in a tetravalent form containing four ACE2 portions; wherein,
    the four ACE2 portions are: four PDs of ACE2 protein shown in SEQ ID NO: 1; or four PD mutants of ACE2 protein shown in SEQ ID NO: 2; or two PDs of ACE2 protein shown in SEQ ID NO: 1 and two PD mutants of ACE2 protein shown in SEQ ID NO: 2; or four ACE2 protein extracellular domains shown in SEQ ID NO: 3; or four ACE2  protein extracellular domain mutants shown in SEQ ID NO: 4; or two ACE2 protein extracellular domains shown in SEQ ID NO: 3 and two ACE2 protein extracellular domain mutants shown in SEQ ID NO: 4;
    the amino acid of the Fc segment is as shown in SEQ ID NO: 5-8;
    two of the four ACE2 portions are respectively linked to the C-terminus of the Fc segment through the same or different linkers, and the other two are respectively directly linked to the N-terminus of the Fc segment;
    the linker is (GGGGS)  n, wherein n is an integer of 2-5.
  12. A fusion protein selected from:
    M01: a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively;
    M02: a bivalent fusion protein consisting of two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively;
    M03: a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively;
    M04: a bivalent fusion protein consisting of two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively;
    M05: a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively;
    M06: a bivalent fusion protein consisting of two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively;
    M05F: a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 3 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively;
    M06F: a bivalent fusion protein consisting of two inactivated ACE2 portions shown in SEQ ID NO: 4 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively;
    M07: a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M08: a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M09: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M10: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein the two inactivated ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two active ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M11: a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M12: a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M13: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M14: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two inactivated ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two active ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M15: a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  3;
    M16: a tetravalent fusion protein consisting of four inactivated ACE2 portions  shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M17: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M18: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M15F: a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 3 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M16F: a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 4 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M17F: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 3, two inactivated ACE2 portions shown in SEQ ID NO: 4 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M18F: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 3, two inactivated ACE2 portions shown in SEQ ID NO: 4 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two inactivated ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two active ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  3;
    M19: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are directly linked to the C-terminus of Fc respectively;
    M20: a tetravalent fusion protein consisting of two active ACE2 portions shown in  SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are linked to the C-terminus of Fc through the linker GS respectively;
    M21: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are linked to the C-terminus of Fc through the linker GGS respectively;
    M22: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are linked to the C-terminus of Fc through the linker GGGS respectively;
    M23: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are linked to the C-terminus of Fc through the linker GGGGS respectively;
    M24: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  2;
    M25: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  4;
    M26: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  5;
    M27: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the  N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  6;
    M28: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  7;
    M29: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  8;
    M30: a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
    M31: a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
    M32: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG2 Fc shown in SEQ ID NO: 6, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  4;
    M33: a tetravalent fusion protein consisting of four active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
    M34: a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
    M35: a tetravalent fusion protein consisting of four activated ACE2 portions shown in SEQ ID NO: 1 and IgG1 Fc shown in SEQ ID NO: 5, wherein two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
    M36: a tetravalent fusion protein consisting of four inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
    M37: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG1 Fc shown in SEQ ID NO: 5, wherein two ACE2 portions are directly linked to the N-terminus of Fc respectively, and the other two are respectively linked to the C-terminus of Fc through (GGGGS)  4;
    Y01: a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 8, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively;
    Y03: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 8, wherein the two active ACE2 portions are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions are respectively linked to the C-terminus of Fc through the linker (GGGGS)  4;
    Y02: a bivalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1 and IgG4 Fc shown in SEQ ID NO: 8, wherein all ACE2 portions have glycosylation mutations: N53A, N90A, N103A, N322A, N432A and N546A, and the two ACE2 portions are directly linked to the N-terminus of Fc respectively;
    Y04: a tetravalent fusion protein consisting of two active ACE2 portions shown in SEQ ID NO: 1, two inactivated ACE2 portions shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 8, wherein the two active ACE2 portions shown in SEQ ID NO: 1 are directly linked to the N-terminus of Fc respectively, and the two inactivated ACE2 portions shown in SEQ ID NO: 2 are respectively linked to the C-terminus of Fc through the linker (GGGGS)  4, wherein all ACE2 portions have glycosylation mutations: N53A, N90A, N103A, N322A, N432A and N546A;
    KIH01: a bivalent fusion protein consisting of one active ACE2 portion shown in SEQ ID NO: 1, one inactivated ACE2 portion shown in SEQ ID NO: 2 and IgG4 Fc shown in SEQ ID NO: 7, wherein the two ACE2 portions are directly linked to the N-terminus of Fc respectively, one chain of IgG4 Fc has T366W mutation, and the other chain of IgG4 Fc has one or more mutations of T366S, L368A and Y407V.
  13. The fusion protein according to any of Claim 1-12, wherein the fusion protein is expressed by yeast.
  14. A nucleic acid molecule selected from:
    (2) the polynucleotide sequence encoding the fusion protein according to any of  Claim 1-13;
    (3) the sequence complementary to the polynucleotide sequence in (1) .
  15. The nucleic acid construct containing the nucleic acid molecules according to Claim 14.
  16. The nucleic acid construct according to Claim 15, wherein the nucleic acid construct is a cloning vector or an expression vector.
  17. The engineered cell, wherein the engineered cell:
    (2) expresses the fusion protein according to any of Claim 1-13; and/or
    (3) contains the nucleic acid molecule according to Claim 14 or the nucleic acid construct according to Claim 15 or 16.
  18. Use of the fusion protein according to any of Claim 1-13, the nucleic acid molecule according to Claim 14, and the nucleic acid construct according to Claim 15 or 16 in the preparation of a medicament for the treatment of coronavirus-induced diseases.
  19. Use of the fusion protein according to any of Claim 1-13, the nucleic acid molecule according to Claim 14, and the nucleic acid construct according to Claim 15 or 16 in the preparation of a medicament for the treatment of acute respiratory distress syndrome, Chronic obstructive pulmonary disease and hypertension.
  20. A pharmaceutical composition containing the fusion protein according to any of Claim 1-13.
PCT/CN2020/112364 2020-03-25 2020-08-31 Ace2-fc fusion proteins and use thereof WO2021189772A1 (en)

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WO2022020353A3 (en) * 2020-07-20 2022-03-10 Dana-Farber Cancer Institute, Inc. Methods and compositions for treatment and prevention of coronavirus infection
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WO2023094507A1 (en) 2021-11-24 2023-06-01 Formycon Ag Improved ace2 fusion proteins
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WO2021203103A3 (en) * 2020-04-03 2021-12-16 Somasekar Seshagiri Ace2 receptor polymorphisms and varying susceptibility to sars-cov-2, methods for diagnosis and treatment
WO2022020353A3 (en) * 2020-07-20 2022-03-10 Dana-Farber Cancer Institute, Inc. Methods and compositions for treatment and prevention of coronavirus infection
WO2022090469A2 (en) 2020-10-29 2022-05-05 Formycon Ag Ace2 fusion proteins and uses thereof
WO2022184854A2 (en) 2021-03-03 2022-09-09 Formycon Ag Formulations of ace2 fc fusion proteins
WO2023006935A3 (en) * 2021-07-30 2023-03-30 Formycon Ag Ace2 fusion proteins and uses thereof
WO2023094507A1 (en) 2021-11-24 2023-06-01 Formycon Ag Improved ace2 fusion proteins
WO2023094571A1 (en) 2021-11-25 2023-06-01 Formycon Ag Stabilization of ace2 fusion proteins
EP4331571A1 (en) 2022-09-02 2024-03-06 Formycon AG Formulations of ace2-igm fusion proteins

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