RU2837537C1 - Soluble ace2, fused protein thereof and methods of using them - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, namely to a fusion protein of soluble ACE2, which binds to a coronavirus, comprising a processed form of soluble ACE2, an Fc domain of an antibody and a tail, wherein the processed form of soluble ACE2 is bonded to the N-terminal region of the Fc-domain, and the tail is located on the C-terminal region of the Fc-domain, the processed form of soluble ACE2 contains or consists of the extracellular domain of ACE2 or a fragment of the extracellular domain of ACE2, which retains the ability to bind to the coronavirus, as well as to the Fc-fusion protein multimer, ACE2-hFc (n), which binds to a coronavirus comprising n polypeptide monomer units, and which is characterized in that each of the polypeptide monomer units is a dimer, in which two processed forms of soluble ACE2 are linked to N-terminal regions of two Fc-domains of heavy chains, respectively, and n polypeptide monomer units are assembled into a multimer by means of a tail located on each of C-ends of two Fc-domains of heavy chains, where n is selected from 4, 5 or 6. Also disclosed is a vector and a cell containing a gene coding said fused protein, and a cell and a vector containing a gene coding said multimer.

EFFECT: invention is effective in preparing a drug for treating or preventing a disease associated with ACE2.

17 cl, 16 dwg, 3 tbl, 12 ex

Description

Field of technology to which the invention relates

The present invention relates to soluble ACE2 (angiotensin converting enzyme 2) and its fusion proteins, as well as to the use thereof.

Prerequisites for the creation of an invention

In December 2019, an outbreak of pneumonia caused by a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), occurred and the coronavirus infection spread rapidly throughout the world. As of February 2021, the number of infected people worldwide has reached more than 100 million, with a mortality rate of approximately 2.2%. The epidemic has not only caused a global health crisis, but also had far-reaching consequences for society, the economy, and other areas of activity.

Coronaviruses belong to the order Torovirus, the family Coronaviridae and the genus Coronaviridae. Coronaviruses are enveloped viruses with a linear single-stranded plus-strand RNA and are a large group of viruses that are widespread in nature. In patients, they cause diseases that have various clinical symptoms - from the common cold to severe lung infection. Over the past two decades, coronaviruses have caused two large-scale epidemics: severe acute respiratory syndrome (SARS) in 2002/2003 and Middle East respiratory syndrome (MERS) in 2012. Since the end of 2019, the new coronavirus epidemic (SARS-CoV2) has significantly surpassed the SARS and MERS epidemics not only in the scale of the epidemic and the cumulative number of infected people, but also in the number of deaths.

The viral genome of SARS-CoV2 is highly similar to the RaTG13 strain isolated from a bat (Chinese horseshoe bat) discovered in 2013 in Yunnan, China, with a sequence identity of up to 96.2% (Zhou et al., 2020). Therefore, it can be concluded that the origin of this novel coronavirus is consistent with the origin of the coronaviruses that caused SARS and MERS, that is, they all originated in bats. The pathway leading to the epidemic of this novel coronavirus, SARS-CoV2, in humans is likely to be similar to SARS and MERS. The virus originated in bats, evolved and amplified in intermediate hosts (e.g., animals with closer contact with humans), and eventually infected humans. The virus continued to evolve in humans and spread rapidly, resulting in an outbreak of viral infection. In nature, SARS-like coronaviruses have long existed in bats in many parts of the world, and most of them are unable to infect humans. However, humans may occasionally become infected with some natural focal diseases through another intermediate host. The severe acute respiratory syndrome epidemic in March 2002 and the SARS-CoV2 outbreak 17 years later demonstrate that as long as a natural host exists, there is a possibility that other pathogenic coronavirus infections may arise in the future.

In November 2020, the US Food and Drug Administration granted emergency use authorization for two monoclonal antibodies, Eli Lilly’s bamlanivimab and Regeneron’s combination casirivimab and imdevimab, to treat SARS-CoV2 infection. Both antibodies were approved for non-hospitalized adults and children 12 years and older with mild to moderate symptoms of SARS-CoV2 and at risk of worsening disease. Monoclonal antibodies typically present a difficult balance between high efficacy and broad-spectrum activity. As the global SARS-CoV2 epidemic has progressed, variants of the virus have emerged and will continue to emerge under selection pressure, such as to further adapt to the human host and human immunity. Variants may have changes in immunodominant sites that render existing neutralizing antibodies ineffective.

Both SARS-CoV and related animal viruses use angiotensin-converting enzyme 2 (ACE2) as a receptor to invade and infect target cells. The coronavirus surface contains multiple S proteins in the form of trimers that have high affinity for ACE2. Thus, to achieve effective virus neutralization, it is necessary to simultaneously block the multivalent, high-affinity binding between S proteins and ACE2. A soluble receptor formed by fusing the extracellular region of ACE2 with the constant region of an antibody has a similar mechanism of action to neutralizing antibodies and is able to block infection by mutated variants that still use ACE2 as a receptor. Soluble ACE2 fusion proteins could be developed as therapeutic agents with broad-spectrum neutralizing activity and would not be limited by virus mutations. Such fusion proteins could not only be used as therapeutic drugs to treat SARS-CoV-2 infections, but could also help stop similar epidemics that may arise in the future.

Brief summary of the invention

Soluble ACE2, soluble ACE2 with active site mutations (NN) and ACE2-Fc fusion protein containing soluble ACE2 or soluble ACE2 with active site mutations (NN) and the Fc segment of human IgG1 are able to effectively neutralize SARS-CoV2 and SARS-CoV and block the formation of multinucleated syncytia that can be induced by binding of the spike protein (S) (containing the furin protease cleavage site) of SARS-CoV2 to its human ACE2 receptor.

According to a first aspect, the present invention provides soluble ACE2 or a processed form thereof. The soluble ACE2 or a processed form thereof may comprise or contain an extracellular domain of ACE2 or a fragment thereof that retains the ability to bind to coronavirus.

In some embodiments of the present invention, the soluble ACE2 or a truncated form thereof may comprise a metalloprotease domain (19-615aa) in the extracellular region of human ACE2. In some embodiments of the present invention, the soluble ACE2 or a truncated form thereof may comprise a metalloprotease domain (1-740aa) in the extracellular region of human ACE2. In some embodiments of the present invention, the soluble ACE2 or a truncated form thereof may comprise Q24, T27, F28, D30, K31, H34, E37, D38, Y41, Q42, L45, M82, Y83, Q325, E329, N330, K353, G354, D355, R357 and R393 of human ACE2, in particular K31 and K353.

In some embodiments of the present invention, the soluble ACE2 or processed form thereof may comprise human ACE2, or any homolog or ortholog thereof, or a fragment thereof that retains the ability to bind to coronavirus.

Soluble ACE2 or its processed form can effectively neutralize a virus that uses ACE2 as a receptor to bind to a host cell. The virus may include SARS-CoV, HCoV-NL63, or SARS-CoV2.

In some embodiments of the present invention, the soluble ACE2 or processed form thereof may comprise an ACE2 comprising a mutation in the active site or a processed form thereof. Preferably, the ACE2 comprising a mutation in the active site or a processed form thereof is human soluble ACE2 or a processed form thereof having mutation(s) H374N and/or H378N at position(s) 374 and/or 378 (ACE2-NN).

Soluble ACE2 or its processed form may be soluble ACE2 or its processed form that has ACE2 enzymatic activity.

Preferably, soluble ACE2 or a processed form thereof may be glycosylated, which may preferably be glycosylated at position(s) 53, 90, 103, 322, 432, 546 and/or 690 at the N-terminus of human ACE2.

Preferably, the soluble ACE2 or processed form thereof may comprise the amino acid sequence represented by SEQ ID NO: 1.

Preferably, the soluble ACE2 or processed form thereof may comprise the amino acid sequence represented by SEQ ID NO: 2.

According to a second aspect, the present invention provides an ACE2-Fc fusion protein which is obtained by fusing soluble ACE2 or a processed form thereof to the Fc domain of an antibody.

In some embodiments of the present invention, soluble ACE2 or a processed form thereof may comprise or contain the extracellular domain of ACE2 or a fragment thereof that retains the ability to bind to coronavirus.

In some embodiments of the present invention, the soluble ACE2 or a truncated form thereof may comprise a metalloprotease domain (19-615aa) in the extracellular region of human ACE2. The soluble ACE2 or a truncated form thereof may comprise a metalloprotease domain (1-740aa) in the extracellular region of human ACE2. The soluble ACE2 or a truncated form thereof may comprise Q24, T27, F28, D30, K31, H34, E37, D38, Y41, Q42, L45, M82, Y83, Q325, E329, N330, K353, G354, D355, R357 and R393 of human ACE2, in particular K31 and K353.

In some embodiments of the present invention, the soluble ACE2 or processed form thereof may comprise human ACE2, or any homologue or orthologue thereof, or a fragment thereof that has the ability to bind to a coronavirus.

Soluble ACE2 or its processed form can effectively neutralize a virus that uses ACE2 as a receptor to bind to a host cell. The virus may include SARS-CoV, HCoV-NL63, or SARS-CoV2.

In some embodiments of the present invention, the soluble ACE2 or its processed form may comprise ACE2 comprising a mutation in the active site or in its processed form. Preferably, the ACE2 comprising a mutation in the active site or in its processed form may comprise human soluble ACE2 or its processed form (ACE2-NN) comprising mutation(s) H374N and/or H378N at position(s) 374 and/or 378.

Soluble ACE2 or its processed form may include soluble ACE2 or its processed form that has ACE2 enzymatic activity.

Preferably, soluble ACE2 or a processed form thereof may be glycosylated, which may preferably be glycosylated at position(s) 53, 90, 103, 322, 432, 546 and/or 690 at the N-terminus of human ACE2.

Preferably, the soluble ACE2 or processed form thereof may comprise the amino acid sequence represented by SEQ ID NO: 1.

Preferably, the soluble ACE2 or processed form thereof may comprise the amino acid sequence represented by SEQ ID NO: 2.

Soluble ACE2 or its processed form can effectively neutralize a virus that uses ACE2 as a receptor to bind to a host cell. The virus may include SARS-CoV, HCoV-NL63, or SARS-CoV2.

In some embodiments of the present invention, the antibody may be an IgG antibody. The antibody may be a human IgG, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG1. The Fc domain of the antibody may be an Fc domain of an antibody comprising two Fc domains of the heavy chains of the antibody. Preferably, each of the Fc domains of the heavy chains has a hinge region at its N-terminus. Preferably, each of the Fc domains of the heavy chains may include a CH3 domain derived from IgG1, IgG2, IgG3 or IgG4. Preferably, each of the Fc domains of the heavy chains may include CH2 and CH3 domains derived from IgG1, IgG2, IgG3 or IgG4. The Fc domains may facilitate dimerization of the two ACE2 domains.

Soluble ACE2 or its processed form can be bound to the C-terminal region of the Fc domain of heavy chains or to the N-terminal region of the Fc domain of heavy chains.

In some embodiments of the present invention, 2n (n is 1, 2 or 3) soluble ACE2 or processed forms thereof may be linked to the C- and/or N-terminal region(s) of two Fc domains of the heavy chains.

In some embodiments of the present invention, two soluble ACE2 or processed forms thereof may be linked, respectively, to the N-terminal region of two heavy chain Fc domains to form a dimer. Alternatively, two soluble ACE2 or processed forms thereof may be linked to the C-terminal region of two heavy chain Fc domains to form a dimer.

