ACE2-fc fusion proteins and uses thereof

Abstract

The present application relates to a fusion protein and its application in treating coronavirus-induced diseases, wherein the fusion protein comprises an ACE2 moiety and an Fc segment; the ACE2 moiety is selected from the group consisting of an ACE2 protein extracellular domain or a mutant thereof, and the extracellular domain comprises a fragment of an ACE2 protein PD domain or a mutant thereof; the Fc segment is selected from the group consisting of an Fc segment of IgG1, IgG2 and IgG4 or a mutant thereof, and the Fc segment of IgG1 has no ADCC activity.

Description

Technical Field

[0001] This invention relates to Fc fusion proteins, particularly ACE2-Fc fusion proteins and their uses. Background Technology

[0002] The proportion of severe cases of COVID-19 caused by the novel coronavirus (SARS-CoV-2) is 13.8%, and the proportion of critical cases is 4.7%, with the mortality rate of critical patients reaching as high as 49%. Currently, there are no specific antiviral drugs for COVID-19, and clinical treatment mainly focuses on symptomatic treatment to support vital signs. Therefore, there is an urgent need to develop effective therapeutic drugs that can inhibit viral infection.

[0003] Studies show that the genome sequence of the novel coronavirus (SARS-CoV-2) maintains 70% homology with that of the SARS virus, and its S protein, which plays an infectious role, shares 76.47% homology. Like SARS, this virus infects through the ACE2 protein (angiotensin-converting enzyme 2), and its affinity for ACE2 reaches 15 nM, which is 10 to 20 times stronger than its binding affinity to the SARS S protein. This high affinity contributes to the high infectivity of 2019-nCoV and suggests that the ACE2 protein could be used as a candidate target for blocking SARS-CoV-2 infection in vivo.

[0004] Traditional neutralizing antibodies against viruses often have a single epitope, which may have the disadvantages of low blocking efficiency and the neutralizing effect being affected by viral mutations. In contrast, the ACE2 protein, as an essential receptor for SARS-CoV-2 infection, is not affected by viral mutations and can be applied to the clinical treatment of SARS and other viruses that also use ACE2 as an infection receptor.

[0005] Fc fusion proteins are a rapidly developing area of ​​biopharmaceutical research in recent years. They utilize the Fc fragment of immunoglobulins as molecular chaperones, fusing functional proteins with them through molecular biology techniques, thereby significantly extending the half-life of the functional protein. Fc fusion proteins retain the activity of the functional protein while also possessing the long half-life and cytotoxicity of immunoglobulins. Summary of the Invention

[0006] The first aspect of the present invention provides a fusion protein comprising an ACE2 portion and an Fc segment; wherein the ACE2 portion is selected from: the extracellular domain of ACE2 protein or a mutant thereof, a fragment of the extracellular domain containing the PD domain of ACE2 protein or a mutant thereof; the Fc segment is selected from the Fc segments of IgG1, IgG2 and IgG4 or mutants thereof, and the Fc segment of IgG1 has no ADCC activity.

[0007] In one or more embodiments, the extracellular domain of the ACE2 protein or a fragment containing the ACE2 protein PD domain is the wild-type extracellular domain or a fragment containing the ACE2 protein PD domain; the mutant of the extracellular domain or fragment is an inactive extracellular domain or a fragment containing the ACE2 protein PD domain, or an extracellular domain or a fragment containing the ACE2 protein PD domain with partial or complete mutations at the glycosylation site, or an inactive extracellular domain or a fragment containing the ACE2 protein PD domain with partial or complete mutations at the glycosylation site, or an active extracellular domain or a fragment containing the ACE2 protein PD domain with partial or complete mutations at the glycosylation site.

[0008] In one or more embodiments, the mutated glycosylation site includes one or more sites selected from N53, N90, N103, N322, N432, N546, and N690. Preferably, the mutated glycosylation site is mutated to A.

[0009] In one or more embodiments, the non-enzymatic extracellular domain or fragment thereof is an extracellular domain or fragment thereof with a mutation at position 374 and / or 378; preferably, it is an extracellular domain or fragment thereof with a mutation at position H374N and / or H378N.

[0010] In one or more embodiments, the mutant of the extracellular domain has a sequence identity of 80% or more with the extracellular domain of the wild-type ACE2 protein, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and more preferably 99% or more; the mutant of the fragment has a sequence identity of 80% or more with the PD domain of the wild-type ACE2 protein, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and more preferably 99% or more.

[0011] In one or more embodiments, the fusion protein is a bivalent form containing two ACE2 moieties, or a tetravalent form containing four ACE2 moieties.

[0012] In one or more embodiments, the fusion protein has reduced ACE2 activity.

[0013] In one or more embodiments, in the bivalent fusion protein, one ACE2 portion is the wild-type ACE2 extracellular domain or a fragment containing the PD domain, and the other ACE2 portion is the non-enzymatic mutant ACE2 protein extracellular domain or a fragment containing the ACE2 protein PD domain.

[0014] In one or more embodiments, in the bivalent fusion protein, both ACE2 portions are either wild-type ACE2 extracellular domains or fragments containing the PD domain, or both ACE2 portions are either non-enzymatic mutant ACE2 protein extracellular domains or fragments containing the PD domain of ACE2 protein.

[0015] In one or more embodiments, in the bivalent fusion protein, at least one ACE2 portion is an extracellular domain without enzymatic activity and with partial or complete mutations at the glycosylation site, or a fragment of the extracellular domain containing the ACE2 protein PD domain, or at least one ACE2 portion is an extracellular domain with enzymatic activity but with partial or complete mutations at the glycosylation site, or a fragment of the extracellular domain containing the ACE2 protein PD domain.

[0016] In one or more embodiments, in the tetravalent form of the fusion protein, at least one ACE2 portion is a wild-type ACE2 extracellular domain or a fragment containing a PD domain thereof, or at least one ACE2 portion is an inactive ACE2 extracellular domain or a fragment containing a PD domain thereof; optionally, at least one ACE2 portion has a partial or complete glycosylation site mutated.

[0017] In one or more embodiments, in the tetravalent fusion protein, at least one ACE2 portion is a wild-type extracellular domain or a fragment containing the ACE2 protein PD domain, or at least one ACE2 portion is an extracellular domain with enzymatic activity but with partial or complete mutations at the glycosylation site or a fragment containing the ACE2 protein PD domain, or at least one ACE2 portion is an extracellular domain without enzymatic activity and without mutations at the glycosylation site or a fragment containing the ACE2 protein PD domain, or at least one ACE2 portion is an extracellular domain without enzymatic activity and with partial or complete mutations at the glycosylation site or a fragment containing the ACE2 protein PD domain.

[0018] In one or more embodiments, the ACE2 portion is linked to Fc via a linker peptide; preferably, the linker peptide is a linker containing G and S.

[0019] In one or more embodiments, the IgG1 Fc contains an N297A glycosylation site mutation, and the IgG4 Fc has an S228P mutation and optionally an N297A mutation.

[0020] In one or more embodiments, the two IgG Fc cells each have a club-forming mutation and a mortise-forming mutation, preferably, the club-forming mutation is a mutation at the T366 site, and the mortise-forming mutation is a mutation selected from one or more sites of T366, L368, and Y407, more preferably, the club-forming mutation is T366W, and the mortise-forming mutation is selected from one or more sites of T366S, L368A, and Y407V.

[0021] In one or more embodiments, the fusion protein is expressed from yeast.

[0022] In one or more embodiments, the ACE2-Fc fusion protein is a bivalent fusion protein composed of two ACE2 moieties and an Fc, with each ACE2 moieties directly linked to the N-terminus of the Fc.

[0023] Preferably, the ACE2 portion has any of the sequences described in SEQ ID NO:1-4, and the Fc portion has any of the sequences described in SEQ ID NO:5-8;

[0024] Preferably, the ACE2 moiety has a glycosylation mutation selected from the following: N53A, N90A, N103A, N322A, N432A, N546A, N690A;

[0025] Preferably, the two ACE2 parts are identical;

[0026] Preferably, the ACE2 portion has the sequence described in SEQ ID NO:1 or 2, and the Fc portion has the sequence described in any of SEQ ID NO:5-7;

[0027] Preferably, the ACE2 portion has the sequence described in SEQ ID NO:3 or 4, and the Fc portion has the sequence described in SEQ ID NO:7;

[0028] Preferably, the ACE2 portion has the sequence described in SEQ ID NO:1, and the Fc portion has the sequence described in SEQ ID NO:8. Optionally, each ACE2 portion has one or more or all of the following glycosylation mutations: N53A, N90A, N103A, N322A, N432A, and N546A.

