Novel coronavirus protection nasal spray as well as preparation and use thereof

Abstract

The invention relates to a fully human ACE2-Fc fusion protein, a pharmaceutical composition, a preparation and a kit containing the fully human ACE2-Fc fusion protein, and a nasal spray containing the fusion protein. The invention also relates to the use of said fusion protein for the broad spectrum prevention of coronavirus infections, such as coronavirus SARS-CoV-2, known and unknown variants thereof, including but not limited to infections of the original type Hu-1, alpha, beta, gamma, delta, muon, Armike, JN.1 and / or other future strains. The invention also relates to a method for producing the fusion protein, and a method for preventing and / or treating infection of the coronavirus SARS-CoV-2 and known and unknown variants thereof by using the fusion protein. Furthermore, the invention also relates to the fusion protein as well as a pharmaceutical composition and a preparation thereof, which are used for preventing the spreading of the coronavirus SARS-CoV-2 and known and unknown variants of the coronavirus SARS-CoV-2 infected with a subject.

Description

Novel coronavirus protective nasal spray and its preparation and use

[0001] This application claims priority to Chinese Patent Application No. 202310217327.3 filed on March 4, 2023, which is hereby incorporated by reference in its entirety. Technical Field

[0002] The present disclosure belongs to the field of biopharmaceuticals and specifically relates to a fully human ACE2-Fc fusion protein for preventing, treating, and preventing coronavirus infection and transmission, as well as pharmaceutical compositions, formulations, and kits comprising the fusion protein, such as a nasal spray comprising the fusion protein. The present disclosure also relates to methods for producing the fusion protein, and using the fusion protein to prevent and / or treat infection by the coronavirus SARS-CoV-2 and its known and unknown variants, as well as to prevent the spread of the coronavirus SARS-CoV-2 and its known and unknown variants in infected subjects. Background Art

[0003] Coronaviruses infect a wide range of birds and mammals, including humans. Coronaviruses may spread among the population every year, usually causing mild respiratory illnesses, although they are more severe in infants, the elderly, and people with compromised immune systems. However, certain coronaviruses, including Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-1), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), are highly pathogenic. Moreover, the new coronavirus mutates rapidly. Since the end of 2020, multiple variant SARS-CoV-2 strains (VOCs) carrying mutations that may lead to immune escape have begun to appear. However, the development and marketing cycle of conventional vaccines is long, which is far from enough to cope with the threats posed by the new coronavirus to work, life and health, and global epidemic prevention work. Therefore, emergency technical equipment with broad-spectrum protection plays an important role in reducing the risk of infection to the public, alleviating the pressure of epidemic prevention work, and improving the vitality of social life. The present disclosure provides methods, uses, and products (articles of manufacture) that meet the above and other needs.

[0004] Summary of the Invention

[0005] Coinciding with the global surge in COVID-19 cases, multiple new variants of SARS-CoV-2 (VOCs) began to emerge in late 2020. These VOCs appear to be associated with mutations in the spike (S) protein that could increase viral transmissibility and / or allow for immune escape from the first wave of COVID-19 vaccinations based on the SARS-CoV-2 Hu-1 strain (also known as the original SARS-CoV-2 strain or the SARS-CoV-2 wild-type strain). The emergence and spread of the B.1.1.7 variant in the United Kingdom (UK), the B.1.351 variant in South Africa, and the P.1 variant in Brazil led to their classification as VOCs. These VOCs all contain the N501Y mutation in the receptor binding domain (RBD) of the S protein, which has been reported to increase transmissibility by 40% to 70%. The B.1.351 and P.1 variants have two additional RBD mutations—E484K and K417—that could allow for immune escape from Hu-1 vaccines and antibodies induced by natural infection.

[0006] Randomized controlled clinical trials of COVID-19 vaccines have shown reduced effectiveness against VOCs compared to the SARS-CoV-2 Hu-1 strain. The adjuvant protein-based COVID-19 vaccine NVX-CoV2373 was 89% effective in the UK (where B.1.1.7 is dominant), but only 49% effective in South Africa (where B.1.351 is dominant). The adenovirus vectored COVID-19 vaccine ChAdOx1 was only 10% effective against the B.1.351 variant. Pfizer vaccine recipients were 75% effective against the B.1.351 variant and 95% effective against Hu-1. Coronavac is an inactivated vaccine based on the Hu-1 strain, and no neutralizing antibody titers against P.1 were detected in subjects who received the vaccine.

[0007] While there is some encouraging evidence that the Hu-1 COVID-19 vaccine may prevent severe illness and death caused by VOCs, lower vaccine effectiveness against any COVID-19 disease with increasing transmission rates may make achieving herd immunity particularly difficult. If not effectively controlled, the rapid global spread of SARS-CoV-2 VOCs may lead to the continued emergence of new target variants or VOCs that may contain new escape mutations, such as the Indian variant (B.1.617), which emerged in conjunction with the large surge in COVID-19 cases in the spring of 2021 and has now been declared a new VOC by the WHO. The B.1.617.2 variant of this lineage has been named Delta. In addition, the B.1.1.529 variant first identified in South Africa has been renamed Omicron.

[0008] In these cases, other broad VOC containment methods must be rapidly evaluated.

[0009] In one aspect, the present disclosure provides a fusion protein comprising multiple recombinant polypeptides, wherein the fusion protein comprises a human ACE2 protein or a fragment thereof and a human IgG Fc protein or a functional variant thereof, wherein the fusion protein specifically binds to a wild-type, variant or mutant human ACE2 receptor through one or more binding sites.

[0010] In some embodiments, the fusion protein is a water-soluble protein.

[0011] The use of fully human antibody-Fc fusion protein technology can significantly increase the level of serum-free expression of soluble proteins in vitro, facilitate affinity purification, and improve the stability of ACE2-Fc proteins in vivo and in vitro. Because ACE2-Fc is a fully human fusion protein and the preferred route of administration is passive immunization via nasal spray, it is expected to have good safety.

[0012] In some embodiments, the human ACE2 protein or fragment thereof comprises a human ACE2 extracellular domain or a fragment thereof.

[0013] In some embodiments, the human ACE2 protein or a fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 5, 7, 11, 12, 13, and 14, or an amino acid sequence or a fragment thereof having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0014] In some embodiments, the human ACE2 protein or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence or a fragment thereof having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0015] In some embodiments, the human IgG Fc protein or a functional variant thereof comprises an Fc region sequence selected from any one or more of human IgG1Fc, IgG2Fc, IgG3Fc, and IgG4Fc, or a functional variant and fragment thereof.

[0016] In some embodiments, the human IgG Fc protein or a functional variant thereof is selected from a human IgG1 Fc region sequence or a functional variant and fragment thereof.

[0017] In some embodiments, the human IgG Fc protein or a functional variant thereof comprises the amino acid sequence of SEQ ID NO: 6 or 8, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a fragment thereof.

[0018] In some embodiments, the human IgG Fc protein or a functional variant thereof comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a fragment thereof.

[0019] In some embodiments, the fusion protein optionally comprises a signal peptide.

[0020] In some embodiments, the fusion protein optionally comprises a peptide linker, wherein the human ACE2 protein or fragment thereof and the human IgG Fc protein or functional variant thereof are directly connected or connected via a peptide linker. For example, the peptide linker is selected from an arginine-serine peptide linker (-RS-), a valine-serine linker (-VS-), and a glycine-serine linker (-GS-). The peptide linker can be a common peptide linker known in the art for connecting different functional polypeptide parts in a fusion protein.

[0021] In some embodiments, the fusion protein optionally includes a mutant sequence. The mutant sequence is used for C-terminal modification to facilitate tracking detection.

[0022] In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-18, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a fragment thereof, or a combination thereof, such as, for example, the amino acid sequence of any one of SEQ ID NOs: 1-4 or 15-18, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a fragment thereof.

[0023] In some embodiments, the fusion protein further comprises a detectable tag.

[0024] In one aspect, the present disclosure provides a pharmaceutical composition comprising a fusion protein of the present disclosure and optionally a pharmaceutically acceptable carrier.

[0025] In some embodiments, the pharmaceutical composition comprises about 0.1 mg / ml to about 100 mg / ml of the fusion protein of any one of claims 1 to 15, such as about 0.50 mg / ml to about 20.00 mg / ml, such as about 1.25 mg / ml, about 2.50 mg / ml, or about 5.00 mg / ml.

[0026] In some embodiments, the pharmaceutical composition is in a dosage form suitable for intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intranasal, sublingual, tonsil, oropharyngeal or other parenteral and mucosal routes of administration, preferably in a dosage form suitable for intranasal administration, such as a nasal spray.

[0027] In one aspect, the present disclosure provides a fusion protein or pharmaceutical composition of the present disclosure for preventing or treating infection by coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects.

[0028] In one aspect, the present disclosure provides the use of the fusion protein or pharmaceutical composition of the present disclosure in the preparation of a medicament for preventing or treating infection by the coronavirus SARS-CoV-2 and its variants and / or preventing the spread of the coronavirus SARS-CoV-2 and its variants in infected subjects.

[0029] In one aspect, the present disclosure provides a method for preventing, treating infection by coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in an infected subject, comprising administering a therapeutically effective amount of a fusion protein or pharmaceutical composition of the present disclosure to the subject.

[0030] In some embodiments, the administration is intranasal.

[0031] In one aspect, the present disclosure provides a kit for preventing or treating infection by coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects, comprising a fusion protein or pharmaceutical composition of the present disclosure;

[0032] Container; and

[0033] Optional package insert or label indicating prophylaxis and / or treatment.

[0034] In the present disclosure, the coronavirus SARS-CoV-2 and its variants include but are not limited to SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Miu, Omicron, JN.1; and unknown variants of the coronavirus SARS-CoV-2.

[0035] In one aspect, the present disclosure provides a nucleic acid encoding a fusion protein or a fragment thereof of the present disclosure, a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or the vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 shows the mechanism by which SARS-CoV-2 virus infects the host through the ACE2 receptor.

[0037] Figures 2A-2H show the affinity test results of ACE2-Fc fusion protein (SCB-719) with S proteins of different strains. Figure 2A shows the affinity test results with the wild-type (Hu-1) S protein; Figure 2B shows the affinity test results with the Beta variant S protein; Figure 2C shows the affinity test results with the Delta variant S protein; Figure 2D shows the affinity test results with the Omicron variant (BA.1) S protein; Figure 2E shows the affinity test results with the Omicron variant (BA 4 / 5) S protein; Figure 2F shows the affinity test results with the Omicron variant (XBB1.5) S protein; 2G shows the affinity test results with the Omicron variant (EG5.1) S protein; Figure 2H shows the affinity test results with the JN.1 variant S protein.