In some embodiments of the present invention, two soluble ACE2 or processed forms thereof may be linked to the N-terminal region of two heavy chain Fc domains, respectively, while two other soluble ACE2 or processed forms thereof may be linked to the C-terminal regions of two heavy chain Fc domains, respectively, thereby forming a tetrameric ACE2-Fc fusion protein. Further, each of the two soluble ACE2 or processed forms thereof at the N-terminus of the tetrameric ACE2-Fc fusion protein is further, at its N-terminus, linked to a soluble ACE or processed form thereof in tandem, thereby forming a hexameric ACE2-Fc fusion protein. The soluble ACE2 or processed forms thereof may be linked in tandem by a linker. The linker may be a cysteine AAA linker. Alternatively, each of the two soluble ACEs or their processed forms at the C-terminus of the tetrameric ACE2-Fc fusion protein then binds at its C-terminus to a soluble ACE or its processed form in tandem, thereby forming a hexameric ACE2-Fc fusion protein. The soluble ACE2s or their processed forms are bound in tandem via a linker. The linker may be a cysteine AAA linker.

In some embodiments of the present invention, each of the two Fc domains of the heavy chains can be linked at its N-terminus to two soluble ACE2 or processed forms thereof, which are linked in tandem, thereby forming a tetrameric ACE2-Fc fusion protein. The two soluble ACE2 or processed forms thereof are linked in tandem by a linker. The linker can be a cysteine AAA linker. Further, a soluble ACE or a processed form thereof can be further linked at each of the N-terminal regions of the tetrameric ACE2-Fc fusion protein to a soluble ACE or a processed form thereof in tandem, thereby forming a hexameric ACE2-Fc fusion protein. The soluble ACE2 or processed forms thereof are linked in tandem by a linker. The linker can be a cysteine AAA linker. Alternatively, each of the two Fc domains of the heavy chains of the tetrameric ACE2-Fc fusion protein can be linked at its C-terminal region to soluble ACE or its processed form, thereby forming a hexameric ACE2-Fc fusion protein.

Alternatively, each of the two heavy chain Fc domains can be linked at its C-terminal region to two soluble ACE2 or processed forms thereof, which are linked in tandem, thereby forming a tetrameric ACE2-Fc fusion protein. The two soluble ACE2 or processed forms thereof can be linked in tandem by a linker. The linker can be a cysteine AAA linker. Further, each of the two heavy chain Fc domains of the tetrameric ACE2-Fc fusion protein can be linked at its N-terminal region to a soluble ACE or processed form thereof, thereby forming a hexameric ACE2-Fc fusion protein.

Preferably, the ACE2-Fc fusion protein may in fact be a dimeric ACE2-Fc fusion protein, wherein one processed form of ACE2 and one heavy chain Fc domain may comprise the amino acid sequence represented by SEQ ID NO: 3.

Preferably, one truncated form of ACE2 and one heavy chain Fc domain in the ACE2-Fc fusion protein may comprise the amino acid sequence represented by SEQ ID NO: 4.

Preferably, the ACE2-Fc fusion protein may further comprise a signal peptide, preferably a CD33 signal peptide.

Preferably, one truncated form of ACE2 and one heavy chain Fc domain in the ACE2-Fc fusion protein may comprise the amino acid sequence represented by SEQ ID NO: 5.

Preferably, one truncated form of ACE2 and one heavy chain Fc domain in the ACE2-Fc fusion protein may comprise the amino acid sequence represented by SEQ ID NO: 6.

Preferably, in a tetrameric ACE2-Fc fusion protein, one processed form of ACE2 may be linked to one heavy chain Fc domain, which is further linked to one ACE2 (ACE2-Fc-ACE2), resulting in an amino acid sequence represented by SEQ ID NO: 13.

Preferably, in a tetrameric ACE2-Fc fusion protein, two ACE2s or truncated forms thereof may be linked to a single Fc domain of the heavy chains (ACE2-ACE2-Fc), resulting in an amino acid sequence represented by SEQ ID NO: 14.

The ACE2-Fc fusion protein is able to effectively neutralize a virus that uses ACE2 as a receptor to bind to the host cell. The virus may include SARS-CoV, HCoV-NL63, or SARS-CoV2.

The ACE2-Fc fusion protein of the present invention is capable of increasing the half-life and yield of soluble ACE2, and can fully meet the needs of rapid process development and emergency use.

According to a third aspect, the present invention provides a multimer of Fc fusion protein (ACE2-hFc(n)) of soluble ACE2 or a truncated form thereof, which may comprise n polypeptide monomeric units, wherein each of the polypeptide monomeric units may be a dimer, in which two soluble ACE2 or truncated forms thereof are linked to the N-terminal regions of two Fc domains of heavy chains, respectively, and the n polypeptide monomeric units are assembled into a multimer via a tail located at each of the C-termini of the Fc domains of the antibody.

In some embodiments of the present invention, each of the heavy chain Fc domains in each polypeptide monomeric unit may be linked at its C-terminus to a tail. Thus, each of the two heavy chain Fc domains of a polypeptide monomeric unit is linked at its C-terminus to one tail, and n polypeptide monomeric units have a total of 2n tails that are linked to each other to form a closed circular multimer.

The tail may comprise any suitable amino acid sequences and may be a tail present in naturally occurring antibodies. Alternatively, said tail may be a modified tail that differs from the native tail in length and/or composition. Alternatively, the tail may be an artificially synthesized tail suitable for multimerization, for example, a tail consisting of a flexible Cys sequence of suitable length. Alternatively, the tail may comprise a variant or fragment of a natural sequence, such as the IgM tail PTLYNVSLVMSDTAGTCY (SEQ ID NO: 15) or the IgA tail PTHVNVSVVVMAEVDGTCY (SEQ ID NO: 16). Alternatively, the IgM or IgA tail variant may typically comprise an amino acid sequence comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acids from the IgM tail PTLYNVSLVMSDTAGTCY (SEQ ID NO: 15) or the IgA tail PTHVNVSVVMAEVDGTCY (SEQ ID NO: 16). The tail may also be a hybrid IgM/IgA tail. Preferably, the tail may comprise the amino acid sequence TGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 17).

In some embodiments of the present invention, soluble ACE2 or a processed form thereof may comprise or contain the extracellular domain of ACE2 or a fragment thereof that retains the ability to bind to coronavirus.

In some embodiments of the present invention, the soluble ACE2 or a truncated form thereof may comprise a metalloprotease domain (19-615aa) in the extracellular region of human ACE2. The soluble ACE2 or a truncated form thereof may comprise a metalloprotease domain (1-740aa) in the extracellular region of human ACE2. The soluble ACE2 or a truncated form thereof may comprise Q24, T27, F28, D30, K31, H34, E37, D38, Y41, Q42, L45, M82, Y83, Q325, E329, N330, K353, G354, D355, R357 and R393 of human ACE2, in particular K31 and K353.

In some embodiments of the present invention, the soluble ACE2 or processed form thereof may comprise human ACE2 or any homologue or orthologue thereof, or a fragment thereof that retains the ability to bind to coronavirus.

Soluble ACE2 or its processed form can effectively neutralize a virus that uses ACE2 as a receptor to bind to a host cell. The virus may include SARS-CoV, HCoV-NL63, or SARS-CoV2.

In some embodiments of the present invention, the soluble ACE2 or its processed form may comprise ACE2 containing a mutation in the active site or in its processed form. Preferably, the ACE2 containing a mutation in the active site or in its processed form may comprise human soluble ACE2 or its processed form (ACE2-NN) having the mutation(s) H374N and/or H378N at position 374 and/or at position 378.

Soluble ACE2 or its processed form may include soluble ACE2 or its processed form that has ACE2 enzymatic activity.

Preferably, soluble ACE2 or a processed form thereof may be glycosylated at position(s) 53, 90, 103, 322, 432, 546 and/or 690 in the N-terminal portion of human ACE2.

Preferably, the soluble ACE2 or processed form thereof may comprise the amino acid sequence represented by SEQ ID NO: 1.

Preferably, the soluble ACE2 or processed form thereof may comprise the amino acid sequence represented by SEQ ID NO: 2.

Soluble ACE2 or its processed form can effectively neutralize a virus that uses ACE2 as a receptor to bind to a host cell. The virus may include SARS-CoV, HCoV-NL63, or SARS-CoV2.

In some embodiments of the present invention, the antibody may be an IgG antibody. The antibody may be a human IgG, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG1. The Fc domain of the antibody may refer to an Fc domain of the antibody comprising two Fc domains of the heavy chains of the antibody. Preferably, each of the Fc domains of the heavy chains may have a hinge region at its N-terminus.

Preferably, each of the heavy chain Fc domains may comprise a CH3 domain derived from IgG1, IgG2, IgG3, or IgG4. Preferably, each of the heavy chain Fc domains may comprise CH2 and CH3 domains derived from IgG1, IgG2, IgG3, or IgG4. The Fc domains may facilitate dimerization of the two ACE2 domains.

In some embodiments of the present invention, the heavy chain Fc domain may comprise a heavy chain Fc domain having an L309C mutation at position 309.

Soluble ACE2 or its processed form can be bound to the C-terminal region of the Fc domain of heavy chains or to the N-terminal region of the Fc domain of heavy chains.

Preferably, in each polypeptide monomeric unit, one processed form of ACE2, one heavy chain Fc domain together with the tail may comprise the amino acid sequence represented by SEQ ID NO: 7.

Preferably, in each polypeptide monomeric unit, one processed form of ACE2, one heavy chain Fc domain together with the tail may comprise the amino acid sequence represented by SEQ ID NO: 8.

Preferably, in each polypeptide monomeric unit, one processed form of ACE2, one heavy chain Fc domain together with the tail may comprise the amino acid sequence represented by SEQ ID NO: 18.

In some embodiments of the present invention, the ACE2-hFc(n) fusion protein multimer may comprise ACE2-hFc5, ACE2-NN-hFc5, or ACE2-NN-hFc5 L309C, wherein:

ACE2-hFc5 is a pentamer assembled from 5 polypeptide monomeric units via 10 tails located at the C-termini of 5 Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 7;

ACE2-NN-hFc5 is a pentamer assembled from 5 polypeptide monomeric units via 10 tails located at the C-termini of 5 Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 8;

ACE2-NN-hFc5-L309C is a pentamer assembled from 5 polypeptide monomeric units via 10 tails at the C-termini of 5 Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 18, and wherein the heavy chain Fc domain comprises the L309C mutation at position 309.

In some embodiments of the present invention, the ACE2-hFc(n) fusion protein multimer may comprise ACE2-hFc6, ACE2-NN-hFc6, or ACE2-NN-hFc6 L309C, wherein:

ACE2-hFc6 is a hexamer assembled from 6 polypeptide monomeric units via 12 tails at the C-termini of 6 Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 7;

ACE2-NN-hFc6 is a hexamer assembled from 6 polypeptide monomeric units via 12 tails at the C-termini of 6 Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 8;

ACE2-NN-hFc6-L309C is a hexamer assembled from 6 polypeptide monomeric units via 12 tails at the C-termini of 6 Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 18.