[0029] In one or more embodiments, the ACE2-Fc fusion protein is a tetravalent fusion protein composed of four ACE2 moieties and an Fc, with two ACE2 moieties directly linked to the N-terminus of the Fc, and the other two ACE2 moieties directly or via (GGGGS) linked to the C-terminus of the Fc. n Connect, where n is a positive integer from 1 to 8.

[0030] Preferably, the two ACE2 portions connected to the N-terminus of Fc have any of the sequences described in SEQ ID NO: 1-4, and the two ACE2 portions connected to the C-terminus of Fc have any of the sequences described in SEQ ID NO: 1-4.

[0031] Preferably, Fc has any of the sequences described in SEQ ID NO:5-8.

[0032] Preferably, n is 3, 4, 7 or 8;

[0033] Preferably, the two ACE2 portions connected to the N end of Fc are identical, and the two ACE2 portions connected to the C end of Fc are identical;

[0034] Preferably, the ACE2 moiety has a glycosylation mutation selected from the following: N53A, N90A, N103A, N322A, N432A, N546A, N690A;

[0035] Preferably, the two ACE2 portions connected to the N end of Fc have the sequence described in SEQ ID NO:1 or 2, the two ACE2 portions connected to the C end of Fc have the sequence described in SEQ ID NO:1 or 2, and Fc has any of the sequences described in SEQ ID NO:5-7;

[0036] Preferably, the two ACE2 portions connected to the N end of Fc have the sequence described in SEQ ID NO:3 or 4, the two ACE2 portions connected to the C end of Fc have the sequence described in SEQ ID NO:3 or 4, and Fc has the sequence described in SEQ ID NO:7;

[0037] Preferably, the two ACE2 portions connected to the N-terminus of Fc have the sequence described in SEQ ID NO:1, the two ACE2 portions connected to the C-terminus of Fc have the sequence described in SEQ ID NO:2, and Fc has the sequence described in SEQ ID NO:8. Optionally, each ACE2 portion has one or more or all of the following glycosylation mutations: N53A, N90A, N103A, N322A, N432A, N546A.

[0038] The present invention also provides a nucleic acid molecule selected from: (1) a polynucleotide sequence encoding the fusion protein described herein; and (2) a complementary sequence of the polynucleotide sequence described herein.

[0039] The present invention also provides a nucleic acid construct containing the nucleic acid molecules described herein.

[0040] In one or more embodiments, the nucleic acid construct is a cloning vector or an expression vector.

[0041] The present invention also provides an engineered cell, characterized in that the engineered cell: (1) expresses the fusion protein described herein; and / or (2) contains the nucleic acid molecule described herein, or the nucleic acid construct described herein.

[0042] This article also provides a pharmaceutical composition comprising the ACE2-Fc fusion protein described herein, the nucleic acid molecule described herein, or the nucleic acid construct described herein, and optionally a pharmaceutically acceptable carrier or excipient.

[0043] The present invention also provides the use of a fusion protein, a nucleic acid molecule containing a nucleic acid sequence encoding the fusion protein or its complementary sequence, and a nucleic acid construct containing the nucleic acid molecule in the preparation of a medicament for treating diseases caused by coronaviruses, wherein the fusion protein contains an ACE2 portion and an Fc segment; wherein the ACE2 portion is selected from: the extracellular domain of the ACE2 protein or a fragment thereof or a mutant of the extracellular domain or a fragment thereof, the fragment of the extracellular domain containing the PD domain of the ACE2 protein or a mutant thereof; the Fc segment is selected from the Fc segments of IgG1, IgG2 and IgG4 or mutants thereof, and the Fc segment of IgG1 has no ADCC activity.

[0044] In one or more embodiments, the coronavirus is selected from one or more of the following: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, 2019-nCoV.

[0045] In one or more implementations, diseases caused by coronaviruses include, but are not limited to, Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARDS), coronavirus pneumonia, pulmonary hypertension, acute respiratory distress syndrome (ARDS), heart failure, and COVID-19.

[0046] In one or more embodiments, the fusion protein is as described in the first aspect herein.

[0047] The present invention also provides the use of an ACE2-Fc fusion protein, a nucleic acid molecule containing a nucleic acid sequence encoding the fusion protein or its complementary sequence, and a nucleic acid construct containing the nucleic acid molecule in the preparation of a medicament for treating acute respiratory distress syndrome (ARDS), wherein the ACE2-Fc fusion protein contains an ACE2 portion and an Fc portion; wherein the ACE2 portion is selected from: the extracellular domain of the ACE2 protein or a fragment thereof or a mutant of the extracellular domain or a fragment thereof, the fragment of the extracellular domain containing the PD domain of the ACE2 protein or a mutant thereof; the Fc portion is selected from the Fc portions of IgG1, IgG2 and IgG4 or mutants thereof, and the Fc portion of IgG1 has no ADCC activity.

[0048] In one or more embodiments, the ACE2-Fc fusion protein is as described in the first aspect herein.

[0049] A method for producing an ACE2-Fc fusion protein includes the steps of introducing the nucleic acid construct described herein into cells capable of expressing the ACE2-Fc fusion protein and incubating the cells under conditions capable of expressing the ACE2-Fc fusion protein.

[0050] In one or more embodiments, the cell is a mammalian cell CHO or a Pichia pastoris cell.

[0051] 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.

[0052] Preferably, the ACE2-Fc fusion protein is a bivalent fusion protein composed of two ACE2 moieties and an Fc, wherein the two ACE2 moieties are directly linked to the N-terminus of the Fc, the ACE2 moieties have the sequence described in SEQ ID NO:1, and the Fc has the sequence described in SEQ ID NO:8. Optionally, each ACE2 moieties has one or more or all of the following glycosylation mutations: N53A, N90A, N103A, N322A, N432A, N546A.

[0053] Preferably, the ACE2-Fc fusion protein is a tetravalent fusion protein composed of four ACE2 moieties and Fc, with two ACE2 moieties directly connected to the N-terminus of Fc, and the other two ACE2 moieties directly connected to the C-terminus of Fc or connected via (GGGGS). n The connection is a positive integer n from 1 to 8, wherein the two ACE2 portions connected to the N end of Fc have the sequence described in SEQ ID NO:1, the two ACE2 portions connected to the C end of Fc have the sequence described in SEQ ID NO:2, and Fc has the sequence described in SEQ ID NO:8. Optionally, each ACE2 portion has one or more or all of the following glycosylation mutations selected from: N53A, N90A, N103A, N322A, N432A, N546A. Attached Figure Description

[0054] Figure 1 : Schematic diagram of the fusion protein of the present invention neutralizing viral toxicity.

[0055] Figure 2 : Schematic diagram of the fusion protein involved in this invention.

[0056] Figure 3 This invention relates to a schematic diagram of the mechanism of action of the fusion protein in treating acute respiratory distress syndrome. Detailed Implementation

[0057] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form preferred technical solutions.

[0058] This invention aims to synthesize an antibody-like molecule that binds to the coronavirus itself, thereby neutralizing viral toxicity rather than protecting cells from infection. Specifically, this invention discloses an ACE2-Fc fusion protein as a blocking agent for inhibiting COVID-19 infection. The Fc fusion protein can be circulated extracellularly via its Fc fragment binding to the FcRn receptor in the endosome, avoiding lysosomal degradation, extending the drug's half-life, reducing the frequency of administration, increasing drug efficacy, and lowering treatment costs.