[0038] Figures 3A-3D illustrate the principle behind the ACE2-Fc fusion protein nasal spray's ability to prevent SARS-CoV-2 infection. Figure 3A shows the ACE2-Fc fusion protein nasal spray being sprayed into the nasal cavity; Figure 3B illustrates the process of airborne viruses or virus-carrying droplets entering the nasal cavity; Figure 3C shows the soluble ACE2-Fc fusion protein nasal spray, developed using Fc fusion protein technology, being sprayed into the human upper respiratory tract, primarily the nasal cavity, forming a first line of defense against viral invasion, like a biologically functional invisible mask. Figure 3D illustrates the process of SARS-CoV-2 virus invading cells and how the ACE2-Fc fusion protein blocks this invasion. When airborne viruses or virus-carrying droplets are present, the first barrier to entry is through the cells of the upper respiratory tract. At this point, the ACE2-Fc fusion protein coating the nasal cavity and upper respiratory tract can competitively bind to any mutant SARS-CoV-2 virus, thereby blocking the site where the viral S protein binds to host cells and preventing the virus from entering the host cell and infecting the body.

[0039] Figures 4A-4C illustrate exemplary purification and characterization of ACE2-Fc fusion proteins according to embodiments of the present disclosure. Figure 4A shows an expression level assay of ACE2-Fc fusion protein in CHO cells; Figure 4B shows affinity purification of ACE2-Fc fusion protein; and Figure 4C shows purity assay of ACE2-Fc fusion protein.

[0040] Figures 5A-5C exemplarily illustrate the neutralization activity test of the ACE2-Fc fusion protein of the embodiment of the present disclosure against the original strain and mutant pseudovirus of the new coronavirus. Figure 5A: The mutant strains detected include alpha, beta, gamma, delta and Omicron strains; Figure 5B: The mutant strains detected include beta, delta, BA.1, BA.2, BA2.12.1, BA.2.75, BA.2.76, BA.2.75.2, BA.4 / 5, BF.7, BQ.1.1, XBB, EG.1, JN.1 and SARS; Figure 5C: The ratio of neutralization activity (neutralization activity against pseudovirus of variant strains / neutralization activity against pseudovirus of original strains). Figures 5A-5C show that the ACE2-Fc fusion protein of the present disclosure has neutralizing activity against pseudoviruses of all currently known variant strains, and the neutralization activity against variant strains is higher than that against the original strain.

[0041] Figures 6A-6B show the design of the challenge experiment. Figure 6A shows a delta challenge test in transgenic mice carrying human ACE2. The 40 mice participating in the experiment were divided into 4 groups, with 10 mice in each group. Samples were taken on the 3rd day (5 mice) and 10th day (5 mice) after the challenge to measure body weight, lung viral load and lung pathology analysis and scoring. The first group in Figure 6B is a control group, in which normal saline / vegetarian is administered to mice by nasal spray; the second group is administered 0.1 ml ACE2-Fc fusion protein to mice by nasal spray at a dose of 5 mg / kg; the third group is administered 0.1 ml ACE2-Fc fusion protein to mice by nasal spray at a dose of 50 mg / kg; the fourth group is administered 0.2 ml ACE2-Fc fusion protein by intraperitoneal injection at a dose of 50 mg / kg.

[0042] Figures 7A-7B exemplify the results of challenge experiments (pharmacodynamic experiments) conducted on transgenic mice using nasal and systemic administration. In indicates nasal administration; ip indicates systemic administration. Figure 7A shows the results of the lung live virus load test. Figure 7B shows the results of the lung virus (RNA) load test. Figure 8A exemplifies the distribution imaging of various organs of test substance B1, solvent B2, and test substance S4 at different time points (n=3, 2), A: liver; B: spleen; C: kidney; D: heart; E: lung; F: brain; G: blood; H: nasal cavity.

[0043] FIG8B exemplarily shows the fluorescence signal intensity of the nasal cavity of the test substance S4 at different time points (p / s / cm 2 / sr) / (μW / cm 2 ), Mean ± SEM., n = 3).

[0044] FIG9 exemplarily shows the PK and in vitro efficacy curves of ACE2-Fc fusion protein (IV / IP). DETAILED DESCRIPTION

[0045] The present disclosure provides a fully human ACE2-Fc fusion protein for preventing, treating, and preventing the spread of coronavirus infection, a pharmaceutical composition and preparation and a kit comprising the same, as well as a method for manufacturing and using the same. In some embodiments, a nasal spray formulation comprising an ACE2-Fc fusion protein shows a surprisingly wide range of effects in preventing coronavirus infection, i.e., by applying the ACE2-Fc fusion protein receptor to the entire nasal surface, the virus is efficiently and competitively prevented from binding to ACE2 on the surface of the nasal mucosal epithelial cells, effectively inhibiting the invasion of the virus. This means that when faced with new variant coronavirus strains and when the existing vaccine has limited protective power, passive immunization with ACE2-Fc soluble receptors administered by nasal spray can flexibly, quickly, and effectively achieve protection against viral invasion, especially for high-risk groups (medical staff, customs entry-exit management personnel), and places facing aggregation, closed environments, and limited air circulation (such as airplanes, high-speed rail, and other public transportation). In addition, the ACE2-Fc fusion protein, by binding to the virus in the body of an infected individual, can prevent the spread of the virus in the body of the infected individual when it appears outside the body (e.g., sneezing and saliva), especially to prevent family transmission. Furthermore, the ACE2-Fc fusion protein reduces the amount of virus in the infected individual by binding to the virus in the infected individual, thereby achieving a therapeutic effect. This therapeutic effect can be achieved through nasal administration or systemic administration.

[0046] The COVID-19 pandemic has now infected over 500 million people worldwide, with a cumulative death toll exceeding 6 million. Even more worrying is that, following previous infections with variants such as the Indian Delta, South African, and Brazilian strains, more contagious mutations, such as the Omicron strain, have been emerging globally since the end of 2021. These strains not only increase infection rates but also evade available vaccines and neutralizing antibodies, forcing the global implementation of various response strategies. However, the mutation of Omicron BA.1 has gradually led to the emergence of a series of more contagious variants, including BA.2, BA.2.12.1, BA.4, BA.5, XBB, EG.5, and JN.1. Therefore, the world urgently needs to quickly develop effective and safe response strategies to address the rapid mutation rate and high immune evasion potential of the COVID-19 virus. Currently, the development of vaccines against these mutant strains lags behind the virus's own mutation rate. We need to develop alternative approaches, beyond vaccines, to block infection by any COVID-19 variant.

[0047] The ACE2-Fc fusion protein disclosed in this disclosure is intended to be a powerful supplement to make up for the shortcomings of current vaccine development and solve the problems of rapid mutation rate, high immune escape and strong infectiousness of the new coronavirus. It is expected to effectively alleviate the current global outbreak of the new coronavirus.

[0048] Key technologies to be addressed: First, how can ACE2-Fc fusion protein, as a receptor that can bind to any mutant strain of the new coronavirus, be applied to the protection against mutant strains of the new coronavirus. ACE2 is the key receptor for the new coronavirus to enter host cells. Faced with the continuous mutation of the virus and its escape from vaccines and neutralizing antibodies, the protective efficacy of vaccines and therapeutic monoclonal antibodies continues to weaken, but the receptor for the virus to enter host cells remains unchanged. At the same time, studies have shown that the more infectious the strain, the higher its affinity for the ACE2 receptor. Therefore, the use of ACE2-Fc fusion protein as a soluble receptor to block viral invasion can effectively respond to any existing and future mutant strains of the new coronavirus. This is determined by the key mechanism of new coronavirus infection. Existing monoclonal antibodies may lose their protective ability against existing and future mutant strains at any time. Secondly, the feasibility of ACE2-Fc fusion protein as a nasal spray preparation in daily use, that is, the applicability of storage and use temperature.

[0049] The novel coronavirus can enter the human body through the nasal mucosa of the upper respiratory tract, leading to infection. Therefore, the nasal cavity is the primary entry point for the novel coronavirus. Using a nasal spray, the ACE2-Fc soluble receptor, which can effectively and competitively block the virus from binding to ACE2 on the surface of nasal mucosal epithelial cells, is applied to the entire nasal surface. This can effectively inhibit viral invasion, especially when faced with new variants and when existing vaccines have limited protection. Passive immunization with the ACE2-Fc soluble receptor administered through a nasal spray can flexibly, rapidly, and effectively protect against viral invasion, especially for high-risk groups (medical staff, customs entry-exit management personnel), and those in crowded, closed environments with limited air circulation (such as airplanes, high-speed rail, and other public transportation).

[0050] Figure 1 illustrates how the ACE2-Fc fusion protein addresses the rapid mutation rate of the novel coronavirus, the strong transmissibility of mutant strains, and the inadequate updating of vaccines against these mutations. ACE2 has been identified as the functional host receptor for severe acute respiratory syndrome coronavirus (SARS-CoV-2). A schematic diagram of how SARS-CoV-2 infects the host through the ACE2 receptor is shown in Figure 1. This is because the receptor used to enter host cells remains unchanged, regardless of how the virus mutates or how it attempts to escape vaccines and neutralizing antibodies. Furthermore, studies have shown that more infectious strains have a higher affinity for the receptor, laying the foundation for the ACE2 receptor to block any SARS-CoV-2 mutant from infecting host cells.

[0051] Figures 3A-3D further illustrate how ACE2-Fc blocks SARS-CoV-2 infection. Using Fc fusion protein technology, a soluble human ACE2-Fc fusion protein was developed and formulated into a nasal spray. This nasal spray delivers the ACE2-Fc fusion protein to the human upper respiratory tract, primarily the nasal cavity, providing the first line of defense against viral invasion, creating a biologically effective invisible mask. When airborne viruses or droplets carrying them are present, the virus's first barrier to entry is through infection of cells in the upper respiratory tract. The ACE2-Fc fusion protein in the nasal cavity and upper respiratory tract can competitively bind to any mutant SARS-CoV-2 virus, thereby blocking the site where the virus's S protein binds to host cells and preventing the virus from entering host cells and infecting the body through the S protein.

[0052] On the one hand, the present disclosure provides a fusion protein comprising a plurality of recombinant polypeptides, the fusion protein comprising a human ACE2 protein or a fragment thereof and a human IgG Fc protein or a functional variant thereof, the fusion protein specifically binding to a wild-type, variant or mutant human ACE2 receptor through one or more binding sites. It can be used for the treatment of coronavirus infection (e.g., prophylactic, therapeutic). The present disclosure also provides methods for its manufacture and use. The nasal spray formulation comprising the ACE2-Fc fusion protein showed a surprisingly wide-ranging effect in preventing coronavirus infection, namely, by applying the soluble ACE2-Fc fusion protein receptor to the entire nasal surface, efficiently and competitively preventing the virus from binding to ACE2 on the surface of nasal mucosal epithelial cells, and effectively inhibiting viral invasion. Figures 2A-2H show that the ACE2-Fc fusion protein of the present disclosure has good affinity for the S protein of different strains.

[0053] In some embodiments, the fusion protein disclosed herein is a soluble protein, which comprises a human ACE2 protein or a fragment thereof and a human IgG Fc protein or a functional variant thereof.

[0054] The fusion protein specifically binds to the wild-type, variant or mutant human ACE2 receptor through one or more binding sites.

[0055] In some embodiments, the human ACE2 protein or a fragment thereof contained in the fusion protein described in the present disclosure comprises the human ACE2 extracellular domain or a fragment thereof.