In some embodiments of the present invention, the fusion protein multimer may be one or more multimers selected from the following fusion protein multimers:

ACE2-hFc4, which is a tetramer assembled from 4 polypeptide monomeric units via tails at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 7;

ACE2-NN-hFc4, which is a pentamer assembled from 4 polypeptide monomeric units with tails at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units includes a dimer consisting of two processed forms of ACE2 and two Fc domains of the heavy chains, wherein one processed form of ACE2, one Fc domain of the heavy chains together with the tail include the amino acid sequence represented by SEQ ID NO: 8;

ACE2-NN-hFc4-L309C, which is a tetramer assembled from 4 polypeptide monomeric units via tails at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence presented in SEQ ID NO: 18, and wherein the heavy chain Fc domain comprises the L309C mutation at position 309;

ACE2-hFc5, which is a pentamer assembled from 5 polypeptide monomeric units via tails at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 7;

ACE2-NN-hFc5, which is a pentamer assembled from 5 polypeptide monomeric units via tails at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 8;

ACE2-NN-hFc5-L309C, which is a pentamer assembled from 5 polypeptide monomeric units via tails at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence presented in SEQ ID NO: 18, and wherein the heavy chain Fc domain comprises the L309C mutation at position 309;

ACE2-hFc6, which is a hexamer assembled from 6 polypeptide monomeric units via tails at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACESARS-CoV22 and two heavy chain Fc domains, wherein one ACE2 processed form, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 7;

ACE2-NN-hFc6, which is a hexamer assembled from 6 polypeptide monomeric units via tails at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 8;

ACE2-NN-hFc6-L309C, which is a hexamer assembled from 6 polypeptide monomeric units via tails at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two Fc domains of heavy chains, wherein one processed form of ACE2, one Fc domain of heavy chains together with the tail comprise the amino acid sequence presented by SEQ ID NO: 18.

Fusion protein multimers can significantly increase the affinity for the viral S protein and at the same time enhance the effector function of the Fc molecule.

According to a fourth aspect, the present invention provides an expression vector comprising a gene encoding soluble ACE2 or a truncated form thereof according to the first aspect, an ACE2-Fc fusion protein according to the second aspect, or a multimer of an Fc fusion protein (ACE2-hFc(n)) of soluble ACE2 or a truncated form thereof according to the third aspect.

According to a fifth aspect, the present invention provides a mammalian cell line comprising a gene encoding soluble ACE2 according to the first aspect, an ACE2-Fc fusion protein according to the second aspect, or a multimer of an Fc fusion protein (ACE2-hFc(n)) of soluble ACE2 or its processed form according to the third aspect. The cell line may include, in particular, a CHO cell line, a 293 cell line, a Vero cell line and a cell line derived therefrom, such as Vero E6 cells or HEK293T cells.

According to a sixth aspect, the present invention provides a method for producing soluble ACE2 according to the first aspect, the ACE2-Fc fusion protein according to the second aspect, or the Fc-fusion protein multimer (ACE2-hFc(n)) of soluble ACE2 or its processed form according to the third aspect, comprising the following steps:

(1) transfecting a mammalian cell line with the expression vector of the fourth aspect to obtain a mammalian cell line expressing soluble ACE2 of the first aspect, the ACE2-Fc fusion protein of the second aspect, or a multimer of Fc fusion protein (ACE2-hFc(n)) of soluble ACE2 or a processed form thereof of the third aspect;

(2) culturing the mammalian cell line obtained in step (1) under culturing conditions to produce soluble ACE2 of the first aspect, the ACE2-Fc fusion protein of the second aspect, or the Fc-fusion protein multimer (ACE2-hFc(n)) of soluble ACE2 or a processed form thereof of the third aspect so as to produce a recombinant protein; and

(3) purification of the recombinant protein obtained in step (2).

According to a seventh aspect, the present invention provides a pharmaceutical composition comprising: soluble ACE2 according to the first aspect, an ACE2-Fc fusion protein according to the second aspect, or a multimer of the Fc fusion protein (ACE2-hFc(n)) of soluble ACE2 or its processed form according to the third aspect; and a pharmaceutically acceptable carrier.

According to an eighth aspect, the present invention provides the use of soluble ACE2 according to the first aspect, the ACE2-Fc fusion protein according to the second aspect, or the ACE2-hFc(n) multimer of the Fc-fusion protein of soluble ACE2 or its processed form according to the third aspect in the preparation of a medicament for the treatment or prevention of a disease associated with ACE2.

The disease may be a disease selected from a group of any other diseases resulting from infection with a virus that uses ACE2 as a receptor. The virus may include a coronavirus, such as SARS-CoV, HCoV-NL63, or SARS-CoV2. The disease may be a disease selected from a group of diseases that include pneumonia, severe acute respiratory infection, renal failure, heart failure, adult respiratory distress syndrome (ARDS), liver failure, gastrointestinal disease, or severe acute respiratory syndrome.

The soluble ACE2 of the first aspect, the ACE2-Fc fusion protein of the second aspect, or the Fc fusion protein multimer (ACE2-hFc(n)) of soluble ACE2 or the truncated form thereof of the third aspect in the present invention can be used for administration in emergency situations, thereby avoiding high levels of morbidity and mortality resulting from infections caused by viruses that use ACE2 as a receptor, in particular coronavirus infections.

The soluble ACE2 of the first aspect, the ACE2-Fc fusion protein of the second aspect, or the Fc-fusion protein multimer (ACE2-hFc(n)) of soluble ACE2 or its processed form of the third aspect in the present invention can also be used for passive immunization of healthcare workers and individuals at risk of infection with a virus, in particular a coronavirus, using ACE2 as a receptor.

Syncytia are multinucleated giant cells that are ultimately formed by cell fusion after viral infection of host cells. In severe patients infected with SARS-CoV2, diffuse damage to the alveolar epithelium may be observed, resulting in the formation of fused multinucleated cells (syncytium). Syncytia is caused by the binding of viral S protein to ACE2, which is the main cause of the cytopathic effect. Meanwhile, cytokine storm can also cause alveolar damage. The multimeric Fc fusion protein (ACE2-NN-hFcn) of soluble ACE2 can effectively prevent the binding of viral S protein to ACE2, thereby avoiding the formation of fused multinucleated cells (syncytium) and the subsequent cytopathic effect.

Preferably, the drug can be administered by inhalation, intranasally or by instillation into the respiratory tract, eye injection and injection into the middle ear, ear instillation, topical, transdermal, parenteral, subcutaneous and intravenous injection, intradermal injection, intramuscular injection, intrapleural instillation, intraperitoneal injection, by intralisional administration, application to the mucosa or transplantation of a sustained-release carrier. Preferably, the drug is administered by inhalation using a nebulizer.

According to a ninth aspect, the present invention provides a method for screening a drug against an infection caused by a virus, in particular a coronavirus, using ACE2 as a receptor, wherein the method comprises: screening a drug using soluble ACE2 according to the first aspect, the ACE2-Fc fusion protein according to the second aspect, the Fc-fusion protein multimer (ACE2-hFc(n)) of soluble ACE2 or its processed form according to the third aspect, the vector according to the fourth aspect or the mammalian cell line according to the fifth aspect.

According to a tenth aspect, the present invention provides a method for screening a drug against an infection caused by a virus, in particular a coronavirus, using ACE2 as a receptor, wherein the method comprises: using the furin protease or the furin cleavage site in the S protein of the coronavirus as a target for screening the drug.

The drug may be an inhibitor of the furin protease.

The drug is able to block the formation of syncytium via the S protein containing the furin cleavage site during infection with a virus, in particular a coronavirus that uses ACE2 as a receptor.

According to an eleventh aspect, the present invention provides the use of a reagent targeting furin protease or a furin cleavage site in the S protein of a coronavirus in the preparation of a medicament for an infection caused by a virus, in particular a coronavirus, using ACE2 as a receptor.

In particular, the present invention relates to the use of a reagent that blocks syncytium formation via an S protein containing a furin cleavage site during an infection caused by a virus, in particular a coronavirus, using ACE2 as a receptor, in the preparation of a medicament for the treatment of an infection caused by a virus using ACE2 as a receptor, in particular a coronavirus.

Preferably, the reagent may be a furin protease inhibitor.

The coronavirus may be SARS-CoV2.

According to a twelfth aspect, the present invention provides a mutant S protein which is a processed form and/or a form in which the furin cleavage site is mutated.

The processed form may include an S protein containing only the extracellular domain (S1+S2), i.e. deleting the transmembrane and intracellular regions.

The furin cleavage site can be mutated by deletion, substitution, or addition of one or more amino acids such that the furin cleavage site is no longer active as a furin cleavage site.

Preferably, the form in which the furin cleavage site is mutated may comprise a mutation from RRAR to SRAS in the furin cleavage site of the S protein.

Preferably, in addition to the mutation at the furin cleavage site, the mutated S protein may contain only the extracellular domain (S1+S2), i.e. deleting the transmembrane and intracellular regions.

The mutated S protein may have an amino acid sequence represented by SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

SEQ ID NO: 10 is the amino acid sequence in which the furin cleavage site of the S protein has been mutated from RRAR to SRAS. SEQ ID NO: 11 is the amino acid sequence of the S protein in a truncated form with the transmembrane and intracellular regions deleted. SEQ ID NO: 12 is the amino acid sequence in which the furin cleavage site has been mutated from RRAR to SRAS and the S protein has a truncated form with the transmembrane and intracellular regions deleted.

According to a thirteenth aspect, the present invention relates to the use of a mutant S protein according to the twelfth aspect in the preparation of a medicament for the treatment or prevention of a disease associated with ACE2.

The disease is an infectious disease caused by a virus that uses ACE2 as a receptor, and preferably the virus may be a coronavirus, preferably SARS-CoV, HCoV-NL63 or SARS-CoV2.

The disease may be selected from a group of diseases including pneumonia, severe acute respiratory infection, renal failure, heart failure, adult respiratory distress syndrome (ARDS), liver failure, gastrointestinal disease, or severe acute respiratory syndrome.

The drug can be used for passive immunization of healthcare workers and individuals at risk of infection with viruses that use ACE2 as a receptor, in particular coronaviruses.

According to a fourteenth aspect, the present invention provides a recombinant vaccine for preventing infection caused by SARS-CoV2, which comprises the S protein of the twelfth aspect.

The furin cleavage site of the SARS-CoV2 S protein and furin protease may be used as targets for drug screening for the treatment of infectious disease caused by a virus that uses ACE2 as a receptor.

The present invention confirms that the furin cleavage site in the SARS-CoV2 S protein is necessary for syncytium formation. In particular, the ARS-CoV 2 spike (S) protein has an RRAR motif near residues 681-685 (near the S1/S2 junction), which can be cleaved by a protease such as furin. Sequence alignment shows that this motif is present in all known SARS-CoV2 strains, but not in the bat strain RaTG13, which is most closely related to SARS-CoV2. In an alignment of bat virus sequences in the database, a similar sequence (RRAT) is present only in the bat SARS-HKU5 virus. We found that 293T cells transfected with plasmids expressing wild-type ARS-CoV2 S protein or furin protease cleavage site-mutated SARS-CoV2 S protein could bind to ACE2-IgG1 Fc, whereas control cells transfected with empty vector did not bind to ACE2-IgG1 Fc. Wild-type ARS-CoV2 S protein and furin protease cleavage site-mutated ARS-CoV2 S protein could be normally expressed on the surface of 293T cells. In addition, furin protease cleavage site-mutated ARS-CoV2 S protein (RRAR mutated to SRAS) could bind more tightly to ACE2-IgG1 Fc.