[0059] Full-length ACE2 is a transmembrane protein composed of an N-terminal peptidase domain (PD) and a C-terminal collectorrin-like domain (CLD), with the CLD ending in a transmembrane helix and an intracellular segment. The peptidase domain (PD) is located in the extracellular domain and is responsible for cleaving angiotensin II and angiotensin I. The crystal structure of ACE2-PD also provides a direct binding site for the coronavirus S protein. PD is crucial in ACE2 for coronavirus cell binding. The ACE2 portion of the ACE2-Fc fusion protein of this invention can be the extracellular domain of ACE2 or a fragment thereof containing the peptidase domain of ACE2, or a mutant of the extracellular domain or the fragment. Specifically, the ACE2 portion can be the extracellular domain of ACE2 or a fragment thereof containing the wild-type ACE2 peptidase domain, or mutants thereof that retain the ability to bind to ligands (e.g., the viral S protein). The mutant can be an extracellular domain of ACE2 that has lost its peptidase activity in cleaving angiotensin II and angiotensin I, or a fragment thereof containing the peptidase domain. For example, this can be achieved by mutating the histidine residue at the active site of ACE2 to another amino acid residue, specifically by mutating histidine (His) at positions 374 and / or 378 to aspartate (Asn). The mutant can also be one that retains peptidase activity. In patients infected with 2019-nCoV or SARS virus, ACE2 expression is downregulated due to viral internalization, leading to impaired function in regulating the renin-angiotensin system, resulting in multiple symptoms such as pulmonary hypertension, acute respiratory distress, and heart failure. Therefore, in clinical use, the ACE2 peptidase activity of the ACE2-Fc fusion protein can be preserved to compensate for the symptoms caused by ACE2 deficiency. The mutant also comprises a fragment of the ACE2 extracellular domain or the ACE2 peptidase domain thereof with or without peptidase activity, wherein some or all glycosylation sites are mutated, and these glycosylation sites comprise one or more sites of asparagine residues selected from positions 53, 90, 103, 322, 432, 546, and 690, as described elsewhere herein. Exemplarily, the active ACE2-PD domain sequence is shown in SEQ ID NO:1, the inactive ACE2-PD domain sequence is shown in SEQ ID NO:2, the active ACE2 extracellular domain sequence is shown in SEQ ID NO:3, and the inactive ACE2 extracellular domain sequence is shown in SEQ ID NO:4. In some embodiments, the mutation in the mutant of the ACE2 extracellular domain occurs outside the PD domain.

[0060] ACE2-Fc fusion proteins are proteins produced by fusing the Fc fragment of an immunoglobulin with the ACE2 moiety. Fc fusion proteins not only exhibit the biological activity of the ACE2 moiety but also possess antibody properties, such as inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). The Fc fragments involved in this invention can be derived from different immunoglobulin subtypes such as human IgG1, IgG2, and IgG4. IgG1 often exhibits strong effector functions, recruiting NK cells or macrophages to kill (ADCC) or phagocytose (ADCP) target cells or pathogens through binding to the Fcγ receptor, thereby exerting a clearance effect. In contrast, molecules such as IgG2 and IgG4 have weaker affinity for the Fcγ receptor, resulting in weaker ADCC or ADCP effects. Studies have shown that the binding of the Fc fragment to the Fcγ receptor depends on glycosylation of aspartate at position 297; mutations at this glycosylation site will cause the loss of its binding ability to the Fcγ receptor, thus eliminating ADCC and ADCP activities. For antiviral therapy, ADCC / ADCP may promote the body's immune system to clear the virus, thereby promoting patient recovery. However, on the other hand, studies have shown that neutralizing antibodies or fusion proteins may mediate the binding of the virus to macrophages, thereby promoting viral infection of macrophages, and subsequently viral amplification and replication within macrophages, leading to an increase in viral load and negatively impacting the patient's disease course. This effect is called antibody-dependent enhancement (ADE). By selecting IgG2 or IgG4 subtypes, or through Fc engineering strategies, including mutations at the N297 glycosylation site, the risk of ADE in patients can be reduced, improving drug safety. Therefore, the Fc segment of the fusion protein of this invention can be the Fc segment of IgG1, IgG2, and IgG4, or a mutant thereof. These Fc segments can eliminate ADCC and ADCP activity by introducing mutations at the N297 site (e.g., mutation to A). In addition, the IgG4 Fc segment can eliminate strand exchange by introducing mutations at the S228 site (e.g., mutation to P). Therefore, the Fc segment of the fusion protein of the present invention is selected from: the Fc segment of IgG1 with or without the N297 mutation, the Fc segment of IgG2 with or without the N297 mutation, and the Fc segment of IgG4 with or without the S228 and / or N297 mutation. In a specific embodiment, the Fc segment of the fusion protein of the present invention is selected from: the Fc segment of IgG1 with the N297 mutation, the Fc segment of wild-type IgG2, and the Fc segment of IgG4 with the S228 and optionally the N297 mutation.For example, 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 both the N297A and S228P mutations is shown in SEQ ID NO:8.

[0061] The ACE2-Fc fusion protein of this invention can have different valences. Through multivalent binding of multiple ACE2s to the 2019-nCoV viral spike protein, it can simultaneously bind multiple S proteins on a single spike, or bind multiple spikes of a single virus, or even bind multiple spikes of multiple viruses, thereby enhancing its neutralizing activity. Multivalent binding can significantly increase intermolecular forces through the affinity effect, thereby improving the neutralizing effect that blocks viral infection. In this paper, the bivalent ACE2-Fc fusion protein includes two ACE2 moieties located at the N-terminus of the Fc segment. In the bivalent form of the fusion protein, both ACE2 moieties may have peptidase activity or neither may have it, or one may have peptidase activity while the other does not. For example, one ACE2 moieties may be the full-length extracellular domain of wild-type ACE2, a fragment containing a peptidase domain, or a mutant of them retaining their enzymatic activity, while the other ACE2 moieties may be an inactive extracellular domain, a fragment containing a peptidase domain, or a mutant of them lacking enzymatic activity. In this study, the tetravalent ACE2-Fc fusion protein comprises two ACE2 moieties located at the N-terminus of the Fc segment and two ACE2 moieties located at the C-terminus of the Fc segment. In the tetravalent form of the ACE2-Fc fusion protein, the enzymatic activity of each ACE2 moieties can be fully or partially retained. That is, in the tetravalent form of the ACE2-Fc fusion protein, only one, two, three, or four ACE2 moieties may possess peptidase activity. Therefore, in the tetravalent form of the ACE2-Fc fusion protein, at least one ACE2 moieties may be the wild-type ACE2 extracellular domain or a fragment containing the PD domain, or a mutant of these with retained enzymatic activity; or at least one ACE2 moieties may be the inactive ACE2 extracellular domain or a fragment containing the PD domain, or a mutant of these with inactive activity. This ensures that the tetravalent form of the ACE2-Fc fusion protein can efficiently block viral infection while preventing the risks of excessive ACE2 peptidase activity, such as a drop in blood pressure. For example, the two ACE2 moieties at the N-terminus of the ACE2-Fc fusion protein are wild-type ACE2 extracellular domains or their PD domains, possessing peptidase activity, while the two ACE2 moieties at the C-terminus are ACE2 extracellular domains or their PD domains that have lost peptidase activity through H374N and / or H378N mutations. Of course, the ACE2-Fc fusion protein of the present invention can also have two ACE2 moieties at the N-terminus that lack peptidase activity, while the two ACE2 moieties at the C-terminus possess peptidase activity.

[0062] The ACE2 portion of the fusion protein of this invention can be directly linked to the Fc or linked via a commonly used adaptor peptide. Exemplarily, the adaptor peptide of this invention is a adaptor containing both G and S, including but not limited to (GS)n, (GGS)n, (GGGS)n, and (GGGGS)n, where G refers to glycine, S refers to serine, and n is a positive integer from 1 to 15, preferably 1 to 10, and more preferably 1 to 8. In a specific embodiment, the adaptor is (GGGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, or 8. Other commonly used adaptor peptides are well known in the art.

[0063] The fusion protein of the present invention may also contain a signal peptide, and the sequence of the signal peptide suitable for expression in various cellular or subcellular structures is well known in the art. For example, the ACE2-Fc fusion protein contains the natural signal peptide of ACE2 at its N-terminus: MSSSSWLLLSLVAVTAA (SEQ ID NO: 9). This natural signal peptide is included when referring to the amino acid number of ACE2 herein, but this is only for convenience of description and is not intended to limit whether the fusion protein contains a signal peptide or the type and sequence of the signal peptide contained therein.