[0056] In some embodiments, the human ACE2 protein or fragment thereof contained in the fusion protein described in the present disclosure can be a wild-type human ACE2 extracellular domain or a fragment thereof, or a human ACE2 extracellular domain variant or mutant or a fragment thereof, as long as the human ACE2 extracellular domain variant or mutant or a fragment thereof retains the ability to bind to the wild-type, variant or mutant human ACE2 receptor.

[0057] In some embodiments, the human ACE2 protein or a fragment thereof contained in the fusion protein described in the present disclosure comprises the amino acid sequence of any one of SEQ ID NOs: 5, 7, 11, 12, 13 and 14, or an amino acid sequence or a fragment thereof having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0058] In some embodiments, the human ACE2 protein or a fragment thereof contained in the fusion protein described in the present disclosure comprises the amino acid sequence described in SEQ ID NO:5 or an amino acid sequence or a fragment thereof having at least about 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% sequence identity thereto.

[0059] In some embodiments, the human IgG Fc protein or a functional variant thereof contained in the fusion protein of the present disclosure comprises any one or more Fc region sequences selected from human IgG1Fc, IgG2Fc, IgG3Fc and IgG4Fc, or functional variants and fragments thereof.

[0060] In some embodiments, the human IgG Fc protein or a functional variant thereof contained in the fusion protein of the present disclosure is selected from the human IgG1 Fc region sequence or a functional variant and fragment thereof.

[0061] In some embodiments, the human IgG Fc protein or a functional variant thereof contained in the fusion protein described in the present disclosure comprises the amino acid sequence described in SEQ ID NO: 6 or 8, or an amino acid sequence or a fragment thereof having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0062] In some embodiments, the human IgG Fc protein or a functional variant thereof comprised by the fusion protein described in the present disclosure comprises the amino acid sequence described in SEQ ID NO:6 or an amino acid sequence or a fragment thereof having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0063] In some embodiments, the fusion protein optionally comprises a signal peptide, for example, the signal peptide is shown in SEQ ID NO.9.

[0064] In some embodiments, the fusion protein optionally comprises a peptide linker, wherein the human ACE2 protein or fragment thereof and the human IgG Fc protein or a functional variant thereof are directly connected or connected through a peptide linker.

[0065] In some embodiments, the fusion protein optionally includes a mutant sequence, for example, the mutant sequence is shown in SEQ ID NO.10.

[0066] In some embodiments, the fusion proteins of the present disclosure comprise the amino acid sequence of any one of SEQ ID NOs: 1-18, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a fragment thereof, or a combination thereof, such as, for example, the amino acid sequence of any one of SEQ ID NOs: 1-4 or 15-18, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a fragment thereof.

[0067] In some embodiments, the fusion protein described in the present disclosure further comprises a detection tag.

[0068] In one aspect, the present disclosure provides a pharmaceutical composition comprising the fusion protein of the present disclosure and an optional pharmaceutically acceptable carrier.

[0069] In some embodiments, the pharmaceutical compositions of the present disclosure comprise about 0.1 mg / ml to about 100 mg / ml of the fusion protein of any one of claims 1 to 15, such as about 0.50 mg / ml to about 20.00 mg / ml, such as about 1.25 mg / ml, about 2.50 mg / ml, or about 5.00 mg / ml.

[0070] In some embodiments, the pharmaceutical composition of the present disclosure comprises a pharmaceutically acceptable carrier selected from a combination of one or more of a buffer and an osmotic pressure regulator.

[0071] In some embodiments, the pharmaceutical compositions of the present disclosure comprise a buffering agent selected from sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, or a combination thereof.

[0072] In some embodiments, the osmotic pressure regulator contained in the pharmaceutical composition of the present disclosure is a combination of one or more selected from the group consisting of sodium chloride, potassium chloride, glycerol, glucose, sorbitol, sucrose, xylitol and mannitol.

[0073] In some embodiments, the pharmaceutical compositions of the present disclosure comprise an osmotic pressure regulator that is sodium chloride and / or sucrose.

[0074] In some embodiments, the pharmaceutically acceptable carrier of the present disclosure optionally includes a stabilizer, and the stabilizer includes one or more combinations selected from the group consisting of proteins, peptides or hydrolysates such as albumin, gelatin; sugars such as sucrose, lactose, sorbitol; and amino acids such as sodium glutamate, for example, the stabilizer includes sucrose.

[0075] In some embodiments, the pharmaceutically acceptable carrier of the present disclosure optionally includes a bacteriostatic agent, which includes a combination of one or more selected from the group consisting of benzoic acid, sorbic acid, thimerosal and phenylethanol, for example, the bacteriostatic agent includes phenylethanol and / or thimerosal.

[0076] In some embodiments, the pharmaceutical compositions described herein are in dosage forms suitable for administration by intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intranasal, sublingual, tonsil, oropharyngeal or other parenteral and mucosal routes, preferably in dosage forms suitable for intranasal administration, such as nasal sprays.

[0077] In one aspect, the present disclosure provides a fusion protein or pharmaceutical composition of the present disclosure for preventing or treating infection by coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects.

[0078] In some embodiments, the fusion protein or pharmaceutical composition of the present disclosure is administered intranasally for the prevention, treatment, and / or prevention of transmission of coronavirus SARS-CoV-2 and its variants in infected subjects.

[0079] In one aspect, the present disclosure provides the use of the fusion protein or pharmaceutical composition of the present disclosure in the preparation of a medicament for preventing or treating infection by the coronavirus SARS-CoV-2 and its variants and / or preventing the spread of the coronavirus SARS-CoV-2 and its variants in infected subjects.

[0080] In one aspect, the present disclosure provides a method for preventing, treating infection by coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in an infected subject, comprising administering a therapeutically effective amount of a fusion protein or pharmaceutical composition of the present disclosure to the subject.

[0081] In some embodiments, the administration is intranasal.

[0082] In one aspect, the present disclosure provides a kit for preventing or treating infection by coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects, comprising a fusion protein or pharmaceutical composition of the present disclosure;

[0083] Container; and

[0084] Optional package insert or label indicating prophylaxis and / or treatment.

[0085] In the present disclosure, the coronavirus SARS-CoV-2 and its variants include but are not limited to SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Miu, Omicron, JN.1; and unknown variants of the coronavirus SARS-CoV-2.

[0086] In one aspect, the present disclosure provides a nucleic acid encoding a fusion protein or a fragment thereof of the present disclosure, a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or the vector.

[0087] In some embodiments, the vector is phFC(IM).

[0088] In some embodiments, the host cell is a CHO cell, such as GH-CHO.

[0089] In one aspect, the present disclosure discloses a method for producing the fusion protein of the present disclosure, the method comprising:

[0090] (a) cultivating a recombinant cell in suspension culture, wherein the recombinant cell contains a vector comprising a nucleic acid molecule encoding a fusion protein of the present disclosure,

[0091] (b) isolating the fusion protein from the suspension culture.

[0092] In some embodiments, the present disclosure discloses a method for producing the fusion protein of the present disclosure, the method comprising: amplifying the cell quantity in one or more stages, and then

[0093] (a) cultivating recombinant cells in suspension culture, wherein the recombinant cells contain a vector expressing a nucleic acid molecule encoding a fusion protein of the present disclosure,

[0094] (b) isolating the fusion protein from the suspension culture.

[0095] In some embodiments, the present disclosure discloses a method for producing the fusion protein of the present disclosure, the method further comprising:

[0096] (1) Rapidly capture samples or enrich the fusion protein,

[0097] (2) purifying the fusion protein in one or more stages, and

[0098] (3) Concentrating and replacing the liquid to obtain the fusion protein stock solution.

[0099] Similar to other enveloped RNA viruses (such as HIV, RSV and influenza), coronaviruses including SARS-CoV-2 have trimeric surface antigens on their viral envelopes so that they can enter different host cells through specific cell surface receptors during infection. Like SARS-CoV-1, SARS-CoV-2 uses its trimeric viral surface antigen S protein to bind to its specific cell surface receptor ACE2 to enter host cells in the respiratory system of mammals. The fusion protein provided by the present disclosure can bind to the human ACE2 receptor, thereby preventing the virus from binding to the ACE2 receptor, thereby preventing and / or eliminating viral infection. The composition containing the ACE2-Fc fusion protein provided by the present disclosure, such as a nasal spray, can widely and effectively inhibit the infection of viruses that enter the human body through the human ACE2 receptor. In some aspects, the composition containing the ACE2-Fc fusion protein can widely and effectively inhibit the invasion of SARS-CoV-2 virus variants and mutants.

[0100] In some embodiments, the present disclosure discloses a method for treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a fusion protein comprising a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0101] The present disclosure also provides pharmaceutical compositions comprising the fusion proteins provided herein, methods for producing the fusion proteins provided herein, methods for treating subjects with the fusion proteins and / or pharmaceutical compositions provided herein, and related kits.

[0102] All publications, including patent documents, scientific articles, and databases, mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were incorporated by reference individually. If definitions set forth in this disclosure contradict or are inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated by reference into this disclosure, the definitions set forth in this disclosure take precedence over the definitions incorporated by reference into this disclosure. Section headings used in this disclosure are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0103] I. ACE2-Fc fusion protein against coronavirus infection

[0104] The present disclosure discloses ACE2-Fc fusion proteins and compositions containing the same for preventing coronavirus infection. In some embodiments, the composition containing the ACE2-Fc fusion protein can be a nasal spray that can cover the nasal cavity and upper respiratory tract. The ACE2-Fc fusion protein therein can competitively bind to SARS-CoV-2 and any mutant strains of SARS-CoV-2, thereby blocking the site where the virus's S protein binds to host cells, blocking the virus from entering host cells through the S protein and infecting the human body. Based on the mechanism by which SARS-CoV-2 enters host cells and causes infection, those skilled in the art will fully understand that the ACE2-Fc fusion protein can competitively bind to SARS-CoV-2 and any mutant strains of SARS-CoV-2. Any mutant strain mentioned includes both known SARS-CoV-2 mutants and unknown SARS-CoV-2 mutants, wherein the unknown SARS-CoV-2 mutants can be SARS-CoV-2 mutants that already exist but have not yet been discovered and identified by humans, or new SARS-CoV-2 mutants that do not yet exist but will arise in the future due to viral mutations.

[0105] Coronaviruses are a family of positive-sense, single-stranded RNA viruses known to cause severe respiratory illness. They possess the largest genomes of any known RNA virus (26-32 kb) and are phylogenetically divided into four genera (α, β, γ, and δ), with the β-coronaviruses further divided into four lineages (A, B, C, and D). Currently, viruses from the coronavirus family known to infect humans are from the α-coronavirus and β-coronavirus genera. Furthermore, it is believed that the γ-coronavirus and δ-coronavirus genera may infect humans in the future. Non-limiting examples of β-coronaviruses include Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Human Coronavirus HKU1 (HKU1-CoV), Human Coronavirus OC43 (OC43-CoV), Murine Hepatitis Virus (MHV-CoV), Bat SARS-like Coronavirus WIV1 (WIV1-CoV), and Human Coronavirus HKU9 (HKU9-CoV). Non-limiting examples of alpha coronaviruses include human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis coronavirus (TGEV). A non-limiting example of a delta coronavirus is porcine delta coronavirus (SDCV).