In the syncytium formation experiment, no cell fusion was observed between empty vector-transfected control cells and full-length human ACE2-expressing cells (natural human ACE2), and thus no multinucleated syncytium formation was observed. In contrast, a large number of multinucleated syncytia were observed after co-culture of SARS-CoV2 wild-type S protein-expressing cells with full-length human ACE2-expressing cells for 3 h. The resulting syncytia died after 24 h of continuous culture. When the furin site of RRAR in SARS-CoV2 S protein was mutated to SRAS, cell-cell fusion was completely inhibited and no multinucleated syncytia formation was observed, confirming that the mutated S protein lost the ability to mediate cell fusion and that the furin site in S protein played a critical role in SARS-CoV2 infection of cells.

Thus, drugs targeting the furin S protein (PRRAR) site and furin protease are able to suppress SARS-CoV2 infection. Moreover, antiviral drugs for the treatment of SARS-Co V2 infection can be developed by inhibiting the fusion of coronavirus during cell entry.

In addition, when the furin protease cleavage site of RRAR in the S protein of ARS-CoV2 was mutated to SRAS, cell fusion was completely inhibited and no multinucleated syncytia was observed. This indicates that the mutated S protein has lost the ability to mediate cell fusion. Therefore, the SARS-CoV2 S with a mutated furin protease cleavage site in the present invention, in particular, the SARS-CoV2 S having a mutation(s) in the furin protease cleavage site in the form of deletion of the transmembrane and intracellular regions, can be used as a recombinant protein preparation to competitively block the binding of a virus having a wild-type S protein to a cellular receptor, thereby blocking infection. In addition, the SARS-CoV2 S protein with a mutated furin protease cleavage site can also be used as a candidate molecule for a recombinant vaccine and is more stable than the wild-type S protein.

SARS-CoV2, a β-coronavirus, is an enveloped virus. SARS-CoV2 virions are round or oval particles with polymorphism, 60-140 nm in diameter. The spike glycoproteins (S protein) on the surface of the envelope are the main antigenic proteins of the coronavirus and play an extremely important role in the occurrence of infection and the spread of viruses. The S protein consists of two subunits, with the S1 subunit binding to a cell surface receptor, while the S2 subunit contains the main motifs required for the membrane fusion process.

The amino acid sequence of the SARS-CoV2 spike (S) protein has approximately 76% homology with the SARS-CoV S protein. That is, the homology is relatively low. Therefore, most SARS-CoV neutralizing antibodies are unable to neutralize SARS-CoV2. However, SARS-CoV2 uses the same host cell receptor as SARS-CoV, i.e., angiotensin-converting enzyme 2 (ACE2). Both SARS-CoV and the infectious SARS-CoV2 must use ACE2 as a receptor to enter target cells. In other words, although there are numerous differences between the amino acid sequence of the SARS-CoV2 S protein and the SARS-CoV S protein, both viruses still use ACE2 as a receptor to enter host cells. This indicates that the ACE2 protein has a high structural compatibility on its surface with the S protein of such coronaviruses. Therefore, ACE2, as a host protein molecule, can be easily used by spike proteins having many differences in sequences. Thus, ACE2 will still be an entry point for such viruses to infect the human body in the future. The soluble ACE2 and soluble ACE2(NN) with a mutated active center obtained according to the present invention are capable of blocking the binding of the virus using ACE2 as a host receptor to the ACE2 receptor, thereby inhibiting the invasion of the virus. Both soluble ACE2 and ACE2(NN) are of great significance for the prevention and control of a possible future epidemic.

Moreover, due to the epidemic caused by SARS-CoV2 in the human population, many virus variants are emerging under the selection pressure to further adapt to the human host and human immunity. These variants may be characterized by changes in virulence and antigenic sites. Such changes have been observed and reported several times in SARS-CoV. The latest sequencing data of SARS-CoV2 indicate that the receptor binding domain (RBD) of the S protein is still in the process of mutation in the sequence. Another advantage of the soluble ACE2 and soluble ACE2(NN) with active site mutated obtained by the present invention is that SARS-CoV2 can be effectively neutralized as long as the virus uses ACE2 as an entry receptor, and even viral proteins, particularly the S protein, are mutated. For the prevention and treatment of infection caused by a virus in the process of evolution, the soluble ACE2 receptor of the present invention, unlike monoclonal neutralizing antibodies to the S protein, can be used as a therapeutic drug having a broad-spectrum neutralizing ability and not limited by virus mutations, without the need for antibody screening. Thus, it is quite applicable to solving urgent problems of preventing viral epidemics at present and in the future. The experimental results show that the pentameric ACE2-NN-hFc5 of the present invention has high neutralizing activity against infections of all existing major variants of pseudoviruses.

In addition, unlike antibody-based drugs, the receptor is a native protein of the human body and therefore does not require tissue cross-reactivity testing. Receptor fusion proteins are used as an emergency drug for acute infectious diseases, have a long half-life, and are usually administered 1-2 times per treatment. Thus, the receptor also avoids drug antibody testing and long-term toxicity, and shortens the development cycle.

The ACE2-Fc fusion protein and the soluble ACE2-Fc fusion protein (ACE2-NN-hFcn) multimers of the present invention can be therapeutic agents that are specific for preventing a new epidemic in the fastest possible way. The ACE2-Fc fusion proteins and their multimers of the present invention mainly have the following advantages:

(1) preventing the formation of viral resistance in the form of neutralizing antibodies;

(2) preventing the formation of SARS-CoV2 and SARS-CoV syncytia;

(3) the ability to recruit via appropriate complement receptors, dendritic cells, macrophages and natural killer cells against virions or infected cells due to the retention of the Fc domain effector function;

(4) prolongation of the half-life of soluble ACE2 molecules in the bloodstream;

(5) ACE2-Fc, ACE2(NN)-Fc or ACE2-NN-hFcn of the present invention are suitable for compassionate use in emergency situations (subsequently subject to formal clinical trials) because recombinant human ACE2 (rhACE2) has been evaluated in a phase II clinical trial, demonstrating good tolerability and safety, although subjects did not experience significant improvement in clinical symptoms;

(6) ACE2-Fc, ACE2(NN)-Fc or ACE2-NN-hFcn of the present invention may find wide application in the coming months or years to help infected patients prior to vaccination;

(7) the soluble ACE2 obtained according to the method of the present invention, useful for blocking viral infection, is independent of the natural enzymatic activity of ACE2 and does not affect the activity of natural ACE2 in patients; and the soluble ACE2 (NN) with a mutated active center even avoids the potential side effect caused by the enzymatic activity of ACE2 in the body, thereby maximizing the safety of use in the human body;

(8) Compared with anti-S protein antibodies, another advantage of soluble ACE2 is that as long as the virus uses ACE2 as an entry receptor, the efficacy of soluble ACE2, including the increased receptor affinity caused by the viral mutation(s), is not affected by the virus mutation(s).

ACE2 is a metalloprotease that catalyzes the degradation of angiotensin I to the nonapeptide angiotensin (1-9) or angiotensin II to the heptapeptide angiotensin (1-7). ACE2 is thought to be involved in the regulation of cardiovascular function and may play a protective role in acute lung injury, such as vasodilation, antiproliferative stress, and antioxidant stress. ACE2 is expressed in the vasculature and in most organs, but mainly in the lungs, heart, liver, kidneys, and testes. Potential drugs that inhibit ACE2 enzymatic activity are therefore not ideal drugs.

Human native ACE2 (called membrane-type ACE2) has a total length of 805 amino acids, in which the region from positions 1-740 is the extracellular domain, with the remaining 65 amino acids serving as short transmembrane and intracellular regions. The enzymatic activity of ACE2 is carried out by the extracellular domain. Soluble ACE2 or its processed form in the present invention does not have a transmembrane domain.

Brief description of the drawings

Fig. 1 shows the peaks of the ACE2-hFc-ACE2, ACE2-ACE2-hFc, ACE2-hFc5, and ACE2-hFc5-L309C fusion proteins obtained after purification by size-exclusion HPLC (SEC-HPLC).

Fig. 2 illustrates that the spike protein of the novel coronavirus can induce syncytia formation.

Figure 3 illustrates that the ACE2-hFc5 fusion protein significantly inhibits S protein-mediated intercellular membrane fusion in vitro.

Fig. 4 shows the amino acid sequence (SEQ ID NO: 9) and the furin site of the spike protein (S protein) of SARS-CoV2.

Fig. 5 shows the amino acid sequence (SEQ ID NO: 10) of the SARS-CoV2 S protein containing mutations in the furin cleavage site.

Fig. 6 shows the expression of SARS-CoV2 S protein on the cell surface and the result of cell surface analysis of SARS-CoV2 S protein containing mutations in the furin cleavage site.

Fig. 7 shows that no cell fusion occurs between the empty vector-transfected control cells (red) and human ACE2-expressing cells (green) without forming multinucleated syncytia; a group of cells expressing wild-type SARS-CoV2 S protein (red) forms a large number of multinucleated syncytia after co-culture with ACE2-expressing cells (green) for 3 hours, and the resulting syncytia died after 24 hours of continuous culture; and the S protein having mutations in the furin site does not form syncytia after mixing with ACE-expressing cells.

Fig. 8 shows the extracellular domain (Ectdomain) (1-1208aa) (SEQ ID NO: 11) of the wild-type SARS-CoV2 S protein and the extracellular domain (1-1208aa) (SEQ ID NO: 12) of the SARS-CoV2 S protein containing mutations in the furin cleavage site.

Fig. 9 shows the affinity and avidity of the ACE2-hFc and ACE2-hFc5 fusion proteins to the RBD region of the SARS-CoV2 spike S protein.

Fig. 10 illustrates that different forms of ACE2 fusion protein multimers inhibit SARS-CoV2 (D614) pseudovirus infection.

Figure 11 illustrates that the ACE2-hFc5 fusion protein suppresses infections caused by SARS-CoV2 pseudoviruses D614 and G614, SARS virus, and pangolin-CoV pseudovirus.

Fig. 12 illustrates that the ACE2-hFc5 fusion protein suppresses live SARS-CoV2 virus infection.

Fig. 13 shows the physicochemical properties of ACE2-hFc5 before and after nebulization analyzed by size-exclusion HPLC (SEC-HPLC).

Fig. 14 shows the results of in vitro evaluation of the neutralizing activity of the ACE2-hFc5 molecule against ARS-CoV2 pseudovirus S infection before and after nebulization.

Fig. 15 shows the results of RT-qPCR analysis of SARS-CoV2 gRNA and sgRNA content in hamster lungs.

Fig. 16 shows schematically the molecular structures of different forms of ACE2-IgG1 fusion proteins.

Detailed description of embodiments of the present invention

The following embodiments are intended to illustrate the present invention, but not to limit the scope of the present invention. All changes or substitutions made in the methods, steps or conditions of the present invention without departing from the spirit and meaning of the present invention are within the scope of the present invention.