[0064] The ACE2 portion, Fc segment, and fusion protein of this invention also include mutants having at least 70% sequence identity and retaining their respective desired activities. For ACE2, the activity is the ability to bind to a viral protein, such as the S protein; for the Fc segment, the activity is the ability to bind to the FcRn receptor, thereby preventing protein degradation via the lysosomal pathway; for the fusion protein, the activity is the ability to bind to both a viral protein, such as the S protein, and the FcRn receptor. The mutants comprise amino acid sequences having at least 70%, at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, preferably at least 99% sequence identity to a reference sequence and retaining the desired activity of the reference sequence (e.g., the ability to bind to a viral protein, such as the S protein, and / or the ability to bind to the FcRn receptor). Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp. The mutants also include amino acid sequences having one or more mutations (insertions, deletions, or substitutions) in the amino acid sequence while still retaining the desired activity of the reference sequence. The number of mutations typically refers to 1-50, such as 1-20, 1-10, 1-8, 1-5, or 1-3. Substitution is preferably conservative. For example, in the art, conservative substitution with amino acids of similar or comparable properties generally does not alter the function of the protein or peptide. "Amino acids with similar or comparable properties" includes, for example, families of amino acid residues having similar side chains. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or more sites in the polypeptides or proteins of the present invention with another amino acid residue from the same side chain class will not substantially affect their activity.

[0065] An exemplary ACE2-Fc fusion protein of the present invention is shown below:

[0066] M01: A bivalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1 and IgG1 Fc shown in SEQ ID NO:5, wherein the two ACE2 moieties are directly linked to the N-terminus of Fc;

[0067] M02: A bivalent fusion protein composed of two inactivated ACE2 moieties shown in SEQ ID NO:2 and IgG1 Fc shown in SEQ ID NO:5, wherein the two ACE2 moieties are directly linked to the N-terminus of Fc;

[0068] M03: A bivalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1 and IgG2 Fc shown in SEQ ID NO:6, wherein the two ACE2 moieties are directly linked to the N-terminus of Fc;

[0069] M04: A bivalent fusion protein composed of two inactivated ACE2 moieties shown in SEQ ID NO:2 and IgG2 Fc shown in SEQ ID NO:6, wherein the two ACE2 moieties are directly linked to the N-terminus of Fc;

[0070] M05: A bivalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1 and IgG4 Fc shown in SEQ ID NO:7, wherein the two ACE2 moieties are directly linked to the N-terminus of Fc;

[0071] M06: A bivalent fusion protein composed of two inactivated ACE2 moieties shown in SEQ ID NO:2 and IgG4 Fc shown in SEQ ID NO:7, wherein the two ACE2 moieties are directly linked to the N-terminus of Fc;

[0072] M05F: A bivalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:3 and IgG4 Fc shown in SEQ ID NO:7, wherein the two ACE2 moieties are directly linked to the N-terminus of Fc;

[0073] M06F: A bivalent fusion protein composed of two inactivated ACE2 moieties shown in SEQ ID NO:4 and IgG4 Fc shown in SEQ ID NO:7, wherein the two ACE2 moieties are directly linked to the N-terminus of Fc;

[0074] M07: A tetravalent fusion protein composed of four active ACE2 moieties shown in SEQ ID NO:1 and IgG1 Fc shown in SEQ ID NO:5, wherein two ACE2 moieties are directly connected to the N-terminus of Fc, and the other two ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0075] M08: A tetravalent fusion protein composed of four inactivated ACE2 moieties shown in SEQ ID NO:2 and IgG1 Fc shown in SEQ ID NO:5, wherein two ACE2 moieties are directly connected to the N-terminus of Fc, and the other two ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0076] M09: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG1 Fc shown in SEQ ID NO:5, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0077] M10: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactivated ACE2 moieties shown in SEQ ID NO:2, and IgG1 Fc shown in SEQ ID NO:5, wherein the two inactivated ACE2 moieties are directly connected to the N-terminus of Fc, and the two active ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0078] M11: A tetravalent fusion protein composed of four active ACE2 moieties shown in SEQ ID NO:1 and IgG2 Fc shown in SEQ ID NO:6, wherein two ACE2 moieties are directly linked to the N-terminus of Fc, and the other two ACE2 moieties are linked to the C-terminus of Fc through a linker (GGGGS)3.

[0079] M12: A tetravalent fusion protein composed of four inactivated ACE2 moieties shown in SEQ ID NO:2 and IgG2 Fc shown in SEQ ID NO:6, wherein two ACE2 moieties are directly connected to the N-terminus of Fc, and the other two ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0080] M13: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG2 Fc shown in SEQ ID NO:6, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0081] M14: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactivated ACE2 moieties shown in SEQ ID NO:2, and IgG2 Fc shown in SEQ ID NO:6, wherein the two inactivated ACE2 moieties are directly connected to the N-terminus of Fc, and the two active ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0082] M15: A tetravalent fusion protein composed of four active ACE2 moieties shown in SEQ ID NO:1 and IgG4 Fc shown in SEQ ID NO:7, wherein two ACE2 moieties are directly linked to the N-terminus of Fc, and the other two ACE2 moieties are linked to the C-terminus of Fc via (GGGGS)3.

[0083] M16: A tetravalent fusion protein composed of four inactivated ACE2 moieties shown in SEQ ID NO:2 and IgG4 Fc shown in SEQ ID NO:7, wherein two ACE2 moieties are directly connected to the N-terminus of Fc, and the other two ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0084] M17: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0085] M18: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0086] M15F: A tetravalent fusion protein composed of four active ACE2 moieties shown in SEQ ID NO:3 and IgG4 Fc shown in SEQ ID NO:7, wherein two ACE2 moieties are directly linked to the N-terminus of Fc, and the other two ACE2 moieties are linked to the C-terminus of Fc through a linker (GGGGS)3.

[0087] M16F: A tetravalent fusion protein composed of four inactivated ACE2 moieties shown in SEQ ID NO:4 and IgG4 Fc shown in SEQ ID NO:7, wherein two ACE2 moieties are directly connected to the N-terminus of Fc, and the other two ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0088] M17F: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:3, two inactive ACE2 moieties shown in SEQ ID NO:4, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0089] M18F: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:3, two inactive ACE2 moieties shown in SEQ ID NO:4, and IgG4 Fc shown in SEQ ID NO:7, wherein the two inactive ACE2 moieties are directly connected to the N-terminus of Fc, and the two active ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

[0090] M19: It has the same structure as M17, but the ACE2 part located at the C end of Fc is directly connected to Fc;

[0091] M20: The structure is the same as M17, but the ACE2 part located at the C end of Fc is connected to Fc through the connector GS;

[0092] M21: The structure is the same as M17, but the ACE2 part located at the C end of Fc is connected to Fc through the connector GGS;

[0093] M22: The structure is the same as M17, but the ACE2 part located at the C end of Fc is connected to Fc through the connector GGGS;

[0094] M23: The structure is the same as M17, but the ACE2 part located at the C end of Fc is connected to Fc through the connector GGGGS;

[0095] M24: The structure is the same as M17, but the ACE2 part located at the C end of Fc is connected to Fc through connector (GGGGS)2;

[0096] M25: The structure is the same as M17, but the ACE2 part located at the C end of Fc is connected to Fc through connector (GGGGS)4;

[0097] M26: The structure is the same as M17, but the ACE2 part located at the C end of Fc is connected to Fc through connector (GGGGS)5;

[0098] M27: The structure is the same as M17, but the ACE2 part located at the C end of Fc is connected to Fc through connector (GGGGS)6;

[0099] M28: The structure is the same as M17, but the ACE2 part located at the C end of Fc is connected to Fc through connector (GGGGS)7;

[0100] M29: The structure is the same as M17, but the ACE2 part located at the C end of Fc is connected to Fc through connector (GGGGS)8;

[0101] Y01: A bivalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1 and IgG4 Fc shown in SEQ ID NO:8, wherein the two ACE2 moieties are directly linked to the N-terminus of Fc;

[0102] Y03: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactivated ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:8, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactivated ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)4.