[0106] This disclosure discloses a list of severe acute respiratory syndrome-related coronaviruses:

[0107] ■ Bat coronavirus Cp / Yunnan2011

[0108] Bat coronavirus RaTG13

[0109] Bat coronavirus Rp / Shaanxi2011

[0110] ○ Bat SARS coronavirus HKU3

[0111] Bat SARS coronavirus HKU3-1

[0112] Bat SARS coronavirus HKU3-10

[0113] Bat SARS coronavirus HKU3-11

[0114] Bat SARS coronavirus HKU3-12

[0115] Bat SARS coronavirus HKU3-13

[0116] Bat SARS coronavirus HKU3-2

[0117] Bat SARS coronavirus HKU3-3

[0118] Bat SARS coronavirus HKU3-4

[0119] Bat SARS coronavirus HKU3-5

[0120] Bat SARS coronavirus HKU3-6

[0121] Bat SARS coronavirus HKU3-7

[0122] Bat SARS coronavirus HKU3-8

[0123] Bat SARS coronavirus HKU3-9

[0124] Bat SARS coronavirus Rp1

[0125] Bat SARS coronavirus Rp2

[0126] Bat SARS CoV Rf1 / 2004

[0127] Bat CoV 273 / 2005

[0128] Bat SARS CoV Rm1 / 2004

[0129] Bat CoV 279 / 2005

[0130] Bat SARS CoV Rp3 / 2004

[0131] Bat SARS-like coronavirus

[0132] Bat SARS-like coronavirus Rs3367

[0133] Bat SARS-like coronavirus RsSHC014

[0134] Bat SARS-like coronavirus WIV1

[0135] Bat SARS-like coronavirus YNLF_31C

[0136] Bat SARS-like coronavirus YNLF_34C

[0137] BtRf-BetaCoV / HeB2013

[0138] BtRf-BetaCoV / JL2012

[0139] BtRf-BetaCoV / SX2013

[0140] BtRs-BetaCoV / GX2013

[0141] BtRs-BetaCoV / HuB2013

[0142] BtRs-BetaCoV / YN2013

[0143] Civet SARS CoV 007 / 2004

[0144] ■Civet SARS CoV SZ16 / 2003

[0145] Civet SARS CoV SZ3 / 2003

[0146] ○Recombinant SARSr-CoV

[0147] SARS coronavirus ExoN1

[0148] SARS coronavirus MA15

[0149] SARS coronavirus MA15ExoN1

[0150] ■SARS coronavirus wtic-MB

[0151] ■ Chinese horseshoe bat coronavirus

[0152] SARS bat coronavirus

[0153] SARS coronavirus A001

[0154] SARS coronavirus A013

[0155] SARS coronavirus A021

[0156] SARS coronavirus A022

[0157] SARS coronavirus A030

[0158] SARS coronavirus A031

[0159] SARS coronavirus AS

[0160] SARS coronavirus B012

[0161] SARS coronavirus B024

[0162] SARS coronavirus B029

[0163] SARS coronavirus B033

[0164] SARS coronavirus B039

[0165] SARS coronavirus B040

[0166] SARS coronavirus BJ01

[0167] SARS coronavirus BJ02

[0168] SARS coronavirus BJ03

[0169] SARS coronavirus BJ04

[0170] SARS coronavirus BJ162

[0171] SARS coronavirus BJ182-12

[0172] SARS coronavirus BJ182-4

[0173] SARS coronavirus BJ182-8

[0174] SARS coronavirus BJ182a

[0175] SARS coronavirus BJ182b

[0176] SARS coronavirus BJ202

[0177] SARS coronavirus BJ2232

[0178] SARS coronavirus BJ302

[0179] SARS coronavirus C013

[0180] SARS coronavirus C014

[0181] SARS coronavirus C017

[0182] SARS coronavirus C018

[0183] SARS coronavirus C019

[0184] SARS coronavirus C025

[0185] SARS coronavirus C028

[0186] SARS coronavirus C029

[0187] SARS coronavirus CDC#200301157

[0188] ■SARS coronavirus civet010

[0189] SARS coronavirus civet014

[0190] SARS coronavirus civet019

[0191] ■SARS coronavirus civet020

[0192] SARS coronavirus CS21

[0193] SARS coronavirus CS24

[0194] SARS coronavirus CUHK-AG01

[0195] SARS coronavirus CUHK-AG02

[0196] SARS coronavirus CUHK-AG03

[0197] SARS coronavirus CUHK-L2

[0198] SARS coronavirus CUHK-Su10

[0199] SARS coronavirus CUHK-W1

[0200] SARS coronavirus cw037

[0201] SARS coronavirus cw049

[0202] SARS coronavirus ES191

[0203] SARS coronavirus ES260

[0204] SARS coronavirus FRA

[0205] SARS coronavirus Frankfurt 1

[0206] SARS coronavirus Frankfurt1-v01

[0207] SARS coronavirus GD01

[0208] SARS coronavirus GD03T0013

[0209] SARS coronavirus GD322

[0210] SARS coronavirus GD69

[0211] SARS coronavirus GDH-BJH01

[0212] SARS coronavirus GZ-A

[0213] SARS coronavirus GZ-B

[0214] SARS coronavirus GZ-C

[0215] SARS coronavirus GZ-D

[0216] SARS coronavirus GZ02

[0217] SARS coronavirus GZ0401

[0218] SARS coronavirus GZ0402

[0219] SARS coronavirus GZ0403

[0220] SARS coronavirus GZ43

[0221] SARS coronavirus GZ50

[0222] SARS coronavirus GZ60

[0223] SARS coronavirus HB

[0224] SARS coronavirus HC / SZ / 61 / 03

[0225] SARS coronavirus HGZ8L1-A

[0226] SARS coronavirus HGZ8L1-B

[0227] SARS coronavirus HGZ8L2

[0228] SARS coronavirus HHS-2004

[0229] SARS coronavirus HKU-36871

[0230] SARS coronavirus HKU-39849

[0231] SARS coronavirus HKU-65806

[0232] SARS coronavirus HKU-66078

[0233] ■SARS coronavirus Hong Kong / 03 / 2003

[0234] SARS coronavirus HPZ-2003

[0235] SARS coronavirus HSR 1

[0236] SARS coronavirus HSZ-A

[0237] SARS coronavirus HSZ-Bb

[0238] SARS coronavirus HSZ-Bc

[0239] SARS coronavirus HSZ-Cb

[0240] SARS coronavirus HSZ-Cc

[0241] SARS coronavirus HSZ2-A

[0242] SARS coronavirus HZS2-Bb

[0243] SARS coronavirus HZS2-C

[0244] SARS coronavirus HZS2-D

[0245] SARS coronavirus HZS2-E

[0246] SARS coronavirus HZS2-Fb

[0247] SARS coronavirus HZS2-Fc

[0248] ■SARS coronavirus JMD

[0249] SARS coronavirus LC1

[0250] SARS coronavirus LC2

[0251] SARS coronavirus LC3

[0252] SARS coronavirus LC4

[0253] SARS coronavirus LC5

[0254] SARS coronavirus LLJ-2004

[0255] SARS coronavirus NS-1

[0256] SARS coronavirus P2

[0257] SARS coronavirus PC4-115

[0258] SARS coronavirus PC4-127

[0259] SARS coronavirus PC4-13

[0260] SARS coronavirus PC4-136

[0261] SARS coronavirus PC4-137

[0262] SARS coronavirus PC4-145

[0263] SARS coronavirus PC4-199

[0264] SARS coronavirus PC4-205

[0265] SARS coronavirus PC4-227

[0266] SARS coronavirus PC4-241

[0267] SARS coronavirus PUMC01

[0268] SARS coronavirus PUMC02

[0269] SARS coronavirus PUMC03

[0270] SARS coronavirus Rs_672 / 2006

[0271] SARS coronavirus sf098

[0272] SARS coronavirus sf099

[0273] SARS coronavirus ShanghaiQXC1

[0274] SARS coronavirus ShanghaiQXC2

[0275] ■SARS coronavirus Shanhgai LY

[0276] SARS coronavirus Sin0409

[0277] SARS coronavirus Sin2500

[0278] SARS coronavirus Sin2677

[0279] SARS coronavirus Sin2679

[0280] SARS coronavirus Sin2748

[0281] SARS coronavirus Sin2774

[0282] SARS coronavirus Sin3408

[0283] SARS coronavirus Sin3408L

[0284] SARS coronavirus Sin3725V

[0285] SARS coronavirus Sin3765V

[0286] SARS coronavirus Sin842

[0287] SARS coronavirus Sin845

[0288] SARS coronavirus Sin846

[0289] SARS coronavirus Sin847

[0290] SARS coronavirus Sin848

[0291] SARS coronavirus Sin849

[0292] SARS coronavirus Sin850

[0293] SARS coronavirus Sin852

[0294] ■SARS coronavirus Sin_WNV

[0295] SARS coronavirus Sino1-11

[0296] SARS coronavirus Sino3-11

[0297] SARS coronavirus SinP1

[0298] SARS coronavirus SinP2

[0299] SARS coronavirus SinP3

[0300] SARS coronavirus SinP4

[0301] SARS coronavirus SinP5

[0302] SARS coronavirus SoD

[0303] SARS coronavirus SZ1

[0304] SARS coronavirus SZ13

[0305] SARS coronavirus, Taiwan

[0306] SARS coronavirus Taiwan JC-2003

[0307] SARS coronavirus Taiwan TC1

[0308] SARS coronavirus Taiwan TC2

[0309] SARS coronavirus Taiwan TC3

[0310] SARS coronavirus TJ01

[0311] ■SARS coronavirus TJF

[0312] SARS coronavirus Tor2

[0313] SARS coronavirus TW

[0314] SARS coronavirus TW-GD1

[0315] SARS coronavirus TW-GD2

[0316] SARS coronavirus TW-GD3

[0317] SARS coronavirus TW-GD4

[0318] SARS coronavirus TW-GD5

[0319] SARS coronavirus TW-HP1

[0320] SARS coronavirus TW-HP2

[0321] SARS coronavirus TW-HP3

[0322] SARS coronavirus TW-HP4

[0323] SARS coronavirus TW-JC2

[0324] SARS coronavirus TW-KC1

[0325] SARS coronavirus TW-KC3

[0326] SARS coronavirus TW-PH1

[0327] SARS coronavirus TW-PH2

[0328] SARS coronavirus TW-YM1

[0329] SARS coronavirus TW-YM2

[0330] SARS coronavirus TW-YM3

[0331] SARS coronavirus TW-YM4

[0332] SARS coronavirus TW1

[0333] SARS coronavirus TW10

[0334] SARS coronavirus TW11

[0335] SARS coronavirus TW2

[0336] SARS coronavirus TW3

[0337] SARS coronavirus TW4

[0338] SARS coronavirus TW5

[0339] SARS coronavirus TW6

[0340] SARS coronavirus TW7

[0341] SARS coronavirus TW8

[0342] SARS coronavirus TW9

[0343] ■SARS coronavirus TWC

[0344] SARS coronavirus TWC2

[0345] SARS coronavirus TWC3

[0346] SARS coronavirus TWH

[0347] ■SARS coronavirus TWJ

[0348] ■SARS coronavirus TWK

[0349] SARS coronavirus TWS

[0350] ■SARS coronavirus TWY

[0351] ■SARS coronavirus Urbani

[0352] SARS coronavirus Vietnam

[0353] SARS coronavirus WF188

[0354] SARS coronavirus WH20

[0355] SARS coronavirus WHU

[0356] SARS coronavirus xw002

[0357] SARS coronavirus ZJ01

[0358] SARS coronavirus ZJ02

[0359] SARS coronavirus ZJ0301

[0360] SARS coronavirus ZMY 1

[0361] SARS coronavirus ZS-A

[0362] SARS coronavirus ZS-B

[0363] SARS coronavirus ZS-C

[0364] SARS-related bat coronavirus RsSHC014

[0365] ■SARS-related beta-coronavirus Rp3 / 2004

[0366] Severe acute respiratory syndrome coronavirus 2

[0367] The table below shows exemplary SARS-CoV-2 strains.