Unless otherwise indicated, the chemical reagents used in embodiments of the present invention are conventional commercially available reagents, while the technical means used in embodiments of the present invention are conventional means well known to those skilled in the art. Fc in all embodiments and in the figures is derived from IgG1. ACE2-hFc in all embodiments and in the figures is ACE2-NN-hFc, in which one processed form of ACE2 and one heavy chain Fc domain have the amino acid sequence shown in SEQ ID NO: 4. Unless otherwise indicated, the various forms of ACE2-hFc refer to all multimers and mutants of ACE2-hFc and ACE2-hFc. ACE2-hFc-ACE2 described in the embodiments of the present invention and in the figures are tetrameric ACE2(NN)-hFc-ACE2(NN), in which one truncated form of ACE2 is linked to one Fc domain of the heavy chains and is further linked to one ACE2 (ACE2-Fc-ACE2), resulting in the amino acid sequence represented by SEQ ID NO: 13. ACE2-ACE2-hFc in all embodiments and in the figures are tetrameric ACE2(NN)-ACE2(NN)-hFc, in which one Fc domain of the heavy chains (ACE2-ACE2-Fc) is linked to two ACE2s or truncated forms thereof that are linked in tandem, resulting in the amino acid sequence represented by SEQ ID NO: 14. ACE2-hFc5s in all embodiments of the present invention and in the figures refers to ACE2(NN)-hFc(5), which is is a tetramer assembled from 5 polypeptide monomeric units via 10 tails located at the C-termini of 5 Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence presented by SEQ ID NO: 8. ACE2-hFc5-L309C in all embodiments of the present invention and in the figures refers to ACE2(NN)-hFc(5)-L309C, which is a tetramer assembled from 5 polypeptide monomeric units via 10 tails located at the C-termini of 5 Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form ACE2, one heavy chain Fc domain together with the tail comprises the amino acid sequence represented by SEQ ID NO: 18, and the heavy chain Fc domain comprises the L309C mutation at position 309. See Fig. 16.

Example 1: Construction of ACE2-hFc Fusion Protein Expression Plasmid, SARS-CoV2 S Protein Expression Plasmid, and Furin Site Mutant SARS-CoV2 S Protein Expression Plasmid

Using the primers shown in Table 1, DNA sequences encoding the CD33 signal peptide, the extracellular region of the ACE2 metalloprotease domain (containing the enzyme inactivation mutations H374N and H378N), and hlgGl Fc were obtained by Overlap PCR. The specific cloning steps were as follows: pcDNA3.0-ACE2-NEMGE was used as a template to construct the ACE2-hFc expression plasmid, and primers 2-FrontIn and 4-MidR were used to PCR obtain DNA fragments encoding the ACE2 extracellular region (19-615aa) containing the enzyme activity inactivation mutations H374N and H378N; pCHOGS-HH009 was used as a template and primers 3-MidF and 5-IgG1Cterm were used to obtain DNA fragments encoding hlgGl Fc by PCR; then, using the obtained products as templates and primers 6-FrontOutXhoI and 5-IgG1Cterm were used to synthesize full-length DNA encoding CD33 signal peptide-extracellular region-hFc ACE2 by overlapping PCR, which was then inserted into the expression vector pCHOGS between the XhoI and Pad cleavage sites to obtain the expression plasmid pCHOGS-ACE2-NN-hIgG1.

For the construction of the plasmid for expressing the coronavirus spike protein pCAGGS-SARS-CoV2 S-C9, _P CMV3-2019-nCoV-Spike(Sl+S2)-long (Sino Biological, Cat#: VG40589-UT) and SB-S-NheI were used as a template:

and SB-S-C9-XhoI:

were used as primers to obtain DNA fragments encoding the SARS-CoV2 spike protein, which were inserted into the human pCAGGS vector between the NheI and XhoI cleavage sites to obtain a plasmid that provided expression of the SARS-CoV2 S protein. In the plasmid, the N-terminal signal peptide was the CD4 signal peptide, and the C-terminus contained a C9 tag.

Based on the specified plasmid, primers SB-S-NheI, SB-S-C9-XhoI, SB-Drs-f were created: and SB-Drs-r:

The furin protease cleavage site PRRAR in the S protein was mutated to PSRAS by overlapping primer PCR to generate an ARS-CoV2 S protein expression plasmid containing the mutated furin site.

Example 2: Construction of plasmids for expression of ACE2-hFc-ACE2 fusion protein, ACE2-ACE2-hFc fusion protein, ACE2-hFc5 fusion protein, and ACE2-hFc5-L309C fusion protein

The construction of plasmids for expressing the ACE2-hFc-ACE2 fusion protein, the ACE2-ACE2-hFc fusion protein, the ACE2-hFc5 fusion protein, and the ACE2-hFc5-L309C fusion protein was similar to the construction of the plasmid for expressing the ACE2-hFc fusion protein and was carried out by a method similar to the method in Example 1.

Example 3: Expression, purification and size-exclusion HPLC analysis of ACE2-hFc fusion protein and ACE2-hFc5 fusion protein

The expression plasmid expressing the ACE2-hFc fusion protein obtained in Example 1 and the expression plasmid expressing the ACE2-hFc5 fusion protein obtained in Example 2 were respectively transfected into 293F cells using PEI. The culture supernatants were collected 5 days after transfection and purified through a Protein A column in one step to obtain purified ACE2-hFc fusion protein and ACE2-hFc5 fusion protein, respectively. After protein quantification using a NanoDrop™ 2000 spectrophotometer, purity analysis was performed by size-exclusion HPLC (Fig. 1A and Fig. 1C). As shown in Fig. 1A, the purity analysis of the obtained ACE2-hFc fusion protein showed only one peak, and the major peak was 99.34%. As shown in Fig. 1C, the purity analysis of the obtained ACE2-hFc5 fusion protein shows only one peak, with the major peak being 66.94%.

Example 4: Expression, purification and size-exclusion HPLC analysis of ACE2-hFc-ACE2 fusion protein, ACE2-ACE2-hFc fusion protein and ACE2-hFc5-L309C fusion protein

The expression plasmids expressing the ACE2-hFc-ACE2 fusion protein, ACE2-ACE2-hFc fusion protein and ACE2-hFc5-L309C fusion protein obtained in Example 2 were respectively transfected into 293F cells using PEI. The culture supernatants were collected 5 days after transfection and purified through a Protein A column and a molecular sieve in two steps to obtain purified ACE2-ACE2-hFc fusion protein, ACE2-hFc-ACE2 fusion protein and ACE2-hFc5-L309C fusion protein, respectively. After quantification of the proteins using a NanoDrop™ 2000 spectrophotometer, each was subjected to purity analysis by size-exclusion HPLC (Figs. IB, 1D and 1E). As shown in Fig. 1B, the major peak of the ACE2-ACE2-hFc fusion protein was 98.96%. As can be seen from Fig. 1D, the major peak of the ACE2-hFc-ACE2 fusion protein was 97.99%. As can be seen from Fig. 1E, the major peak of the ACE2-hFc5-L309C fusion protein was 97.99%.

Example 5: Expression, purification and size-exclusion HPLC analysis of ACE2-hFc5 fusion protein

The expression plasmid expressing the ACE2-hFc5 fusion protein obtained in Example 2 was transfected into 293F cells using PEI. The culture supernatant was collected 5 days after transfection and purified in several steps to obtain purified ACE2-hFc5 fusion protein. After protein quantification using a NanoDrop™ 2000 spectrophotometer, purity analysis was performed by size-exclusion HPLC (Fig. 1F). As shown in Fig. 1F, the major peak of the ACE2-hFc5 fusion protein was 99.2%.

Example 6: In Vitro Intercellular Membrane Fusion Inhibition Experiment

6.1 Method

Experiment using ACE2 fusion proteins to inhibit SARS-CoV2 syncytia formation

The pCAGGS control vector and the plasmids expressing SARS-CoV2 S protein and SARS S protein were co-transfected with pEGFP-Nl into 293T cells, respectively. The hACE2-C9 expression plasmid (maintained in our laboratory) and the pmCherry-Cl vector were co-transfected into 293T cells using PEI. The cells were digested 24 h after transfection, washed once with complete DMEM (10% FBS, 1×PS), and counted. Then, 10 μg/mL of the control protein and ACE2-hFc5 fusion proteins were coincubated with 2.5E5/well of pCAGGS control vector-transfected cells, SARS-CoV2-S-transfected cells, and SARS-S cells, respectively, at 37°C for 30 min. Then, 2.5E5/well of hACE2-C9-transfected cells were added. After co-culture in a cell incubator under 5% _CO2 at 37°C for 3 hours, the inhibitory activity of ACE2-hFc5 fusion proteins on coronavirus (SARS-CoV2) syncytia formation was observed under a fluorescence microscope. The experimental results were photographed and recorded.

6.2 It can be seen from Fig. 2 that a large number of syncytia were formed in both the SARS-CoV2 virus group and the SARS virus group. The largest number of syncytia was formed in the SARS-CoV2 virus group. In contrast, no cell fusion was observed in the control vector group. This result indicates that the SARS-CoV2 S protein is similar to the SARS S protein and has the ability to induce cell fusion to form syncytia. The SARS-CoV2 S protein has a stronger inducing ability than the SARS S protein (upper panel in Fig. 3). This also indirectly confirms that the SARS-CoV2 S protein has a stronger affinity for the hACE2 receptor than the SARS S protein. When the ACE2-hFc5 fusion protein was added, it was observed that, compared with the control protein, the ACE2-hFc5 fusion protein could significantly inhibit the syncytium formation induced by the S protein of the SARS-CoV2 virus with an inhibition rate of up to 90% or more, and even completely inhibit the syncytium formation induced by the SARS virus.

In addition, it can be seen (lower panel in Fig. 3) that a large number of syncytia were formed when the cells expressing coronavirus S protein were coincubated with the cells expressing ACE2 receptor. This indicates that coronavirus S protein can induce the membrane fusion process after binding to ACE2 receptor. Multinucleated syncytia were more clearly evident after 20 hours. When 10 μg/mL ACE2-hFc5 was added to the experimental system, the number of syncytia was significantly reduced with an inhibition rate of up to 90% or more. While the inhibitory effect of ACE2-hFc under similar conditions was not obvious. In addition, it can be seen that ACE2-hFc5 inhibited the formation of syncytia in a dose-dependent manner. ACE2-hFc5 still had significant inhibitory activity against syncytia formation at a low concentration of 0.3 μg/mL.

The process of fusion of enveloped virus (including coronaviruses) with the cell (inner) membrane is a critical factor for viral infection. Syncytia formation is a prominent pathological change that occurs in the lungs after human infection with the SARS-CoV2 virus. Our experimental results show that the ACE2-hFc5 fusion protein has high syncytia formation inhibition activity and can block infection caused by coronaviruses, in particular SARS-CoV2.

Example 7: Cell surface expression and syncytia formation assays for SARS-CoV2 wild-type S protein and mutated S protein containing mutations in the furin protease cleavage site

Empty pCAGGS vector (control) and plasmids expressing SARS-CoV2 S protein and SARS-CoV2 S protein mutated with furin site (in which PRRAR was mutated to PSRAS) were respectively co-transfected by PEI with pmCherry-C1 vector into 293T cells. Full-length ACE2 expression plasmid having extracellular, transmembrane and intracellular regions and pEGFP-N1 vector were co-transfected into 293T cells by PEI. Cells were digested 24 h after transfection, washed once with complete DMEM (10% FBS, 1×PS) and counted. A portion of empty vector-transfected control cells and cells expressing ARS-CoV2 S protein (SEQ ID NO: 9) (Fig. 4) and furin-mutated ARS-CoV2 S protein (SEQ ID NO: 10) (Fig. 5) were then analyzed for cell surface expression of SARS-CoV2 S proteins. The remaining cells were used to assay syncytia formation.

For the cell surface expression study of SARS-CoV2 S protein, 20 μg/mL of ACE2-NN-Fc fusion protein were respectively incubated with the above cells on ice for 45 minutes, then the cells were washed three times with FACS buffer (PBS, 0.5% BSA), followed by the addition of anti-human Fc secondary antibody FITC (F9512, Sigma) diluted at 1:300 and incubated on ice for 30 minutes. The surface expressions of SARS-CoV2 S protein and mutated SARS-CoV2 S protein at the furin site were analyzed by flow cytometer after the cells were washed three times with FACS buffer (PBS, 0.5% BSA). The results were analyzed by FlowJo V10 software and are shown in Fig. 6.