[0103] To improve expression in protein expression systems (e.g., Pichia pastoris systems), the ACE2 moiety in the fusion protein of the present invention can also be an ACE2 moiety with partial or complete glycosylation sites mutated. These glycosylation sites contain asparagine residues at positions 53, 90, 103, 322, 432, 546, and 690, and threonine or serine residues at positions 55, 92, 105, 324, 434, 548, and 692. Mutating it to a glycosylated form (e.g., alanine) can significantly improve the yield, purity, and enzyme activity of the fusion protein of the present invention produced by the Pichia pastoris system. In the present invention, the ACE2 moiety with partial or complete glycosylation sites mutated may or may not have peptidase activity, for example, by reducing or eliminating peptidase activity through mutations described elsewhere herein.

[0104] In multivalent fusion proteins, at least one, two, three, or four ACE2 moieties, partially or completely, are mutated. Therefore, this invention also includes the ACE2-Fc fusion protein as described below:

[0105] Y02: A bivalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1 and IgG4 Fc shown in SEQ ID NO:8, wherein each ACE2 moieties have glycosylation mutations: N53A, N90A, N103A, N322A, N432A, N546A, and the two ACE2 moieties are directly linked to the N-terminus of Fc;

[0106] Y04: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:8. The two active ACE2 moieties shown in SEQ ID NO:1 are directly linked to the N-terminus of Fc, and the two inactive ACE2 moieties shown in SEQ ID NO:2 are linked to the C-terminus of Fc through a linker (GGGGS)4. Each ACE2 moieties have glycosylation mutations: N53A, N90A, N103A, N322A, N432A, and N546A.

[0107] As described above, the fusion protein of the present invention can also be a heterodimeric ACE2-Fc fusion protein having ACE2 moieties with different activities at the N-terminus (or C-terminus) of the Fc. Further, the heterodimeric ACE2-Fc fusion protein may also have a club-and-mortar mutation in the Fc. For example, by introducing a club mutation (e.g., a mutation at the T366 site) into one IgG Fc and a mortar mutation (e.g., a mutation at the T366 and / or L368 and / or Y407 sites) into another IgG Fc, a stable club-and-mortar complementary heterodimeric structure can be generated. Therefore, the present invention also includes the ACE2-Fc fusion protein as described below:

[0108] KIH01: A bivalent fusion protein consisting of an active ACE2 moiety shown in SEQ ID NO:1, an inactive ACE2 moiety shown in SEQ ID NO:2, and an IgG4 Fc moiety shown in SEQ ID NO:7, wherein the two ACE2 moieties are directly linked to the N-terminus of the Fc moiety, and one strand of the IgG4 Fc moiety has a T366W mutation, while the other strand of the IgG4 Fc moiety has one or more mutations selected from T366S, L368A, and Y407V.

[0109] In some embodiments, this document also provides a pharmaceutical composition comprising the ACE2-Fc fusion protein described herein or its encoding nucleic acid as an active ingredient and optionally a pharmaceutically acceptable carrier or excipient. In this document, the pharmaceutically acceptable carrier, excipient, or stabilizer is non-toxic to the recipient of the protein or nucleic acid at the dose and concentration used, and may include various types of carriers or excipients commonly used in therapeutics to deliver proteins or nucleic acids, as known in the art. The protein or nucleic acid in the pharmaceutical composition is present in a therapeutically effective amount. The therapeutically effective amount and dosage form of the protein or nucleic acid described herein may be determined based on the different types of active ingredients, the age, sex, and severity of the disease of the patient receiving the protein or nucleic acid. The protein or nucleic acid described herein may be administered via conventional routes of administration, such as oral or injectable. Routes of administration for different dosage forms are known in the art.

[0110] The fusion protein or pharmaceutical composition of the present invention can treat diseases caused by coronaviruses. As described above, the fusion protein of the present invention can bind to the coronavirus itself to neutralize viral toxicity and has an extended half-life. The coronaviruses described herein are enveloped, single-stranded positive-sense RNA viruses of the genus *Coronavirus*, including but not limited to HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, and 2019-nCoV (COVID-19). The S protein (spike protein S) of coronaviruses is a key protein for coronavirus infection of cells, playing an important role in viral tissue or host tropism and virulence. Figure 1 As shown, the fusion protein of the present invention binds to the S protein of coronaviruses via its ACE moiety, thereby preventing or reducing the binding of coronavirus proteins to host cells and thus neutralizing toxicity. Therefore, the fusion protein of the present invention can treat diseases caused by coronaviruses. These diseases include, but are not limited to, Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARDS), Acute Respiratory Distress Syndrome (ARDS), coronavirus-induced pneumonia, pulmonary hypertension, acute respiratory distress syndrome, heart failure, and COVID-19. Based on this, this document also provides a method for treating diseases caused by coronaviruses, comprising administering a therapeutically effective amount of the fusion protein or pharmaceutical composition of the present invention to a subject in need. In this document, "individual," "subject," or "patient" refers to mammals, particularly humans.

[0111] This invention also includes coding sequences and complementary sequences of the various amino acid sequences, peptides, proteins, or mutants thereof described herein, as well as nucleic acid constructs containing said coding sequences or complementary sequences. The coding sequences described herein include sequences altered through codon optimization, provided the encoded amino acid sequence remains unchanged. Codon-optimized sequences may exhibit more suitable expression for a specific species. Methods for codon optimization of coding sequences are well known in the art. In this invention, a nucleic acid construct is an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. This nucleic acid construct contains the coding sequences described herein or their complementary sequences, and one or more regulatory sequences operatively linked to these sequences. The regulatory sequence may be a suitable promoter sequence. The promoter sequence is typically operatively linked to the coding sequence of the amino acid sequence to be expressed. The promoter may be any nucleotide sequence exhibiting transcriptional activity in a selected host cell, including mutant, truncated, and heterozygous promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to that host cell. The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is attached to the 3' end of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in a selected host cell can be used in this study.

[0112] In some embodiments, the nucleic acid construct is a vector. Specifically, the coding sequences described herein can be cloned into many types of vectors, including but not limited to plasmids, phage particles, phage derivatives, viruses, and granules. The vector can be an expression vector or a cloning vector.

[0113] Typically, a suitable vector contains an origin of replication functioning in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers. Representative examples of these promoters include: the *lac* or *trp* promoter of *E. coli*; the *lambda* phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, the Pichia pastoris methanol oxidase promoter, and several other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. Marker genes can be used to provide phenotypic traits for selecting host cells for transformation, including but not limited to dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for *E. coli*. When the polynucleotides described herein are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription.

[0114] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells. Expression vectors containing the polynucleotide sequences described herein and suitable transcription / translation control signals can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques.

[0115] The fusion protein of the present invention can be prepared by chemical synthesis or biosynthesis. The processes and conditions for conventional protein chemical synthesis are well known in the art. The biosynthetic method described herein is a cell expression method, comprising the 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 conducive to expression of the fusion protein. The cells include, but are not limited to, mammalian cells (CHO) or Pichia pastoris cells. The vectors, promoters, and other components required for expression in Pichia pastoris are known in the art. For better expression in the Pichia pastoris system, the ACE2 portion of the fusion protein of the present invention may also be an ACE2 portion with partial or complete glycosylation site mutations. An exemplary ACE2-Fc fusion protein with glycosylation site mutations of the present invention is described in Y02 and Y04 above.

[0116] This article also includes host cells or engineered cells that express the proteins described herein and contain the polynucleotide sequences described herein or their nucleic acid constructs. Host cells or engineered cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; filamentous fungal cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: *Escherichia coli*, *Streptomyces*; bacterial cells of *Salmonella typhimurium*; fungal cells such as yeast, filamentous fungi, and plant cells; insect cells of *Drosophila* S2 or Sf9; animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells, etc.

[0117] The vector described in this paper can be introduced into host cells using conventional methods, including microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, and calcium phosphate precipitation.