[0368] The coronavirus genome is capped, polyadenylated, and covered with a nucleocapsid protein. The coronavirus particle comprises a viral envelope containing a type I fusion glycoprotein called the spike (S) protein. Most coronaviruses share a common genomic structure, with the replicase gene contained in the 5' portion of the genome and the structural genes in the 3' portion of the genome.

[0369] The coronavirus spike (S) protein is a class I fusion glycoprotein initially synthesized as a precursor protein. The single precursor S polypeptide forms a homotrimer and is glycosylated and processed within the Golgi apparatus to remove the signal peptide. It is then cleaved by cellular proteases to produce the separate S1 and S2 polypeptide chains, which remain associated within the homotrimer as the S1 / S2 protomer, thus forming a heterodimer. The S1 subunit, located distal to the viral membrane, contains the receptor-binding domain (RBD), which mediates attachment of the virus to its host receptor. The S2 subunit comprises the fusion protein machinery, including the fusion peptide, two heptad repeats (HR1 and HR2) characteristic of fusion glycoproteins, and the central helix, transmembrane domain, and cytosolic tail domain.

[0370] ACE2 serves as the receptor for SARS-CoV-2. Its extracellular domain connects to the spike protein on the SARS-CoV-2 envelope. At this time, the transmembrane serine protease (TMPRSS2) on the cell membrane surface cuts the S protein into two subunits, S1 and S2, and exposes the fusion peptide, achieving the purpose of fusing with the cell membrane and releasing genetic material for replication.

[0371] The viral antigen or immunogen includes a protease cleavage site, where the protease can optionally be furin, trypsin, factor Xa, or cathepsin L. During viral assembly, furin protease can cleave the S protein into different subunits (S1 and S2), which are then released to infect other cells. SARS-CoV lacks the furin protease recognition site, so perhaps due to the different S protein cleavage and assembly mechanisms, SARS-CoV-2 has a stronger affinity for membrane fusion and a higher efficiency of cell entry. Studies have found that SARS-CoV-2's binding ability to ACE2 is 10-20 times that of SARS, resulting in the high infectivity of SARS-CoV-2.

[0372] In some embodiments, the ACE2-Fc fusion protein comprises an ACE2 protein or polypeptide. In some embodiments, the ACE2 protein is a primate ACE2 protein, such as a human ACE2 protein or a fragment thereof. In some embodiments, the human ACE2 protein or fragment thereof is the full-length extracellular region of human ACE2 or a fragment thereof. In some embodiments, the non-dimerizing Fc domain is an IgG, IgM, IgD, IgA, or IgE Fc region, or a variant, mutant, or fragment thereof. In some embodiments, the non-dimerizing Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc region, or a variant, mutant, or fragment thereof. In some embodiments, the Fc protein is a primate Fc protein, such as a human IgG Fc protein or a functional variant thereof. In some embodiments, the human IgG Fc or fragment thereof is the full-length extracellular region of human IgG Fc or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein comprises a human ACE2 protein or a fragment thereof and a human IgG Fc protein or a functional variant thereof. In some embodiments, the ACE2-Fc fusion protein comprises the full-length protein / polypeptide of the extracellular region of human ACE2 or its fragment protein / polypeptide and human IgG Fc or its fragment protein / polypeptide.

[0373] In some embodiments, the ACE2-Fc fusion protein comprises the sequence shown in SEQ ID NO: 1 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein comprises an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 as shown below, or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein comprises the amino acid fragment RS, linking the ACE2 and Fc sequences.

[0374] In some embodiments, the ACE2-Fc fusion protein comprises the sequence shown in SEQ ID NO: 2 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein comprises an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2 as shown below, or a fragment thereof.

[0375] In some embodiments, the ACE2-Fc fusion protein comprises the sequence shown in SEQ ID NO: 3 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein comprises an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3 as shown below, or a fragment thereof.

[0376] In some embodiments, the ACE2-Fc fusion protein comprises the sequence shown in SEQ ID NO: 4 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein comprises an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4 as shown below, or a fragment thereof.

[0377] In some embodiments, the ACE2-Fc fusion protein comprises the sequence shown in SEQ ID NO: 15 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein comprises an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15 as shown below, or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein comprises the amino acid fragment VS, linking the ACE2 and Fc sequences.

[0378] In some embodiments, the ACE2-Fc fusion protein includes the sequence shown in SEQ ID NO: 16 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16 as shown below, or a fragment thereof.

[0379] In some embodiments, the ACE2-Fc fusion protein includes the sequence shown in SEQ ID NO: 17 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence or a fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17 as shown below.

[0380] In some embodiments, the ACE2-Fc fusion protein comprises the sequence shown in SEQ ID NO: 18 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein comprises an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 18 as shown below, or a fragment thereof.

[0381] In some embodiments, the ACE2-Fc fusion protein includes the ACE2 extracellular domain sequence shown in SEQ ID NO: 5 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence or a fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5 as shown below.

[0382] In some embodiments, the ACE2-Fc fusion protein includes the ACE2 extracellular domain sequence shown in SEQ ID NO: 11 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence or a fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11 as shown below.

[0383] In some embodiments, the ACE2-Fc fusion protein includes the ACE2 extracellular domain sequence shown in SEQ ID NO: 12 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12 as shown below, or a fragment thereof.

[0384] In some embodiments, the ACE2-Fc fusion protein includes the ACE2 extracellular domain sequence shown in SEQ ID NO: 13 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13 as shown below, or a fragment thereof.

[0385] In some embodiments, the ACE2-Fc fusion protein includes the ACE2 extracellular domain sequence shown in SEQ ID NO: 14 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14 as shown below, or a fragment thereof.

[0386] In some embodiments, the ACE2-Fc fusion protein includes the ACE2 extracellular domain sequence shown in SEQ ID NO: 7 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence or a fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7 as shown below.

[0387] In some embodiments, the ACE2-Fc fusion protein includes the Fc sequence shown in SEQ ID NO: 6 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6 as shown below, or a fragment thereof.

[0388] In some embodiments, the ACE2-Fc fusion protein includes an Fc tag sequence as shown in SEQ ID NO: 8, or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8, as shown below, or a fragment thereof.

[0389] In some embodiments, the fusion protein of the present disclosure further comprises a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:9, or a fragment thereof. In some embodiments, the signal peptide comprises an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9, or a fragment thereof.

[0390] In some embodiments, the fusion protein of the present disclosure further comprises a mutant sequence for C-terminal modification to facilitate tracking and detection. The mutant sequence included in the ACE2-Fc fusion protein of the present disclosure includes the sequence shown in SEQ ID NO: 10 or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein comprises an amino acid sequence or a fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10.

[0391] In some embodiments, the fusion proteins of the present disclosure comprise a non-dimerizing Fc domain comprising a sequence having one or more cysteine ​​substitutions compared to human IgG1 Fc, optionally wherein the cysteine ​​substitutions are selected from C5S, C11S, and C14S, wherein the position of the substitution is relative to SEQ ID NO: 6, and optionally wherein the cysteine ​​is substituted with serine. In some embodiments, the fusion proteins of the present disclosure comprise a non-dimerizing Fc domain comprising a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0392] In some embodiments, the ACE2 protein peptide is produced by a codon-optimized nucleic acid sequence. In some embodiments, the ACE2 protein peptide is produced by a non-codon-optimized nucleic acid sequence.

[0393] It is contemplated that the ACE2 provided herein can form a fusion protein with an Fc protein, or can be combined with other proteins or peptides, such as by ligation, to form further recombinant polypeptides, including fusion peptides. In some embodiments, the ACE2-Fc fusion protein comprises ACE2 and an Fc protein or polypeptide. In some embodiments, the ACE2 is a primate ACE2, such as a human ACE2 protein or a fragment thereof. In some embodiments, the human ACE2 protein or fragment thereof is the full-length extracellular region of human ACE2 or a fragment thereof. In some embodiments, the Fc is a primate Fc, such as a human IgG Fc protein or a functional variant thereof. In some embodiments, the human IgG Fc or fragment thereof is the full-length extracellular region of human IgG Fc or a fragment thereof. In some embodiments, the ACE2-Fc fusion protein comprises the human ACE2 protein or fragment thereof and a human IgG Fc protein or a functional variant thereof. In some embodiments, the ACE2-Fc fusion protein comprises the full-length extracellular region of human ACE2 protein / polypeptide or a fragment thereof and a human IgG Fc protein or a functional variant thereof. In some embodiments, the ACE2 protein / polypeptide is directly linked to the Fc protein / polypeptide. In some embodiments, the ACE2 and Fc protein / polypeptide are linked via one or more amino acids.

[0394] In some embodiments, one or more peptide linkers (e.g., glycine-serine linkers, e.g., arginine-serine peptide linkers) can be used to connect ACE2 to Fc. In some embodiments, the linker comprises a non-dimerizing Fc domain or a fragment thereof that is capable of binding to an Fc receptor such as FcRn, thereby increasing the half-life of the fusion protein and its complex (e.g., human serum half-life). In some embodiments, as long as the recombinant ACE2-Fc maintains the desired properties (e.g., pre-fusion conformation), it can be a dimer or multimer, e.g., a trimer, a tetramer, a pentamer, a hexamer, and may include any stabilizing mutation (or combination thereof) provided herein. In some embodiments, the non-dimerizing Fc domain or its fragment in the trimeric complex exists as a monomer - that is, the Fc domain or its fragment does not form an interchain covalent bond such as a disulfide bond, and does not form an oligomer because of direct non-covalent Fc interactions between the domains. In some embodiments, the recombinant polypeptide or fusion protein comprises a first sequence described in any one of SEQ ID NOs: 5, 7, 11, 12, 13, or 14, which is linked to a second sequence described in any one of SEQ ID NOs: 6 and 8. In some embodiments, the recombinant polypeptide or fusion protein comprises a first sequence described in any one of SEQ ID NOs: 11 or 13, which is linked to a second sequence described in any one of SEQ ID NOs: 6 and 8. In some embodiments, the first and second sequences are connected by a linker, which is one or more amino acids, such as two amino acids: -RS- or -VS-. In some embodiments, the linker comprises a sequence comprising a glycine-XY repeat sequence.