For the syncytium formation experiment assay, empty vector-transfected control cells and cells transfected with SARS-CoV2 S plasmid and SARS-CoV2 S plasmid mutated at the furin site were separately seeded in a 48-well cell culture plate at 2.5E5/well. After 30 minutes, ACE2-transfected cells were added to the above-mentioned cell wells at 2.5E5/well. After continuous co-culture in a 5% _CO2 cell incubator at 37°C for 3 hours, the formation of viral syncytia was observed under a fluorescence microscope. The experimental results were photographed and recorded. As shown in Fig. 7, neither cell fusion nor multinucleated syncytia formation were observed between empty vector-transfected control cells (red) and full-length ACE2-expressing cells (green). In contrast, a large number of multinucleated syncytia were formed after co-culture of wild-type S protein-expressing cells (red) with human full-length ACE2-expressing cells (green) for 3 h. After the furin cleavage site of RRAR in SARS-CoV2 S protein was mutated to SRAS, cell membrane fusion was completely inhibited and multinucleated syncytia were not observed. This indicates that the mutated S protein lost the ability to mediate cell membrane fusion. Thus, the furin site in SARS-CoV2 S protein is essential for S protein-mediated cell fusion.

Moreover, the SARS-CoV2 S protein mutated at the furin protease cleavage site has a significant effect on inhibiting virus-cell fusion and the formation of multinucleated syncytia (see the far right image in Fig. 7).

Example 8: Affinity and avidity analysis of different forms of ACE2-hFc fusion proteins

8.1 Methods

The affinity (BIAcore T200) and avidity (Fortebio Octet RED384) of the ACE2-NN-hFc and ACE2-NN-hFc5 fusion proteins to the receptor-binding domain (RBD) (aa331-527) of the S protein of coronavirus (SARS-CoV-2) were determined by surface plasmon resonance (SPR) and biolayer interferometry (BLI), respectively.

For the affinity assay, the ACE2-hFc and ACE2-hFc5 fusion proteins were first immobilized on the surface of the CM5 biosensor chip coated with anti-human Fc antibody. Then, 2-fold serial dilutions from 200 nM to 6.25 nM of the SARS-CoV-2 RBD protein, which has a His6-Avi tag at the C-terminus, were passed through the chip at 30 μl/min to determine the kinetics of intermolecular binding and dissociation of the proteins. The 1:1 Langmuir binding model (ΒΙΑ Evaluation Software) was used to calculate the association constant (Ka), dissociation constant (Kd), and equilibrium dissociation constant (KD). For the avidity assay, 20 μg/mL of the SARS-CoV-2 RBD protein with a C-terminal His6-Avi tag was first immobilized on the surface of the streptavidin biosensor. Then, different concentrations of ACE2-hFc (0 nM, 2-fold serial dilutions in the range of I, 65-105.3 nM) and ACE2-hFc5 (0 nM, 2-fold serial dilutions in the range of 8.22-526.3 nM) fusion proteins were used as analytes to bind to the sensor surface linked to the RBD (receptor binding domain) for 180 seconds, followed by dissociation for 300 seconds. The 1:1 binding model (Fortebio data Analysis 11.1-knetics software) was used to calculate the binding constants Ka, Kd, and KD.

8.2 Results

Using the monovalent binding affinity assay (BIAcore T200) (Fig. 9), ACE2-hFc and ACE2-hFc5 fusion proteins were found to have similar affinities for the SARS-CoV-2 RBD, i.e., 26.4 nM and 29.1 nM, respectively. Fortebio software was used to analyze the affinity of ACE2-NN-hFc and ACE2-NN-hFc5 fusion proteins for the S protein RBD. In the experiments, 20 μg/mL of RBD-His-avi protein was immobilized on the surface of the streptavidin biosensor, while different concentrations of ACE2-NN-hFc and ACE2-NN-hFc5 fusion proteins were used as mobile phases. Octet DataAnalysis 11.0 software was used to analyze the experimental data, and 0 nM was used as the background value for subtraction. The results showed (Fig. 9) that, compared with the ACE2-hFc fusion protein, the multimerized ACE2-hFc5 fusion protein significantly increased the affinity for the RBD-His-avi spike protein, and the KD values (<1.0 E-12 M) were beyond the detection range of the instrument.

Example 9: In Vitro Coronavirus Pseudovirus Neutralization Experiment

9.1 Methods

9.1.1 Packaging of the pseudo-coronavirus

For the pseudovirus packaging of the coronavirus strain (D614), HEK293T cells were inoculated into a 10 cm cell culture dish. When the cells reached 80% confluency, the full-length coronavirus S protein expression plasmid pSARS-CoV2 S-C9 (D614) was co-transfected with the packaging plasmid psPAX2 and the fluorescein expression plasmid pHFV-Luc at a ratio of 1:3:4 using Lipofactamine 3000 reagent. The medium was removed 6 h after transfection, fresh DMEM medium containing 2% FBS and penicillin was added, and continuous culture was performed for 48 h. Then, the culture supernatant containing pseudovirus particles was collected, centrifuged and filtered to remove cellular debris, and frozen at -80 °C for further use. For packaging other SARS-CoV-2 variants, SARS, and pangolin coronaviruses, the preparation conditions were similar, except that pSARS-CoV2 S-C9 (D614) was replaced with a plasmid expressing the S proteins of the variants. The coronavirus pseudoviruses used in the present invention include: the original D614SARS-CoV2 strain; the original D614 SARS-CoV2 strain having a mutated furin site; the main epidemic G614 SARS-CoV2 strain; the D614 variant (L18F; A22V; V367F; N439K; Y453F; N501Y; T478I; P1263L); SARS-CoV2; SARS; and pangolin coronavirus.

9.1.2 Experiments to neutralize the coronavirus pseudovirus

For the neutralization experiments of coronavirus pseudoviruses, 293T-ACE2 cells stably expressing human ACE2 were first seeded in an opaque 96-well cell culture plate at 1E5/well and cultured in a _CO2 incubator at 37°C for 20 h to perform the neutralization experiment. On the day of the experiment, 75 μl of coronavirus pseudoviruses were uniformly mixed with 25 μl of different forms of serially diluted soluble ACE2 fusion proteins, followed by incubation at room temperature for 30 min. Then, the cell culture supernatant in the 96-well cell culture plate was removed. Next, the premixed mixtures of pseudoviruses and ACE2 fusion proteins were added to the 293T-ACE2 cells. After incubation in _{a CO2} incubator at 37°C for 24 hours, fresh DMEM containing 2% FBS (fetal bovine serum) was added instead of continuing the culture. After 24 hours, luciferase activity was measured using the Bright-Glo™ Luciferase Gene Assay Kit and a microplate luminometer. At least two duplicate wells and a control well with fetal bovine serum were included in the experiment.

9.2 Results

9.2.1 Neutralization of coronavirus pseudovirus infection by different forms of ACE2-hFc fusion protein multimers

For the comparison of different forms of ACE2-hFc fusion proteins for neutralizing pseudocoronavirus infection, 293T cells stably expressing human ACE2 were used as the host cells, and serially diluted ACE2-NN-hFc fusion proteins were mixed with SARS-CoV-2 pseudoviruses to infect 293T-ACE2. The intracellular luciferase activity (RLU) was determined on the second day after infection. The percentage inhibition of ACE2-NN-hFc fusion proteins at different concentrations was calculated based on the intracellular luciferase activity of the virus-infected phosphate-buffered saline control group. As shown in Fig. 10, different forms of ACE2-hFc fusion proteins had neutralizing activity against SARS-CoV-2 pseudovirus D614 infection. In particular, ACE2-hFc5 and ACE2-hFc5 L309C exhibited the highest in vitro neutralizing activity with IC50 of 9.86 ng/mL (half-maximal inhibitory concentration) and 12.4 ng/mL, respectively, followed by ACE2-hFc-ACE2 tetramer and ACE2-ACE2-hFc tetramer with IC50 of 39.31 ng/mL and 260.8 ng/mL, respectively, while ACE2-NN-hFc exhibited the lowest neutralizing activity. This result indicates that the activity of ACE2 fusion proteins to neutralize SARS-CoV-2 infection is proportional to their affinity for the RBD of S protein, i.e., the higher the degree of multimerization, the higher the neutralizing effect. Thus, the neutralizing activity of the ACE2-hFc fusion protein is significantly enhanced by modification of multimerization.

9.2.2 Neutralizing activity of ACE2-NN-hFc5 against broad-spectrum coronavirus infection

To evaluate the broad-spectrum anti-infective activity of ACE2-NN-hFc5 against SARS-CoV-2 variants and related coronaviruses, several single-point mutant SARS-CoV-2 pseudoviruses based on the predominant variants present in the population were packaged and the neutralizing activity of ACE2-NN-hFc5 was assessed using the 293T-ACE2 stable cell line infection model. The results showed that ACE2-NN-hFc5 has potent neutralizing activity and broad-spectrum antiviral activity against the parent strain D614, the major epidemic strain G614, and other SARS-CoV-2 variants, as well as the SARS virus and the pangolin coronavirus pseudovirus. ACE2-NN-hFc5 has a neutralizing activity with an IC50 of 9.56 ng/mL against the major epidemic strain G614 pseudovirus (Figure 1 and Table 2 below). Regarding the neutralizing activity against pseudovirus infection of the main SARS-CoV-2 variants (see Table 3), the results show that the ACE2-NN-hFc5 multimer has a strong neutralizing capacity in vitro. In particular, ACE2-NN-hFc5 has a higher neutralizing activity with an IC50 of 0.036 ng/mL for the N501Y variant. These results indicate that ACE2-NN-hFc5 has a highly effective and broad-spectrum anticoronavirus activity in vitro.

Example 10: In vitro live virus neutralization assay for SARS-CoV-2

10.1 Method

Vero cells were seeded in a 96-well plate at a density of approximately 2 x 10 ⁴ cells/well. The next day, the cell culture medium was changed to 2% FBS-DMEM. The ACE2-hFc fusion protein was diluted in 2% FBS-DMEM to working concentrations of 20 μg/mL, 2 μg/mL, and 0.2 μg/mL, in triplicate wells. 2019-nCoV virus (virus strain: Wuhan 01 C-Tan-nCoV) was diluted to 200 TCID50/100 μL with 2% FBS-DMEM. An equal volume of 200 TCID50 virus was added to 50 μL of the diluted sample and incubated at 37°C for 1 hour. Then, 100 μl of the antibody-virus complex was added to the cells and incubated at 37°C. The cytopathic effect was observed after incubation at 37°C for 48 h. 100 μl of the culture supernatant was aspirated after 48 h for nucleic acid extraction, and 80 μl of the eluate was used for final elution. 5 μl of the nucleic acid extracts were used to prepare the fluorescent real-time RT-PCR reaction mixture, which was analyzed using the ABI Q5 fluorescence quantitative PCR system. A standard curve was used to determine the TCID50 of the virus in the samples based on the measured PCR cycle thresholds of the samples according to the following formula: viral replication inhibition ratio (%) = (control TCID50 - fusion protein TCID50) / control TCID50 × 100%. The above experiment was conducted in a biosafety level 3 laboratory.