[0118] Furthermore, this document also includes applications of the various products described herein, including pharmaceutical applications. Specifically, this document includes the use of the fusion protein, nucleic acid molecule, and nucleic acid construct described herein in the preparation of medicaments for treating diseases caused by coronaviruses or acute respiratory distress syndrome (ARDS). Diseases caused by coronaviruses include, but are not limited to, Middle East Respiratory Syndrome (MERS), severe acute respiratory syndrome (SERS), coronavirus-induced pneumonia, pulmonary hypertension, acute respiratory distress syndrome (ARDS), heart failure, and COVID-19. The causes of ARDS include, but are not limited to, pneumonia, aspiration, pulmonary contusion, drowning, inhalation of toxic substances; severe systemic infections (bacteria, viruses, fungi, atypical pathogens), malignant tumors, severe multiple injuries (multiple fractures, flail chest, severe traumatic brain injury, and burns), shock, high-risk surgeries (cardiac surgery, aortic surgery, etc.), massive blood transfusions, drug poisoning, pancreatitis, and post-cardiopulmonary bypass surgery. Based on this, this document also provides a method for treating diseases caused by coronaviruses, comprising administering a therapeutically effective amount of the fusion protein or pharmaceutical composition of the present invention to a subject in need.

[0119] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the invention. Unless otherwise stated, the materials and methods used in the embodiments are conventional materials and methods in the art.

[0120] The sequences involved in this invention are as follows:

[0121] Active ACE2-PD sequence, SEQ ID NO:1

[0122] QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD;

[0123] Inactivated ACE2-PD sequence, SEQ ID NO: 2

[0124] QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHNEMGNIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD;

[0125] Active ACE2 extracellular domain sequence, SEQ ID NO: 3

[0126] QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSR)SRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS;

[0127] Inactivated ACE2 extracellular domain sequence, SEQ ID NO: 4

[0128] QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHNEMGNIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSR)SRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS;

[0129] IgG1 containing the N297A mutation, SEQ ID NO: 5

[0130] PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0131] Wild-type IgG2, SEQ ID NO: 6

[0132] RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0133] IgG4 containing the S228P mutation, SEQ ID NO: 7

[0134] SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0135] IgG4 containing the N297A and S228P mutations, SEQ ID NO: 8

[0136] SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0137] Example 1: Expression of different forms of ACE2-Fc series fusion proteins in HEK293 cells

[0138] A series of different forms of ACE2-Fc fusion proteins were designed, with the following variations:

[0139] 1) ACE2 is selected from either the peptidase domain (PD domain, which is numbered M + number, where M indicates expression in mammalian cells) or the full-length extracellular domain (which is numbered M + number + F, where F represents full length).

[0140] 2) Whether ACE2 retains its activity, i.e., the active form and the inactive form, in which the inactive form mutates histidine (His) at positions 374 and 378 into asparagine (Asn).

[0141] 3) The Fc segment was selected from human IgG1 Fc, IgG2 Fc, and IgG4 Fc. IgG1 Fc contains the N297A glycosylation site mutation to eliminate binding to the Fcγ receptor and thus eliminate effector function. IgG4 Fc introduces the S228P mutation to eliminate the generation of strand exchange.

[0142] 4) Different valence positions: When ACE2 molecules are simultaneously linked to both the N-terminus and C-terminus of Fc, with a (GGGGS)3 linker peptide added to the C-terminus to link with ACE2, a tetrameric ACE2-Fc molecule is formed. The total number of designed molecules is shown in the table below.

[0143]

[0144]

[0145] The amino acid sequences of the above 24 molecules were codon optimized for mammalian cells by Suzhou Genewiz Company, and synthesized and constructed into the pTT5 vector. The signal peptide sequence was selected from the natural ACE2 signal peptide (MSSSSWLLLSLVAVTAA). The above 24 molecules were extracted from the plasmid and transfected with polyethyleneimine (PEI) reagent into 200 ml of HEK293 cells in logarithmic growth phase at a density of 1E6 / ml. 24 hours after transfection, 0.5% Peptone was added to promote cell growth. Seven days after transfection, the cells were harvested, centrifuged at 4000 rpm for 30 min, and the supernatant was purified using Protein A packing material (Borglon) after passing through a 0.22 μm filter membrane. The purified supernatant was then dialyzed to PBS and sterilely filtered again through a 0.22 μm filter membrane for storage.

[0146] The 24 ACE2 series fusion proteins were quantified using the BCA protein concentration quantification kit (Yisheng Biotechnology) according to the operating procedures provided in the kit's instructions. Aggregate and purity analyses were performed using SEC-HPLC (G3000SWXL, TOSOH). The results are as follows:

[0147]

[0148]

[0149] Example 2: ELISA experiment on the binding of ACE2-Fc series fusion proteins to the RBD domain of 2019-nCoV S protein

[0150] Enzyme-linked immunosorbent assay (ELISA) was performed on the above 24 molecules to characterize the binding ability of different molecules to the 2019-nCoV S protein. 96-well ELISA plates (Maxisorp, NUNC) were incubated overnight at 4°C with 100 μL of 1 μg / mL 2019-nCoV RBD (COV-VM4BD, Kaika Biotechnology), then blocked with 1% BSA (bovine serum albumin) solution at room temperature for 2 hours, and washed three times with PBST. 100 μL of different concentrations of human ACE2-Fc fusion proteins were added to each 96-well plate and bound to the coated 2019-nCoV RBD at room temperature for 1 hour, followed by washing three times with PBST. Then, HRP enzyme conjugated with anti-human IgG (Goat anti-Human IgG Fc Highly Cross-Adsorbed Secondary Antibody, HRP, A18829, 1:5000, Thermo Fisher Scientific China Co., Ltd.) was added and bound at room temperature for 0.5 hours, followed by washing three times with PBST. Finally, 100 μL of TMB substrate (Solarbio, PR1200) was added to each well for color development, and 50 μL of ELISA stop solution (Solarbio, C1058) was added to terminate the reaction. The absorbance was measured at 450 nm using a BioTek Elx800 microplate reader. Data processing was performed using GraphPad Prism software, and the half-maximal effective concentration (EC50) was calculated using a four-parameter regression model. The results are summarized below:

[0151]

[0152]

[0153] The experimental results above show that the binding force of the tetrameric ACE2-Fc fusion protein is about 10 times higher than that of the dimer form. This may be due to the affinity addition effect formed between the tetramer and the 2019-nCoV S protein, suggesting that it has stronger anti-2019-nCoV virus infection activity.

[0154] Example 3: Competitive blocking of the binding of 2019-nCoV S protein RBD to ACE2 protein by ACE2-Fc series fusion proteins

[0155] Competitive enzyme-linked immunosorbent assays were performed on the above 24 molecules to characterize their blocking ability against the 2019-nCoV S protein. 96-well ELISA plates (Maxisorp, NUNC) were incubated overnight at 4°C with 100 μL of 1 μg / mL 2019-nCoV RBD (COV-VM4BD, Kaika Biotechnology), then blocked with 1% BSA (bovine serum albumin) solution at room temperature for 2 hours, and washed three times with PBST. 50 μL of different concentrations of human ACE2-Fc series fusion proteins were added to each well of the 96-well plate, along with 50 μL of 0.3 nM ACE2 His-Biotin (ACE-HM401B, Kaika Biotechnology) to each well. The mixture was allowed to bind with the coated 2019-nCoV RBD at room temperature for 1.5 hours, followed by washing three times with PBST. Then, [the following was added]... High-sensitivity Streptavidin-HRP (21130, 1:15000, Thermo Fisher Scientific China Co., Ltd.) was added and incubated at room temperature for 1 hour, followed by washing three times with PBST. Finally, 100 μL of TMB substrate (Solarbio, PR1200) was added to each well for color development, and the reaction was terminated with 50 μL of ELISA stop solution (Solarbio, C1058). Absorbance was measured at 450 nm using a BioTek Elx800 microplate reader. Data were processed using GraphPad Prism software, and the half-maximal inhibitory concentration (IC50) was calculated using a four-parameter regression model. The results are summarized below:

[0156] 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

[0157] Example 4: Determination of peptidase activity of ACE2-Fc series fusion proteins

[0158] Peptidase activity assays were performed on the above 24 molecules to characterize the enzymatic cleavage activities of different molecules. Human ACE2-Fc fusion proteins were prepared into 0.2 ng / μL solutions using Assay Buffer (75 mM Tris, 1 M NaCl, 10 μM ZnCl2, pH 7.5). The enzyme reaction substrate Mca-YVADAPK(Dnp)-OH Fluorogenic Peptide Substrate VI (ES007, R&D) was diluted to 40 μM with the same Assay Buffer. 250 μL of the 0.2 ng / μL human ACE2-Fc fusion protein solution was added to a cuvette, followed by 250 μL of the 40 μM reaction substrate solution. The reaction was initiated, with control wells containing Assay Buffer but without protein. Readings were taken for 5 minutes in dynamic mode using an F-180 fluorescence spectrophotometer (Tianjin Gangdong Technology Development Co., Ltd.) at excitation and emission wavelengths of 320 nm and 405 nm, respectively. The specific activity of human ACE2-Fc series fusion proteins was calculated using the specific activity calculation formula:

[0159] Specific activity (pmol / min / μg) = corrected Vmax (RFU / min) x conversion factor (pmol / RFU) / enzyme in each well (μg). The conversion factor was obtained by constructing a standard curve using MCA-Pro-Leu-OH (Bachem, Catalog #M-1975) at different concentrations. The enzyme amount refers to human ACE2-Fc series fusion proteins.