[0395] Papain digestion of antibodies produces two identical antigen-binding fragments, called Fab fragments, and a residual Fc fragment (a name reflecting the ability to crystallize readily). The Fab fragment consists of an intact light chain, the variable region of the heavy chain, and the first constant domain (CH1) of a heavy chain. Each Fab fragment is monovalent with respect to antigen binding, meaning it has one antigen-binding site. The Fc fragment contains the carboxyl-terminal portions of the two heavy chains linked together by disulfide bonds. The effector functions of an antibody are determined by the sequence of the Fc region, which is also recognized by Fc receptors (FcRs) on certain cell types.

[0396] The Fc domain of the Fc fusion protein can bind to the Fc receptor on the surface of immune cells and exert a variety of biological functions, such as mediating the passage through the placenta and mucosal barriers, inflammatory response, antibody-dependent cell-mediated phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), promoting dendritic cell (DC) maturation, regulating cytokine secretion, regulating B cell proliferation and differentiation, etc.

[0397] Table 1: Major Fc receptors and their corresponding biological functions

[0398] The ability of antibodies to interact with neonatal Fc receptors (FcRn) in a pH-dependent manner through the Fc (fragment, crystallizable) region can confer them with extended serum half-lives. In some embodiments, the present disclosure provides engineered Fc fusion molecules, wherein the Fc domain can prolong the half-life of therapeutic proteins or peptides in serum. Due to the homodimeric nature of antibody Fc, naturally occurring IgG antibodies and engineered Fc fusion molecules comprising naturally occurring IgG sequences are typically bivalent and monospecific. For certain therapeutic applications disclosed herein, engineered Fc polypeptides and fusion polypeptides thereof can retain certain positive attributes conferred by monomeric, non-dimerized Fc while achieving flexibility and specificity.

[0399] The most abundant immunoglobulin class in human serum is IgG. The IgG structure consists of two light chains and two heavy chains, each light chain comprising two domains and each heavy chain comprising four domains. The antigen-binding site is located in the Fab region (fragment antigen-binding), which contains the variable light (VL) and variable heavy (VH) chain domains, as well as the constant light (CL) and constant heavy (CH1) chain domains. The Fc region contains the CH2 and CH3 domains of the heavy chains, with the two heavy chains linked together by disulfide bonds (-SS-) at the hinge region.

[0400] The FcRn (neonatal Fc receptor) binding site of IgG is located in the Fc region of the antibody, so the serum half-life characteristics of the antibody extension are retained in the Fc fragment. A separate Fc fragment can be considered to be a homodimer of the heavy chain comprising CH2 and CH3 domains. In some embodiments, the monomeric non-dimerizing Fc domain of the present disclosure comprises one or more mutations in the heavy chain Fc region to stabilize the Fc domain or its fusion polypeptide in aqueous solution / serum. In some embodiments, compared with the corresponding wild-type Fc domain, the monomeric non-dimerizing Fc domain comprises three or more mutations in the Fc amino acid sequence. In some embodiments, compared with the corresponding wild-type Fc domain, the monomeric non-dimerizing Fc domain comprises two mutations in the Fc amino acid sequence.

[0401] In some embodiments, the Fc domains of the present disclosure or its fusion polypeptide include an immunoglobulin portion (e.g., hinge, CH2, and / or CH3 domain), wherein any or all of the cysteine ​​residues are located in the hinge domain of the immunoglobulin portion and are mutated to eliminate the disulfide bond formation involving one or more or all of these cysteine ​​residues. For example, according to the EU index in Kabat, any one or more cysteines at positions 220, 226, and 229 in human IgG1Fc domains (corresponding to the cysteine ​​at positions 233, 239, and 242, respectively, according to Kabat numbering) can be replaced by another amino acid residue such as serine. In some embodiments, the Fc domains of the present disclosure or its fusion polypeptide include the cysteine ​​at position 220, which is replaced by, for example, serine. In some embodiments, the Fc domains of the present disclosure or its fusion polypeptide include the cysteine ​​at position 226, which is replaced by, for example, serine. In some embodiments, the Fc domains of the present disclosure or its fusion polypeptide include the cysteine ​​at position 229, which is replaced by, for example, serine. In some embodiments, the Fc domain or fusion polypeptide thereof of the present disclosure further comprises one or more other amino acid substitutions, insertions and / or deletions. In some embodiments, the Fc domain or fusion polypeptide thereof of the present disclosure further comprises a proline at position 238 (corresponding to a proline at position 251 according to Kabat numbering) that is substituted with, for example, serine.

[0402] In some embodiments, the Fc domain or fusion polypeptide disclosed herein exhibits increased affinity for FcRn within a first pH range compared to a corresponding human IgG1 Fc domain or fusion polypeptide thereof. In some embodiments, the Fc domain or fusion polypeptide disclosed herein exhibits rapid dissociation of the corresponding human IgG1 Fc domain or fusion polypeptide thereof from FcRn within a second pH range. In some embodiments, the first pH range is lower than the second pH range. In some embodiments, the first pH range is lower than about 7.0 and the second pH range is higher than about 7.0. In some embodiments, the first pH range is between about 5.5 and about 6.5 and the second pH range is between about 7.0 and about 8.0. In some embodiments, the first pH range is about 6.0 and the second pH range is about 7.4.

[0403] In some embodiments, the Fc domain of the present disclosure or its fusion polypeptide comprises at positions 217, 228, 243, 262, 273, 274, 262, 273, 274, 288, 290, 298, 305, 309, 310, 321, 326, 344, 353, 356, 363, 364, 368, 375, 388, 389, 390, 397, 398, 399, 401, 405, 407, 409, a human IgG1 Fc domain according to the EU index as in Kabat. 410, 413, 424, 438, and 442 in the domain correspond to positions 227, 241, 256, 275, 286, 287, 305, 307, 317, 324, 328, 329, 340, 345, 365, 374, 377, 386, 387, 391, 398, 416, 417, 418, 425, 426, 427, 430, 436, 438, 440, 441, 444, 455, 469, and 473, respectively, according to Kabat numbering. In some embodiments, the Fc domain or fusion polypeptide thereof of the present disclosure comprises one or more amino acid substitutions selected from the group consisting of P217R, P228K, F243V, V262I, V273L, K274V, K288V, K288D, K288I, K288F, K290L, S298N, V305I, L309E, H310S, C321V, K326G, R344T, P353K, P35 3D, D356P, V363N, S364N, L368W, L368G, S375Y, E388G, N389V, N390L, V397L, L398W, D399K, D401 G, F405E, F405K, F405Q, F405R, F405V, Y407S, K409N, K409D, L410N, D413R, S424G, Q438S and S442K.

[0404] In some embodiments, the Fc domains or fusion polypeptides thereof of the present disclosure comprise one or more amino acid substitutions selected from the group consisting of N421H and N421H according to the EU index in Kabat, and positions 221, 234, 297, 306, 312, 315, 306, 312, 315, 325, 343, 356, 401, 406, and 421 in a human IgG1 Fc domain correspond to positions 234, 247, 314, 325, 331, 334, 344, 364, 377, 430, 437, and 452, respectively, according to Kabat numbering. In some embodiments, the Fc domains or fusion polypeptides thereof of the present disclosure comprise one or more amino acid substitutions selected from the group consisting of N421H and N421H.

[0405] In some embodiments, the Fc domains of the present disclosure or fusion polypeptides thereof are at 221, 224, 270, 271, 273, 290, 271, 273, 290, 294, 305, 315, 319, 332, 343, 349, 357, 364, 368, 391, 405, 409, 424, 426, 435, 437, 438, 441 and 447, according to the index in EU Kabat, corresponding to positions 234, 237, 283, 284, 286, 307, 311, 324, 334, 338, 341, 364, 370, 378, 387, 391, 419, 436, 440, 455, 457, 466, 468, 469, 472 and 478, respectively, according to the Kabat numbering. In some embodiments, the Fc domain or fusion polypeptide thereof of the present disclosure comprises one or more amino acid substitutions selected from the group consisting of D221A, H224Y, D270Y, P271S, V273L, K290L, E294D, V305F, N315D, Y319S, I332M, P343W, Y349E, E357V, S364N, L368G, Y391R, F405E, K409V, S424G, S426G, H435P, T437G, Q438S, Q438P, Q438K, L441T, and K447F.

[0406] In some embodiments, the Fc domain or fusion polypeptide thereof of the present disclosure comprises one or more amino acid substitutions, insertions and / or deletions at any one or more of positions 252, 254, 256, 428 and 434 of a human IgG1 Fc domain according to the EU index in Kabat, which correspond to positions 265, 267, 269, 459 and 465, respectively, according to Kabat numbering. In some embodiments, the Fc domain or fusion polypeptide thereof of the present disclosure comprises one or more amino acid substitutions selected from the group consisting of M252Y, S254T, T256E, M428L and N434S.

[0407] In some embodiments, the fusion proteins described herein may include an Fc domain monomer or a fragment of an Fc domain of an immunoglobulin to increase the serum half-life of the polypeptide. In some embodiments, the fusion proteins described herein cannot form dimers (e.g., homodimers or heterodimers) through the interaction between any two Fc domain monomers in the non-covalent complex disclosed herein; instead, the fusion proteins described herein can trimerize (this trimerization is independent of the Fc domain therein) to form a trimeric complex comprising three Fc domain monomers, which may have the same sequence or different sequences.

[0408] In some embodiments, the Fc domain can be mutated to lack effector function, typically a "dead" Fc domain that does not bind to an Fc receptor, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and / or FcγRIV. For example, the Fc domain can include specific amino acid substitutions known to minimize the interaction between the Fc domain and the Fcγ receptor. In some embodiments, the Fc domain is from an IgG1 antibody and includes the amino acid substitutions L234A, L235A, and G237A. In some embodiments, the Fc domain is from an IgG1 antibody and includes the amino acid substitutions D265A, K322A, and N434A. The above amino acid positions are defined according to Kabat (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The Kabat numbering of amino acid residues of a given antibody can be determined by aligning homologous regions of the antibody sequence with a "standard" Kabat numbering sequence. Furthermore, in some embodiments, the Fc domain does not induce any immune system-related response. For example, the Fc domain in a polypeptide dimer can be modified to reduce interaction or binding between the Fc domain and Fcγ receptors.

[0409] In some cases, the fusion protein described in the present disclosure comprises an Fc region of a human immunoglobulin or a fragment or variant thereof. In some embodiments, the Fc domain of the fusion protein is an Fc region of human IgG1 or a fragment or variant thereof. In some embodiments, the Fc domain of the fusion protein has an N-terminal truncation compared to a native human immunoglobulin Fc region. In some embodiments, the Fc domain of the fusion protein does not comprise the CH1 domain of a native human immunoglobulin Fc region.