10.2 Results

The neutralization effect of ACE2-hFc5 on the live 2019-nCoV virus (Wuhan C-Tan-nCoV 01 strain) was evaluated in a biosafety laboratory using Vero cells as the host cells. The experimental results showed (Fig. 12) that both ACE2-hFc5 and ACE2-hFc had significant neutralizing activity against coronavirus infection with IC50 of 0.02-0.06 μg/mL and 5.3-6.8 μg/mL, respectively. ACE2-NN-hFc5 achieved a higher neutralization effect in vitro (hundreds of times that of ACE2-NN-hFc), and 80% virus inhibition was still observed at a concentration of 0.2 μg/mL. The above results fully confirmed that ACE2-NN-hFc5 has effective anti-coronavirus activity in vitro.

Example 11: Evaluation of nebulized ACE2-hFc5 fusion protein in hamsters 11.1 Methods

11.1.1 Analysis of ACE2-NN-hFc5 deposition and distribution in the airways and lungs after nebulized inhalation

To deliver nebulized ACE2-hFc5, we selected a small animal systemic exposure nebulizer delivery system that included an air pump, air mass flow meter, and systemic exposure cabinet. The nebulizer delivery system was interfaced with an Aerogen® Solo nebulizer, adapter, and aerosol collection device. To analyze drug deposition and distribution in the airways, nasal lavage fluids from hamsters were collected by flushing with normal saline at 0 h, 6 h, and 24 h after drug delivery, respectively. Tracheas, main bronchi, and alveoli were collected, and a portion of each was lysed with tissue lysis buffer. The supernatant was removed by centrifugation to detect ACE2-hFc5 multimers. For lung deposition dose analysis, the lungs of each hamster were lavaged with 4 ml normal saline to collect bronchoalveolar lavage fluid (BALF) at 15 min, 30 min, and 60 min after nebulizer inhalation. The lungs were then removed and homogenized. A portion of the lung homogenate was lysed with tissue lysis buffer, and the supernatant was then separated by centrifugation to determine ACE2-hFc5 multimer content.

11.1.2 ACE2-hFc5 ELISA

The ACE2-hFc5 multimer content was determined using an enzyme-linked immunosorbent assay (ELISA) for binding SARS-CoV-2 RBD. Specifically, streptavidin at a concentration of 2 μg/mL was applied to fix 2 μg/mL biotin-labeled SARS-CoV-2 RBD. The tested dilutions of lavage fluid or tissue lysis supernatant were added, while purified ACE2-NN-hFc5 was used as a standard. Anti-hFc secondary antibody labeled with horseradish peroxidase (HRP) was used for the detection. The OD450-OD630 values were read using a microplate reader.

11.1.3 Analysis of ACE2-hFc5 multimeric forms by size-exclusion HPLC before and after nebulization

ACE2-hFc5 multimers before and after nebulization were analyzed for aggregation and degradation by HPLC. An Agilent 1260 high-performance liquid chromatography system with a G4000 TSK G4000SWxl analytical column and a TSK-GEL SWxl guard column were used. The buffer included 50 mM phosphate buffer and 300 mM NaCl at a pH of 6.7 ± 0.1. The analysis was performed for 20 or 25 min at a flow rate of 0.8 mL/min.

11.2 Result: ACE2-NN-hFc5 could be efficiently deposited into hamster alveoli by nebulized administration.

The respiratory tract administration route by inhalation with a nebulizer was studied because SARS-CoV-2 mainly causes viral infection of the respiratory tract and the infection site is mainly in the lungs. ACE2-hFc5 was nebulized with an Aerogen nebulizer and the nebulized droplets were collected with a matched glass tube set in an ice bath with a recovery rate of 90% or more. The physicochemical properties of ACE2-hFc5 before and after nebulization and its neutralizing activity against SARS-CoV-2 pseudovirus infection were analyzed by size-exclusion HPLC. The results showed that nebulization did not cause the aggregation and degradation of ACE2-NN-hFc5 (Fig. 13). Furthermore, ACE2-NN-hFc5 was shown to be able to significantly suppress SARS-CoV-2 pseudovirus infection, and its antiviral activity was not significantly changed before and after nebulization (Fig. 14).

The small animal nebulizer delivery system was then used to administer 5 mg/mL ACE2-NN-hFc5 to hamsters via nebulizer inhalation over 50 min. Nasal lavage fluid, trachea, main bronchi, and alveoli of the hamsters were collected to analyze the deposition and distribution of ACE2-NN-hFc5 in each part of the airways at 0 h, 6 h, and 24 h after nebulization. It was found that inhaled ACE2-hFc5 was mainly distributed in the alveoli (about 75%) at 0 h, 6 h, and 24 h after inhalation. The distribution of ACE2-hFc5 in nasal lavage fluid rapidly decreased from 17.33% at 0 h to 0.7% at 24 h after inhalation. The distribution of ACE2-hFc5 in the trachea was smaller and ranged from 0.35% to 3.4%. The distribution of ACE2-hFc5 in the bronchi gradually increased from 5% to 19%. These results indicate that inhaled ACE2-NN-hFc5 can effectively reach the alveoli and mainly distribute in the alveoli within 24 hours after inhalation.

Next, the relationship between inhalation doses of ACE2-hFc5 and lung deposition in hamsters was investigated, and the neutralizing activity against pseudoviruses in bronchoalveolar lavage fluid was analyzed. Bronchoalveolar lavage fluid and lung homogenate were collected from hamsters after nebulizer inhalation of ACE2-hFc5 at a concentration of 5 mg/mL for 15 min, 30 min and 60 min. Then, the content of ACE2-hFc5 was determined by ELISA analysis. The amount of ACE2-NN-hFc5 in bronchoalveolar lavage fluid and lung homogenate was calculated as the total amount of lung deposition. The results show that the amount of lung deposition in hamsters increases with the increase of the dose after nebulizer inhalation of ACE2-hFc5. The deposition amounts were 6.48 μg, 18.69 μg, and 33.35 μg when inhaled for 15 min, 30 min, and 60 min, respectively. The deposition amounts of ACE2-hFc5 in the lungs of hamsters decreased rapidly after inhalation. At 12 hours after inhalation, the deposition amounts were 14.73% to 46% of those immediately after inhalation. However, when inhaled for 15 min and 30 min, the bronchoalveolar lavage fluid obtained from hamsters whose respiratory organs were each washed with 4 ml of normal saline 12 hours after inhalation retained 90% or higher neutralizing activity against SARS-CoV-2 pseudoviruses after 4-fold dilution.

Example 12: Evaluation of the efficacy of ACE2-NN-hFc5 in a hamster model of novel coronavirus (SARS-CoV-2) infection

12.1 Method

12.1.1 Experimental design

All animal experiments were approved by the Animal Ethics Review Committee of Kunyaming Institute of Biomedical Sciences, Chinese Academy of Medical Sciences and complied with the laboratory practices and guidelines of the National Kunyaming High-Level Biosafety Laboratory, Yunnan, China.

Adult specific-pathogen-free hamsters were transferred to a high-level biological safety laboratory and raised individually in their respective cages. The hamsters were divided into three groups according to body weight: a control group without treatment; a group receiving nebulizer therapy for 15 minutes twice a day; and a group receiving nebulizer therapy for 30 minutes twice a day, with 7 hamsters in each group. In the experiment, the hamsters were inoculated with ¹⁰⁴ PFU of SARS-CoV-2 viruses (GD108#) through the nasal cavities. After 2 hours, the first nebulizer inhalation was performed with nebulization for 15 min (25 mg ACE2-hFc5) and for 30 min (50 mg ACE2-hFc5), respectively. Nebulizer inhalation was performed once every 12 hours for a total of 6 times. Hamsters were weighed before each nebulizer inhalation. The experiment was terminated 62 hours after virus infection. Lung tissues were collected for SARS-CoV-2 viral genomic load (gRNA) and subvirus (sgRNA) analysis. Per 100 mg of lung tissue (left lung), 1 ml of phosphate-buffered saline (PBS) was added. 200 μl was taken for RNA extraction after rapid homogenization, then RT-qPCR analysis was performed to determine the gRNA and sgRNA viral load of SARS-CoV-2. Statistical analysis was performed using GraphPad Prism 8 software. Two-tailed Mann-Whitney U test was used to analyze the differences between two groups.

12.2 Results: Nebulized administration of ACE2-hFc5 effectively reduces viral load in a hamster model of S ARS-CoV-2 infection.

Based on the established nebulizer inhalation delivery method, we evaluated the in vivo antiviral ability of ACE2-NN-hFC5 against SARS-CoV-2 infection in a hamster model. To evaluate the efficacy of ACE2-hFC5, two dose groups were used in the experiment: one group was nebulizer inhalation for 15 minutes, the other was nebulizer inhalation for 30 minutes. The virus titers peaked on the third day after hamster infection with SARS-CoV-2, and the viremia level gradually increased over time. Therefore, the third day after virus infection was chosen as the endpoint of the experiment. There was some improvement trend in the body weight of hamsters after treatment with ACE2-NN-hFc5. According to the results of gRNA and sgRNA quantification in lung tissues, nebulized inhalation of ACE2-hFc5 could significantly inhibit the replication of SARS-CoV-2 virus in lung tissues (Fig. 15). Fig. 15 shows the results of qPCR analysis performed at the end of the experiment (62 hours after infection) to determine the changes in the SARS-CoV-2 genome (gRNA) and subgenome (sgRNA) in the lungs of hamsters. denotes a control group without treatment; denotes the ACE2-hFc5 group-15 min-twice a day; represents the ACE2-hFc5-30 min-twice group; the horizontal line represents the median. The Mann-Whitney U test was used to statistically analyze the differences between the two groups. In conclusion, ACE2-hFc5 nebulizer inhalation can effectively reduce the viral load in the hamster model of SARS-CoV-2 infection and has potential for the prevention and treatment of SARS-CoV-2 pneumonia.

Although the present invention has been described in detail above based on a general description, it should be obvious to those skilled in the art that specific embodiments and experiments can be made, and some changes or improvements can be made. Therefore, all such changes or improvements made without departing from the essence of the present invention fall within the scope of protection of the present invention.