[0160] The results are summarized below:

[0161]

[0162]

[0163] Example 5: Screening of tetrameric ACE2-Fc fusion protein linker peptides

[0164] Based on the M17 molecule, linker peptides between ACE2 and the C-terminus of Fc were screened, and a series of linker peptides of different lengths were designed as follows:

[0165] M17 N-terminal active form, C-terminal inactive form IgG4,S228P Quadrivalent (GGGGS)3 M19 N-terminal active form, C-terminal inactive form IgG4,S228P Quadrivalent none M20 N-terminal active form, C-terminal inactive form IgG4,S228P Quadrivalent GS M21 N-terminal active form, C-terminal inactive form IgG4,S228P Quadrivalent GGS M22 N-terminal active form, C-terminal inactive form IgG4,S228P Quadrivalent GGGS M23 N-terminal active form, C-terminal inactive form IgG4,S228P Quadrivalent GGGGS M24 N-terminal active form, C-terminal inactive form IgG4,S228P Quadrivalent (GGGGS)2 M25 N-terminal active form, C-terminal inactive form IgG4,S228P Quadrivalent (GGGGS)4 M26 N-terminal active form, C-terminal inactive form IgG4,S228P Quadrivalent (GGGGS)5 M27 N-terminal active form, C-terminal inactive form IgG4,S228P Quadrivalent (GGGGS)6 M28 N-terminal active form, C-terminal inactive form IgG4,S228P Quadrivalent (GGGGS)7 M29 N-terminal active form, C-terminal inactive form IgG4,S228P Quadrivalent (GGGGS)8

[0166] The different molecular sequences described above underwent codon optimization, gene synthesis, and cloning into the pTT5 vector. Expression and purification were performed using the methods described in Examples 1-4 above. SEC purity analysis, combined with activity assays, blocking activity assays, and enzymatic activity assays were conducted. The results are summarized below:

[0167]

[0168]

[0169] Example 6: Expression of ACE2-Fc series fusion proteins using the Pichia pastoris system

[0170] Based on the screening results above, four molecules were selected for expression in the Pichia pastoris system. To avoid the impact of high-mannose glycosylation modifications in the yeast system on drug half-life and immunogenicity, the Fc glycosylation sites of all yeast-based molecules were mutated. The glycosylation sites of the ACE2 molecule were either retained or mutated. A total of four molecules were designed:

[0171]

[0172] The ACE2 glycosylation mutation sites were asparagine residues at positions 53, 90, 103, 322, 432, and 546, which were mutated to alanine to form a glycosylated form. The four molecules were codon-optimized using Pichia pastoris, and the resulting genes were synthesized and cloned into the Invitrogen pPICZα vector. Following the Invitrogen EasySelect Pichia Expression Kit instructions, the linearized plasmid was transformed into GS115 competent cells via electroporation. Positive clones were screened for Zeocin resistance. Ten clones were selected on 3000 μg / ml Zeocin-resistant plates and induced to express at low concentrations for three days in 200 mL BMMY medium with 0.5% methanol. The supernatant was used for Protein A purification, and the clone with the highest yield for each molecule was subjected to the SEC purity analysis described above, combined with activity analysis, inhibitory activity analysis, and enzymatic activity analysis.

[0173] The results are summarized below:

[0174]

[0175] Example 7: Investigating the neutralizing effect of ACE2-Fc series fusion proteins on 2019-nCoV infection using a pseudovirus infection experiment.

[0176] Thirteen ACE2-Fc fusion protein molecules selected in Examples 1-7 were subjected to 2019-nCoV S pseudovirus neutralization experiments to characterize the neutralizing and blocking ability of different molecules against 2019-nCoV S protein infection. 293T cells were transiently co-transfected with pCMVR8.2 lentiviral packaging plasmid, pHRCMV-Luc reporter gene plasmid, and CMVR-2019-nCoV-S virus eukaryotic expression plasmid. The supernatant was collected 48 hours after transfection, centrifuged, and sterilized using a 0.45 μm filter to obtain packaged 2019-nCoV S pseudovirus. Some pseudoviruses were aliquoted and stored at -4°C, while others were frozen at -80°C for later use. The p24 content was detected and calculated using an HIV-1 p24 detection kit to determine the required dose of pseudovirus for the neutralization assay. 293T cells were transiently transfected with the pcDNA3-ACE2 eukaryotic expression plasmid to obtain 293T-ACE2 cells that transiently express ACE2. Cells were grown at 1 x 10⁻⁶ cells per well. 4 293T-ACE2 cells were seeded in 96-well cell culture plates, 100 μL per well, and cultured overnight. Different concentrations of human ACE2-Fc fusion protein were mixed with a 2019-nCoV S pseudovirus solution standardized using p24 content at a 1:1 volume ratio and incubated at room temperature for 10 minutes. 100 μL of the fusion protein and virus mixture was added to the 96-well cell culture plates, and incubated for 16 hours. The medium was then replaced with fresh medium (200 μL per well, without virus), and cultured for another 48 hours. The cells were then analyzed using a firefly luciferase assay kit and a microplate reader (TACAN). The expression level of luciferase in fireflies was detected using a multi-wavelength microplate reader (M1000). Data processing was performed using GraphPad Prism software, and the half-maximal inhibitory concentration (IC50) was calculated using a four-parameter regression model. The results are summarized below:

[0177]

[0178]

[0179] Example 8: Generating heterodimer ACE2-Fc using mortar and pestle mutation

[0180] By introducing the T366W mutation (“pallet” mutation) into one strand of IgG4 Fc (containing the S228P mutation) and the T366S / L368A / Y407V mutation (“mortar” mutation) into the other strand of IgG4 Fc, a stable heterodimeric structure based on palmetto-mortar complementarity can be generated. The active and inactive ACE2 PD domains were fused into the aforementioned IgG4 Fc (containing the S228P) fragments containing palmetto and mortar mutations, respectively. The heterodimer molecule was named KIH01. Plasmid co-transfection and expression purification were performed according to the method described in Example 1, and its binding activity, competitive inhibition activity, peptidase activity, and viral neutralization activity were tested. The results are summarized below:

[0181] SEC purity (monomer percentage) 92％ Combined with ELISA EC50(nM) 0.194 Competing with ELISA IC50 (nM) 7.7 Peptidase activity (pmol / min / μg) 589 Inhibitory activity IC50 (nM) in virus neutralization assay 4.7 sequence list <110> Shanghai Inmed Medical Technology Co., Ltd. <120> ACE2-Fc fusion protein and its applications <130> 201760 <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 598 <212> PRT <213> Artificial Sequence <400> 1 Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn 1 5 10 15 His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30 Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala 35 40 45 Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln 50 55 60 Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu 65 70 75 80 Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser 85 90 95 Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr 100 105 110 Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125 Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135 140 Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg 145 150 155 160 Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala 165 170 175 Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190 Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp 195 200 205 Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220 Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser 225 230 235 240 Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255 Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270 Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280 285 Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300 Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly 305 310 315 320 Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335 Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350 Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365 Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380 Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His 385 390 395 400 Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu 405 410 415 Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430 Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445 Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460 Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr 465 470 475 480 Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490 495 Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510 Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525 Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly 530 535 540 Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys 545 550 555 560 Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575 Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp 580 585 590 Trp Ser Pro Tyr Ala Asp 595 <210> 2 <211> 598 <212> PRT <213> Artificial Sequence <400> 2 Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn 1 5 10 15 His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30 Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala 35 40 45 Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln 50 55 60 Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu 65 70 75 80 Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser 85 90 95 Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr 100 105 110 Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125 Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135 140 Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg 145 150 155 160 Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala 165 170 175 Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190 Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp 195 200 205 Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220 Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser 225 230 235 240 Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255 Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270 Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280 285 Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300 Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly 305 310 315 320 Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335 Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350 Leu Thr Ala His Asn Glu Met Gly Asn Ile Gln Tyr Asp Met Ala Tyr 355 360 365 Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380 Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His 385 390 395 400 Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu 405 410 415 Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430 Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445 Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460 Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr 465 470 475 480 Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490 495 Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510 Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525 Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly 530 535 540 Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys 545 550 555 560 Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575 Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp 580 585 590 Trp Ser Pro Tyr Ala Asp 595 <210> 3 <211> 723 <212> PRT <213> Artificial Sequence <400> 3 Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn 1 5 10 15 His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30 Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala 35 40 45 Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln 50 55 60 Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu 65 70 75 80 Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser 85 90 95 Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr 100 105 110 Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125 Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135 140 Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg 145 150 155 160 Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala 165 170 175 Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190 Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp 195 200 205 Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220 Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser 225 230 235 240 Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255 Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270 Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280 285 Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300 Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly 305 310 315 320 Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335 Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350 Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365 Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380 Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His 385 390 395 400 Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu 405 410 415 Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430 Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445 Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460 Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr 465 470 475 480 Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490 495 Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510 Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525 Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly 530 535 540 Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys 545 550 555 560 Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575 Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp 580 585 590 Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605 Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620 Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys 625 630 635 640 Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala 645 650 655 Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys 660 665 670 Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg 675 680 685 Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser 690 695 700 Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro 705 710 715 720 Pro Choice Watch <210> 4 <211> 723 <212> PRT <213> Artificial Sequence <400> 4 Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn 1 5 10 15 His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30 Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala 35 40 45 Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln 50 55 60 Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu 65 70 75 80 Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser 85 90 95 Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr 100 105 110 Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125 Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135 140 Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg 145 150 155 160 Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala 165 170 175 Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190 Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp 195 200 205 Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220 Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser 225 230 235 240 Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255 Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270 Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280 285 Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300 Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly 305 310 315 320 Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335 Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350 Leu Thr Ala His Asn Glu Met Gly Asn Ile Gln Tyr Asp Met Ala Tyr 355 360 365 Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380 Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His 385 390 395 400 Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu 405 410 415 Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430 Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445 Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460 Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr 465 470 475 480 Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490 495 Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510 Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525 Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly 530 535 540 Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys 545 550 555 560 Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575 Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp 580 585 590 Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605 Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620 Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys 625 630 635 640 Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala 645 650 655 Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys 660 665 670 Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg 675 680 685 Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser 690 695 700 Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro 705 710 715 720 Pro Val Ser <210> 5 <211> 231 <212> PRT <213> Artificial Sequence <400> 5 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 1 5 10 15 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 65 70 75 80 Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105 110 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 130 135 140 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 145 150 155 160 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170 175 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220 Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 6 <211> 227 <212> PRT <213> Artificial Sequence <400> 6 Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 7 <211> 228 <212> PRT <213> Artificial Sequence <400> 7 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Leu Gly Lys 225 <210> 8 <211> 228 <212> PRT <213> Artificial Sequence <400> 8 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Leu Gly Lys 225 <210> 9 <211> 17 <212> PRT <213> Artificial Sequence <400> 9 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala

Claims

1. A fusion protein, characterized in that, The fusion protein consists of four ACE2 moieties and an Fc segment. Two ACE2 moieties are directly linked to the N-terminus of the Fc segment, and the other two ACE2 moieties are linked to the C-terminus of the Fc segment via adaptor peptides. The two N-terminal ACE2 moieties are wild-type PD domains, and the two C-terminal ACE2 moieties are PD domains that have lost peptidase activity due to H374N and / or H378N mutations; or the two C-terminal ACE2 moieties are wild-type PD domains, and the two N-terminal ACE2 moieties are PD domains that have lost peptidase activity due to H374N and / or H378N mutations. The Fc segment is the Fc segment of IgG1 or IgG4. The sequence of the wild-type ACE2 PD domain is shown in SEQ ID NO:1, the sequence of the ACE2 PD domain that has lost peptidase activity due to H374N and / or H378N mutation is shown in SEQ ID NO:2, the sequence of the IgG1 Fc segment is shown in SEQ ID NO:5, and the sequence of the IgG4 Fc segment is shown in SEQ ID NO:

5. NO:7 or 8, and the linker peptide is GS, GGS, GGGS or (GGGGS)n, where G refers to glycine, S refers to serine, and n is a positive integer from 1 to 8.

2. The fusion protein as described in claim 1, characterized in that, The fusion protein is selected from any one of the following: M09: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG1 Fc shown in SEQ ID NO:5, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3. M10: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactivated ACE2 moieties shown in SEQ ID NO:2, and IgG1 Fc shown in SEQ ID NO:5, wherein the two inactivated ACE2 moieties are directly connected to the N-terminus of Fc, and the two active ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3. M17: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3. M18: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two inactive ACE2 moieties are directly connected to the N-terminus of Fc, and the two active ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3. M20: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactivated ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactivated ACE2 moieties are connected to the C-terminus of Fc through the GS adapter. M21: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through the adapter GGS. M22: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactivated ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactivated ACE2 moieties are connected to the C-terminus of Fc through the adapter GGGS. M23: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactivated ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactivated ACE2 moieties are connected to the C-terminus of Fc through the adapter GGGGS. M24: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactivated ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:

7. The two active ACE2 moieties are directly linked to the N-terminus of the Fc, and the two inactivated ACE2 moieties are linked to the C-terminus of the Fc via linkers (GGGGS). )2 connect; M25: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactivated ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactivated ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)4. M26: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)5. M27: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)6. M28: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)7. M29: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactivated ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactivated ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)8. Y03: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:8, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)4. Y04: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:

8. The two active ACE2 moieties shown in SEQ ID NO:1 are directly linked to the N-terminus of Fc, and the two inactive ACE2 moieties shown in SEQ ID NO:2 are linked to the C-terminus of Fc through a linker (GGGGS)4. The glycosylation mutations of each ACE2 moieties are: N53A, N90A, N103A, N322A, N432A, and N546A.

3. A fusion protein, characterized in that, The fusion protein is selected from any one of the following: M17F: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:3, two inactive ACE2 moieties shown in SEQ ID NO:4, and IgG4 Fc shown in SEQ ID NO:7, wherein the two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3. M18F: A tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:3, two inactive ACE2 moieties shown in SEQ ID NO:4, and IgG4 Fc shown in SEQ ID NO:

7. The two inactive ACE2 moieties are directly connected to the N-terminus of Fc, and the two active ACE2 moieties are connected to the C-terminus of Fc through a linker (GGGGS)3.

4. A fusion protein, characterized in that, The fusion protein is a tetravalent fusion protein composed of two active ACE2 moieties shown in SEQ ID NO:1, two inactive ACE2 moieties shown in SEQ ID NO:2, and IgG4 Fc shown in SEQ ID NO:

7. The two active ACE2 moieties are directly connected to the N-terminus of Fc, and the two inactive ACE2 moieties are directly connected to the C-terminus of Fc.

5. The fusion protein according to any one of claims 1-4, characterized in that, The fusion protein was expressed by yeast.

6. A nucleic acid molecule encoding the fusion protein of any one of claims 1-5.

7. A nucleic acid construct comprising the nucleic acid molecule of claim 6.

8. The nucleic acid construct as described in claim 7, wherein it is a cloning vector or an expression vector.

9. Engineered cells, characterized in that, The engineered cells: (1) Expressing the fusion protein of any one of claims 1-5; and / or (2) Contains the nucleic acid molecule of claim 6, or the nucleic acid construct of claim 7 or 8.

10. The use of the fusion protein of any one of claims 1-5, the nucleic acid molecule of claim 6, or the nucleic acid construct of claim 7 or 8 in the preparation of a medicament for treating diseases caused by coronavirus 2019-nCoV.

11. The use of the fusion protein of any one of claims 1-5, the nucleic acid molecule of claim 6, or the nucleic acid construct of claim 7 or 8 in the preparation of a drug for inhibiting COVID-19 infection.

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