[0410] In some cases, the Fc domain of the fusion protein comprises one or more amino acid substitutions, deletions, or insertions compared to the native human IgG1 sequence. In some embodiments, the Fc domain of the fusion protein comprises substitutions at one or more amino acid positions in the CH2 domain. In some embodiments, the Fc domain of the fusion protein comprises substitutions at one or more amino acid positions at the DE turn. In some embodiments, the Fc domain of the fusion protein comprises substitutions at naturally occurring amino acids at position 297, wherein the substitutions detectably reduce and / or eliminate the glycosylation at position 297. In specific embodiments, the Fc domain of the fusion protein comprises substitutions of asparagine with cysteine ​​at position 297 of the antibody heavy chain. In other embodiments, the Fc domain of the fusion protein lacks glycosylation at position 297. In some embodiments, the Fc domain of the fusion protein comprises an N297Q substitution. In each of these, the numbering system for the constant region is the numbering system of the EU index specified in Kabat.

[0411] In some embodiments, amino acid substitutions, deletions, or insertions can result in improved expression, purification, or stability profiles of the fusion protein. In some embodiments, amino acid substitutions, deletions, or insertions can result in improved binding affinity and specificity profiles of the fusion protein. In some cases, the Fc domain of the fusion protein does not contain any cysteine ​​residues capable of forming interchain disulfide bonds.

[0412] In some embodiments, the Fc domain of the fusion protein or complex comprises the sequence set forth in SEQ ID NO: 5. In some embodiments, the Fc domain of the fusion protein or complex comprises an amino acid sequence having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5, or a fragment thereof.

[0413] In some embodiments, the recombinant fusion protein comprises an ACE2 protein or a fragment thereof as described in Section 1. In some embodiments, the recombinant fusion protein comprises an ACE2 as described in Section 1 linked to an Fc protein or a functional variant thereof as described in Section 1.

[0414] In some embodiments, the Fc protein is produced from a codon-optimized nucleic acid sequence. In some embodiments, the Fc protein is produced from a non-codon-optimized nucleic acid sequence.

[0415] In some embodiments, the ACE2-Fc fusion protein provided herein is further linked to a coupling moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein, or a VLP, or a combination thereof. In some embodiments, the ACE2-Fc fusion protein is linked to the coupling moiety via a coupling.

[0416] II. Polynucleotides and Vectors

[0417] The present disclosure also provides polynucleotides (nucleic acid molecules) encoding the ACE2-Fc fusion protein provided by the present disclosure, and vectors for genetically engineering cells to express the ACE2-Fc fusion protein.

[0418] In some embodiments, polynucleotides encoding the fusion proteins provided herein are provided. In some aspects, the polynucleotide comprises a single nucleic acid sequence, such as a nucleic acid sequence encoding an ACE2-Fc fusion protein polypeptide. In other examples, the polynucleotide comprises a first nucleic acid sequence encoding a recombinant polypeptide, particularly an ACE2, and a second nucleic acid sequence encoding a recombinant polypeptide of an Fc.

[0419] In some embodiments, the polynucleotide encoding the fusion protein comprises at least one promoter operably linked to control the expression of the recombinant polypeptide. In some embodiments, the polynucleotide comprises two, three or more promoters operably linked to control the expression of the recombinant polypeptide.

[0420] In some embodiments, for example, when a polynucleotide comprises two or more nucleic acid coding sequences, for example, a first nucleic acid sequence encoding an ACE2 and a second nucleic acid sequence encoding a recombinant polypeptide of Fc, at least one promoter is operably linked to control the expression of the two or more nucleic acid sequences. In some embodiments, a polynucleotide comprises two, three, or more promoters that are operably linked to control the expression of a recombinant polypeptide.

[0421] In some embodiments, the expression of the recombinant polypeptide is inducible or conditional. Therefore, in some aspects, the polynucleotide encoding the recombinant polypeptide comprises a conditional promoter, an enhancer or a transactivator. In some such aspects, the conditional promoter, the enhancer or the transactivator is an inducible promoter, an enhancer or a transactivator or is a repressible promoter, an enhancer or a transactivator. For example, in some embodiments, an inducible or conditional promoter can be used for limiting the expression of the recombinant polypeptide to a specific microenvironment. In some embodiments, the expression driven by an inducible or conditional promoter is regulated by exposure to an exogenous agent (such as heat, radiation or a drug).

[0422] In the case where the polynucleotide comprises more than one nucleic acid sequence encoding a recombinant polypeptide, the polynucleotide may further include a nucleic acid sequence encoding a peptide between one or more nucleic acid sequences. In some cases, the nucleic acid encoding peptide located between the nucleic acid sequences separates the translation product of the nucleic acid sequence during or after translation. In some embodiments, the peptide comprises an internal ribosome entry site (IRES), a self-cleaving peptide, or a peptide that causes ribosome skipping, such as a T2A peptide.

[0423] In some embodiments, a polynucleotide encoding a recombinant polypeptide is introduced into a composition containing cultured cells (e.g., host cells), e.g., by retroviral transduction, transfection, or transformation. In some embodiments, this allows expression (e.g., production) of the recombinant polypeptide. In some embodiments, the expressed recombinant polypeptide is purified.

[0424] In some embodiments, the polynucleotides (nucleic acid molecules) provided by the present disclosure encode the nucleic acid sequence of the fusion protein of ACE2-Fc as described in the present disclosure. In some embodiments, the polynucleotides (nucleic acid molecules) provided by the present disclosure encode the nucleic acid sequence of the fusion protein of ACE2-Fc as described in Disclosure 1. In some embodiments, the polynucleotides (nucleic acid molecules) provided by the present disclosure encode the recombinant polypeptide comprising ACE2 as described in the present disclosure. In some embodiments, the polynucleotides (nucleic acid molecules) provided by the present disclosure encode the recombinant polypeptide comprising Fc as described in the present disclosure. A vector or construct containing the nucleic acid molecule as described in the present disclosure is also provided. In some embodiments, the vector or construct comprises one or more promoters, which are operably linked to the nucleic acid molecule encoding the recombinant polypeptide to drive its expression. In some embodiments, the promoter is operably linked to one or more nucleic acid molecules, for example, a nucleic acid molecule encoding a polypeptide containing ACE2.

[0425] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the retroviral vector is a gamma-retroviral vector.

[0426] In some embodiments, carrier or construct include single promoter, which drives the expression of one or more nucleic acid molecules of polynucleotide. In some embodiments, such promoter can be polycistronic (bicistronic or tricistronic, referring to, for example, U.S. Patent number 6,060,273). For example, in some embodiments, transcription unit can be engineered to include a bicistronic unit comprising IRES (internal ribosome entry site), which allows co-expression of gene products (for example, encoding different recombinant polypeptides) by the message from a single promoter. In some embodiments, the carrier provided by the present disclosure is a bicistronic, which allows the carrier to include and express two nucleotide sequences. In some embodiments, the carrier provided by the present disclosure is a tricistronic, which allows the carrier to include and express three nucleotide sequences.

[0427] In some embodiments, a single promoter directs the expression of RNA that contains two or three genes (e.g., encoding a chimeric signaling receptor and encoding a recombinant receptor) in a single open reading frame (ORF) that are separated from each other by sequences encoding self-cleaving peptides (e.g., 2A sequences) or protease recognition sites (e.g., furin). Thus, the ORF encodes a single polypeptide that is processed into a single protein during translation (for 2A) or after translation. In some cases, a peptide (e.g., T2A) can cause the ribosome to skip (ribosome skipping) the synthesis of the C-terminal peptide bond of the 2A element, resulting in separation between the end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe. Genetic Vaccines and Ther. 2: 13 (2004) and de Felipe et al. Traffic 5: 616-626 (2004), incorporated by reference in their entirety for all purposes). Many 2A elements are known in the art. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, but are not limited to, 2A sequences from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine teschovirus-1 (P2A), as described in U.S. Patent Publication No. 20070116690.

[0428] III. ACE2-Fc Fusion Protein Compositions and Formulations

[0429] In some embodiments, the present disclosure provides ACE2-Fc fusion protein compositions, i.e., compositions (pharmaceutical compositions) comprising an ACE2-Fc fusion protein comprising a sequence selected from the group consisting of SEQ ID NOs: 1-18, or any combination thereof. In some embodiments, the present disclosure provides compositions comprising a recombinant fusion protein having the sequence described in SEQ ID NO: 1, or a fragment, variant, or mutant thereof. The fusion protein compositions mentioned in the present disclosure, comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 5-8, or a fragment, variant, or mutant thereof, can be used in a formulation.

[0430] In some embodiments, a single dose of the ACE2-Fc fusion protein composition may include about 0.1 mg to about 100 mg of the ACE2-Fc fusion protein, preferably about 0.25 mg to about 35 mg of the ACE2-Fc fusion protein, preferably about 0.25 mg to about 15 mg of the ACE2-Fc fusion protein. In some embodiments, a single dose comprises 0.25, 0.3, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05, 1.10, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 2.00, 2.05, 2.10, 2.15, 2.20, 2.25, 2.30, 2.35, 2.40, 2.45, 2.50, 2.75, 3.00, 3.25, 3.5, 3.75, or 4 mg. ACE2-Fc fusion protein. In some embodiments, a single dose comprises 0.5 mg of ACE2-Fc fusion protein. In other embodiments, a single dose comprises 1 mg of ACE2-Fc fusion protein. In further embodiments, a dose comprises 2.0 mg of ACE2-Fc fusion protein.

[0431] In some embodiments, the ACE2-Fc fusion protein compositions provided herein may include about 0.1 mg / ml to about 100 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein compositions may include about 0.1 mg / ml to about 95 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein compositions may include about 0.1 mg / ml to about 85 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein compositions may include about 0.1 mg / ml to about 75 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein compositions may include about 0.1 mg / ml to about 65, 55, 45, 35, 25, or 15 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 0.1 mg / ml to about 5, 6, 7, 8, 9 or 10 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mg / ml to about 5 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mg / ml to about 4 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mg / ml to about 3 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / ml to about 2 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / ml to about 1.5 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 1 mg / ml to about 5 mg / ml ACE2-Fc fusion protein.Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, 4.50, 4.75, 5.00, 5.25, 5.50, 5.75, 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, or 10.00 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 1.00 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 1.25 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 2.50 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 5.00 mg / ml ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may include about 10.00 mg / ml ACE2-Fc fusion protein.

[0432] In some cases, it may be desirable to combine the disclosed fusion proteins with other agents (e.g., vaccines) or other pharmaceutical products that induce a protective response. For example, a nasal spray formulation of a quadrivalent influenza vaccine can protect against influenza A (H1N1), influenza A (H3N2), and two B viruses. Fusion proteins including those described herein can be administered simultaneously or sequentially with vaccines or other pharmaceutical products targeting other viruses or influenza that infect the human body through the ACE2 pathway in the oral, nasal, and upper respiratory tracts.