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</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>TIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDK

WSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNP

QECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYE

VNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNL

YSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTA

WDLGKGDFRILMCTKVTMDDFLTAHNEMGNIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHL

KSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVV

EPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNML

RLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADEPKSSDKTHTCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ

KSLSLSPGK</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="5">

<INSDSeq>

<INSDSeq_length>844</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..844</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q8">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial Sequence</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial

sequence</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..844</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q9">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MPLLLLLPLLWAGALATIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNT

NITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNT

MSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMAR

ANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL

PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSM

LTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHE

AVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQ

WMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKC

DISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSP

YADEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP

PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="6">

<INSDSeq>

<INSDSeq_length>844</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..844</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q10">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial Sequence</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial

sequence</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..844</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q11">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MPLLLLLPLLWAGALATIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNT

NITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNT

MSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMAR

ANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL

PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSM

LTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHNEMGNIQYDMAYAAQPFLLRNGANEGFHE

AVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQ

WMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKC

DISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSP

YADEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP

PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="7">

<INSDSeq>

<INSDSeq_length>862</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..862</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q12">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial Sequence</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial

sequence</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..862</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q13">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MPLLLLLPLLWAGALATIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNT

NITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNT

MSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMAR

ANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL

PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSM

LTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHE

AVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQ

WMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKC

DISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSP

YADEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP

PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSTGKPTLYNVSLVMSDTAGTCY</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="8">

<INSDSeq>

<INSDSeq_length>862</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..862</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q14">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial Sequence</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial

sequence</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..862</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q15">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MPLLLLLPLLWAGALATIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNT

NITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNT

MSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMAR

ANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL

PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSM

LTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHNEMGNIQYDMAYAAQPFLLRNGANEGFHE

AVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQ

WMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKC

DISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSP

YADEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP

PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSTGKPTLYNVSLVMSDTAGTCY</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="9">

<INSDSeq>

<INSDSeq_length>1273</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1273</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q16">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Coaxana purpurea</INSDQualifier_value>

<NonEnglishQualifier_value>Coaxana

purpurea</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLH

STQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLI

VNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNL

REFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWT

AGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRF

PNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS

FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIS

TEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCV

NFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVA

VLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTN

SPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTEC

SNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIE

DLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFG

AGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQAL

NTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM

SECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNG

THWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI

SGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTS

CCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="10">

<INSDSeq>

<INSDSeq_length>1273</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..1273</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q17">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial Sequence</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial

sequence</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1273</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q18">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLH

STQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLI

VNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNL

REFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWT

AGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRF

PNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS

FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIS

TEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCV

NFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVA

VLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTN

SPSRASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTEC

SNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIE

DLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFG

AGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQAL

NTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM

SECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNG

THWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI

SGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTS

CCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="11">

<INSDSeq>

<INSDSeq_length>1208</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..1208</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q19">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial Sequence</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial

sequence</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1208</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q20">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLH

STQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLI

VNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNL

REFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWT

AGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRF

PNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS

FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIS

TEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCV

NFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVA

VLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTN

SPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTEC

SNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIE

DLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFG

AGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQAL

NTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM

SECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNG

THWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI

SGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="12">

<INSDSeq>

<INSDSeq_length>1208</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..1208</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q21">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial Sequence</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial

sequence</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1208</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q22">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLH

STQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLI

VNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNL

REFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWT

AGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRF

PNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS

FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIS

TEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCV

NFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVA

VLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTN

SPSRASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTEC

SNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIE

DLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFG

AGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQAL

NTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM

SECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNG

THWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI

SGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="13">

<INSDSeq>

<INSDSeq_length>1443</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..1443</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q23">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial</NonEnglishQualifier_v

alue>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1443</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q24">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MPLLLLLPLLWAGALATIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNT

NITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNT

MSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMAR

ANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL

PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSM

LTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHNEMGNIQYDMAYAAQPFLLRNGANEGFHE

AVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQ

WMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKC

DISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSP

YADEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP

PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGKAAATIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQN

MNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGK

VCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGD

YWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMW

GRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMMLTDPGNVQK

AVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHNEMGNIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLS

AATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMK

REIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAG

QKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD</INSD

Seq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="14">

<INSDSeq>

<INSDSeq_length>1440</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..1440</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q25">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial</NonEnglishQualifier_v

alue>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1440</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q26">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MPLLLLLPLLWAGALATIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNT

NITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNT

MSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMAR

ANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL

PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSM

LTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHNEMGNIQYDMAYAAQPFLLRNGANEGFHE

AVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQ

WMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKC

DISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSP

YADTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQ

EIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSL

DYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDV

EHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDA

MVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTM

DDFLTAHNEMGNIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEI

NFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHV

SNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGA

KNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADEPKSSDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG

KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ

PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK</INSDSeq

_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="15">

<INSDSeq>

<INSDSeq_length>18</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..18</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q27">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial</NonEnglishQualifier_v

alue>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..18</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q28">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>PTLYNVSLVMSDTAGTCY</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="16">

<INSDSeq>

<INSDSeq_length>18</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..18</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q29">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial</NonEnglishQualifier_v

alue>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..18</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q30">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>PTHVNVSVVMAEVDGTCY</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="17">

<INSDSeq>

<INSDSeq_length>21</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..21</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q31">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial</NonEnglishQualifier_v

alue>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..21</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q32">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>TGKPTLYNVSLVMSDTAGTCY</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="18">

<INSDSeq>

<INSDSeq_length>862</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..862</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q33">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Artificial</INSDQualifier_value>

<NonEnglishQualifier_value>Artificial</NonEnglishQualifier_v

alue>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..862</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q34">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>Synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MPLLLLLPLLWAGALATIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNT

NITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNT

MSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMAR

ANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL

PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSM

LTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHNEMGNIQYDMAYAAQPFLLRNGANEGFHE

AVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQ

WMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKC

DISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSP

YADEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVCHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP

PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSTGKPTLYNVSLVMSDTAGTCY</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (42)

1. A fusion protein of soluble ACE2 that binds to coronavirus, comprising the processed form of soluble ACE2, the Fc domain of the antibody and the tail, wherein the processed form of soluble ACE2 is bound to the N-terminal region of the Fc domain, and the tail is located at the C-terminal region of the Fc domain, the processed form of soluble ACE2 contains or consists of the extracellular domain of ACE2 or a fragment of the extracellular domain of ACE2 that retains the ability to bind to coronavirus, and the processed form of soluble ACE2 comprises the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and the tail is a tail derived from IgM and/or IgA and comprising the sequence represented by SEQ ID NO: 17. 2. The fusion protein of claim 1, wherein the antibody is an IgG antibody, preferably a human IgG antibody, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG1, preferably, the Fc domain of the antibody comprises two Fc domains of the heavy chains of the antibody, preferably, each of the Fc domains of the heavy chains has a hinge region at its N-terminus, preferably, each of the heavy chain Fc domains comprises a CH3 domain derived from IgG1, IgG2, IgG3 or IgG4, preferably, each of the heavy chain Fc domains comprises CH2 and CH3 domains derived from IgG1, IgG2, IgG3 or IgG4, and Preferably, each of the Fc domains of the heavy chains includes a hinge region, a CH2 domain, and a CH3 domain. 3. The fusion protein according to claim 1, characterized in that 2n processed forms of soluble ACE2 are linked to the N-terminal region of the Fc domain, where n is selected from 1, 2 or 3, preferably, two processed forms of soluble ACE2 are bound to the N-terminal regions of two heavy chain Fc domains to form a dimer; Preferably, the processed forms of soluble ACE2 are linked in tandem by a linker, preferably a cysteine AAA linker. 4. The fusion protein of claim 1, wherein the processed form of soluble ACE2 is glycosylated, preferably at position(s) 53, 90, 103, 322, 432, 546 and/or 690 of human ACE2. 5. A multimer of an Fc fusion protein, ACE2-hFc(n), that binds to a coronavirus, comprising n polypeptide monomeric units, wherein each of the polypeptide monomeric units is a dimer in which two processed forms of soluble ACE2 are linked to the N-terminal regions of two heavy chain Fc domains, respectively, and the n polypeptide monomeric units are assembled into a multimer via a tail located at each of the C-termini of the two heavy chain Fc domains, wherein the processed form of soluble ACE2 comprises an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, the tail is a tail derived from IgM and/or IgA and comprising the sequence represented by SEQ ID NO: 17, and n is selected from 4, 5 or 6. 6. A fusion protein multimer according to claim 5, characterized in that each of the C-terminal regions of the two Fc domains of the heavy chains is linked to a tail in each polypeptide monomeric unit and n polypeptide monomeric units have a total of 2n tails linked to each other and forming a multimer. 7. A fusion protein multimer according to claim 5, characterized in that the fusion protein multimer comprises one or more multimers selected from the following group: ACE2-hFc4, which is a tetramer assembled from 4 polypeptide monomeric units via tails located at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 7; ACE2-NN-hFc4, which is a tetramer assembled from 4 polypeptide monomeric units via tails located at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 8; ACE2-NN-hFc4-L309C, which is a tetramer assembled from 4 polypeptide monomeric units via tails located at the C-termini of the Fc domains, each of the polypeptide monomeric units comprising a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 18, and the heavy chain Fc domain comprises the L309C mutation at position 309; ACE2-hFc5, which is a pentamer assembled from 5 polypeptide monomeric units via tails located at the C-termini of the Fc domains, each of the polypeptide monomeric units includes a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail include the amino acid sequence represented by SEQ ID NO: 7; ACE2-NN-hFc5, which is a pentamer assembled from 5 polypeptide monomeric units via tails located at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 8; ACE2-NN-hFc5-L309C, which is a pentamer assembled from 5 polypeptide monomeric units via tails located at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence presented by SEQ ID NO: 18, and wherein the heavy chain Fc domain comprises the L309C mutation at position 309; ACE2-hFc6, which is a hexamer assembled from 6 polypeptide monomeric units via tails located at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 7; ACE2-NN-hFc6, which is a hexamer assembled from 6 polypeptide monomeric units via tails located at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units comprises a dimer consisting of two processed forms of ACE2 and two heavy chain Fc domains, wherein one processed form of ACE2, one heavy chain Fc domain together with the tail comprise the amino acid sequence represented by SEQ ID NO: 8; ACE2-NN-hFc6-L309C, which is a hexamer assembled from 6 polypeptide monomeric units via tails located at the C-termini of the Fc domains, wherein each of the polypeptide monomeric units includes a dimer consisting of two processed forms of ACE2 and two Fc domains of heavy chains, wherein one processed form of ACE2, one Fc domain of the heavy chain together with the tail include the amino acid sequence presented by SEQ ID NO: 18. 8. An expression vector comprising a gene encoding a fusion protein according to any one of claims 1-4. 9. An expression vector comprising a gene encoding a multimer of the fusion protein according to any one of claims 5-7. 10. A mammalian cell for expressing the fusion protein of any one of claims 1-4, comprising a gene encoding the fusion protein of any one of claims 1-4. 11. A mammalian cell according to claim 10, characterized in that the cell comprises, in particular, a CHO cell, a 293 cell, a Vero cell and a cell obtained therefrom; preferably comprises a Vero E6 cell or a HEK293T cell. 12. A mammalian cell for expressing a fusion protein multimer according to any one of claims 5-7, comprising a gene encoding a fusion protein multimer according to any one of claims 5-7. 13. A mammalian cell according to claim 12, characterized in that the cell comprises, in particular, a CHO cell, a 293 cell, a Vero cell and a cell obtained therefrom; preferably comprises a Vero E6 cell or a HEK293T cell. 14. Use of a fusion protein according to any one of claims 1-4 or a fusion protein multimer according to any one of claims 5-7 in the preparation of a medicinal product for the treatment or prevention of a disease associated with ACE2, wherein said disease is selected from the group consisting of infection caused by a virus that uses ACE2 as a receptor, pneumonia, severe acute respiratory infection, renal failure, heart failure, adult respiratory distress syndrome (ARDS), liver injury, gastrointestinal disease, or severe acute respiratory syndrome, and the virus is a coronavirus. 15. The use according to paragraph 14, characterized in that the coronavirus is selected from the group including SARS-CoV, HCoV-NL63 or SARS-CoV2. 16. Use according to paragraph 14, characterized in that the medicinal product is used for passive immunization of healthcare workers and individuals at risk of infection with a virus, in particular a coronavirus that uses ACE2 as a receptor. 17. The use according to any one of claims 14 to 16, characterized in that the medicinal product is administered by inhalation, intranasally or by instillation into the respiratory tract, eye injection and injection into the middle ear, ear drops, topical, transdermal, parenteral, subcutaneous and intravenous injection, intradermal injection, intramuscular injection, intrapleural instillation, intraperitoneal injection, by intralisional administration, application to the mucous membrane or by transplantation of a prolonged-release carrier; preferably the medicinal product is administered by inhalation using a nebulizer.