[0433] The combined use provides protection against multiple pathogens. In some aspects, the combined use can protect against multiple strains of the same pathogen. The combined use is essential for minimizing the number of vaccinations required to confer protection against multiple pathogens or virus strains, thereby reducing administrative costs and increasing coverage. This may be particularly useful, for example, when vaccinating infants or children.

[0434] Also provided are compositions comprising the disclosed ACE2-Fc fusion protein and a pharmaceutically acceptable carrier. In some embodiments, the disclosed compositions include the ACE2-Fc fusion protein provided herein and an optional pharmaceutically acceptable carrier, such as water, a buffer, or saline. In some embodiments, the disclosed compositions include the ACE2-Fc fusion protein provided herein and PBS buffer or saline. In some embodiments, the disclosed compositions include the ACE2-Fc fusion protein provided herein and PB buffer or saline. In some embodiments, the disclosed composition includes the ACE2-Fc fusion protein provided by the present disclosure and disodium hydrogen phosphate dihydrate, disodium hydrogen phosphate monohydrate, sodium chloride, sucrose, phenylethanol and water, specifically, 0.1-15 mg / ml ACE2-Fc fusion protein, and 0.5-10 mg disodium hydrogen phosphate dihydrate, 0.01-5 mg / ml disodium hydrogen phosphate monohydrate, 0.1-10 mg / ml sodium chloride, 10-100 mg / ml sucrose, 0.25-5 mg / ml phenylethanol and an appropriate amount of water. In some embodiments, the disclosed composition comprises 1-5 mg / ml ACE2-Fc fusion protein, and 2.13 mg disodium hydrogen phosphate dihydrate, 0.82 mg / ml disodium hydrogen phosphate monohydrate, 2.92 mg / ml sodium chloride, 50 mg / ml sucrose, 2.50 mg / ml phenylethanol and an appropriate amount of water. In some embodiments, the disclosed composition comprises 1.25 mg / ml ACE2-Fc fusion protein, 2.13 mg of disodium hydrogen phosphate dihydrate, 0.82 mg / ml of disodium hydrogen phosphate monohydrate, 2.92 mg / ml of sodium chloride, 50 mg / ml of sucrose, and 2.50 mg / ml of phenylethyl alcohol. In some embodiments, the disclosed composition comprises 2.0 mg / ml ACE2-Fc fusion protein, 2.13 mg of disodium hydrogen phosphate dihydrate, 0.82 mg / ml of disodium hydrogen phosphate monohydrate, 2.92 mg / ml of sodium chloride, 50 mg / ml of sucrose, and 2.50 mg / ml of phenylethyl alcohol. In some embodiments, the disclosed composition comprises 2.5 mg / ml ACE2-Fc fusion protein, 2.13 mg of disodium hydrogen phosphate dihydrate, 0.82 mg / ml of disodium hydrogen phosphate monohydrate, 2.92 mg / ml of sodium chloride, 50 mg / ml of sucrose, and 2.50 mg / ml of phenylethyl alcohol. In some embodiments, the disclosed composition comprises 5.0 mg / ml ACE2-Fc fusion protein, 2.13 mg disodium hydrogen phosphate dihydrate, 0.82 mg / ml disodium hydrogen phosphate monohydrate, 2.92 mg / ml sodium chloride, 50 mg / ml sucrose, and 2.50 mg / ml phenylethanol. In some embodiments, the disclosed composition comprises the ACE2-Fc fusion protein provided herein and disodium hydrogen phosphate, such as disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate, such as disodium hydrogen phosphate monohydrate, sodium chloride, and Tween 80.

[0435] In some embodiments, the ACE2-Fc fusion protein composition comprises the protein nanoparticles provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the ACE2-Fc fusion protein composition comprises the VLPs provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the ACE2-Fc fusion protein composition comprises the isolated nucleic acid provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the ACE2-Fc fusion protein composition comprises the vector provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the ACE2-Fc fusion protein composition comprises the virus provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the ACE2-Fc fusion protein composition comprises the pseudovirus provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the ACE2-Fc fusion protein composition comprises the cells provided herein and an optional pharmaceutically acceptable carrier.

[0436] In some embodiments, the ACE2-Fc fusion protein is prophylactic. In some embodiments, the ACE2-Fc fusion protein is therapeutic. In some embodiments, the ACE2-Fc fusion protein is a prophylactic and therapeutic drug. Such pharmaceutical compositions can be administered to a subject by a variety of modes of administration known to those of ordinary skill in the art, for example, intramuscularly, intradermally, subcutaneously, intravenously, intraarterially, intraarticularly, intraperitoneally, intranasally, sublingually, intratonsillarly, oropharyngeally, or other parenteral and mucosal routes. Preferably, the ACE2-Fc fusion protein composition is administered nasally. Actual methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in publications such as Remingtons Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pa., 1995.

[0437] Therefore, the ACE2-Fc fusion protein described in the present disclosure can be formulated with a pharmaceutically acceptable carrier to help maintain biological activity while also promoting increased stability during storage within an acceptable temperature range. Potential carriers include, but are not limited to, physiologically balanced culture media, phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, antifreeze additives or stabilizers, such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate) or other protective agents. The resulting aqueous solution can be packaged for use as is or lyophilized. The lyophilized formulation is mixed with a sterile solution before single or multiple dose administration. In some embodiments, the phosphate-buffered saline solution includes sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate dihydrate.

[0438] In some embodiments, the ACE2-Fc fusion protein compositions of the present invention include an aqueous carrier as a solvent. Suitable carriers include, for example, sterile water, saline, phosphate-buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic. In some embodiments, the ACE2-Fc fusion protein compositions of the present invention include phosphate-buffered saline (PBS), such as NaCl, KCl, Na2HPO4, and KH2PO4, or further such as NaCl, Na2HPO4, and NaH2PO4.

[0439] Formulated compositions, particularly liquid preparations, may contain antibacterial agents to inhibit the growth of microorganisms to prevent or minimize degradation during storage, including but not limited to benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben at effective concentrations (typically 1% w / v). The antibacterial agents may include one or more of essential oils, high salt, high sugar, high acid, and anaerobic types. In some embodiments, the antibacterial agents described in the present disclosure include one or more of benzoic acid, sorbic acid, thimerosal, and phenylethyl alcohol. In some embodiments, the antibacterial agents include thimerosal and / or phenylethyl alcohol. Some patients may be contraindicated with antibacterial agents; therefore, the lyophilized preparation can be reconstituted in a solution containing or not containing such ingredients.

[0440] The composition of the present invention may contain pharmaceutically acceptable carrier substances required to approximate physiological conditions, such as pH adjusters and buffers, tonicity adjusters, isotonic agents, wetting agents, and the like, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. The isotonic agent is selected from one or more of glucose, dextrose, sucrose, glycerol, mannitol, sodium chloride, and potassium chloride, preferably dextrose.

[0441] In some embodiments, the ACE2-Fc fusion protein composition includes a pharmaceutically acceptable excipient, including, for example, a solvent, a filler, a buffer, a tonicity regulator, and a preservative (Pramanick et al., Pharma Times, 45:65-77, 2013, incorporated by reference in its entirety for all purposes). In some embodiments, the fusion protein composition of the present disclosure may include an excipient that acts as one or more of a solvent, a filler, a buffer, and a tonicity regulator (e.g., sodium chloride in saline can be used as both an aqueous carrier and a tonicity regulator).

[0442] The ACE2-Fc fusion protein composition described in the present disclosure may include a buffer. The buffer controls the pH to inhibit degradation of the active agent during processing, storage, and optionally reconstitution. Suitable buffers include, for example, salts containing acetate, citrate, phosphate, or sulfate. Other suitable buffers include, for example, amino acids such as arginine, glycine, histidine, and lysine. The buffer may further include hydrochloric acid or sodium hydroxide. In some embodiments, the buffer maintains the pH of the composition within the range of 6 to 9. In some embodiments, the pH is greater than (lower limit) 6, 7, or 8. In some embodiments, the pH is less than (upper limit) 9, 8, or 7. That is, the pH is within the range of about 6 to 9, wherein the lower limit is less than the upper limit. In some embodiments, the pH value is between about 5.8 and 6.8.

[0443] The ACE2-Fc fusion protein compositions disclosed herein may include a tonicity regulator. Suitable tonicity regulators include, for example, glucose, glycerol, sodium chloride, sucrose, and mannitol.

[0444] The ACE2-Fc fusion protein composition disclosed herein may include an osmotic pressure regulator selected from one or more of sodium chloride, potassium chloride, glycerol, glucose, sorbitol, sucrose, xylitol, and mannitol, such as sodium chloride and / or sucrose. The ACE2-Fc fusion protein composition disclosed herein may include a solvent. The solvent may be selected from one or more of ethanol, propylene glycol, and purified water, such as water.

[0445] The ACE2-Fc fusion protein compositions disclosed herein may include fillers. Fillers are particularly useful when the pharmaceutical composition is lyophilized prior to administration. In some embodiments, the filler is a preservative that helps stabilize and prevent degradation of the active agent during freeze or spray drying and / or during storage. Suitable fillers are sugars (mono-, di-, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0446] The ACE2-Fc fusion protein composition disclosed herein may include a preservative. Suitable preservatives include, for example, antioxidants and antimicrobial agents. Suitable preservatives include, but are not limited to, one or more of benzalkonium chloride, methylparaben, propylparaben, potassium sorbate, and sodium benzoate, preferably potassium sorbate. The suitable antioxidant may be selected from the group consisting of one or more of EDTA-2Na, butylated hydroxyanisole, 2,6-di-tert-butyl-4-methylphenol, sodium metabisulfite, potassium metabisulfite, butylated hydroxyanisole, L-ascorbyl palmitate, sodium thiosulfate, and vitamin E. However, in a preferred embodiment, the fusion protein composition disclosed herein is prepared under sterile conditions and in a disposable container, and therefore does not need to include a preservative.

[0447] The ACE2-Fc fusion protein compositions disclosed herein may include antibiotics. Antibiotics are substances or drugs that can kill bacteria or microorganisms or inhibit their activity. Suitable antibiotics and / or antibacterial agents may include one or more of the following: β-lactams, including penicillins, cephalosporins, carbapenems, β-lactams containing enzyme inhibitors, and monocyclic amides; aminoglycosides; tetracyclines; fluoroquinolones; folate pathway inhibitors; chloramphenicol; glycopeptides, including vancomycin and teicoplanin; and macrolides. Antibiotics may also include one or more synthetic drugs, such as sulfonamides, furans, and quinolones. The ACE2-Fc fusion protein compositions disclosed herein may or may not include antibacterial agents / antibiotics.

[0448] The ACE2-Fc fusion protein composition disclosed herein may include pharmaceutically acceptable flavors or other ingredients that impart flavor or aroma.

[0449] In some embodiments, the composition can be provided as a sterile composition. The pharmaceutical composition generally comprises an effective amount of the disclosed ACE2-Fc fusion protein and can be prepared by conventional techniques. Typically, the amount of ACE2-Fc fusion protein composition per dose is selected to be an amount that blocks viral invasion as a soluble receptor without significant adverse side effects. In some embodiments, the composition can be provided in unit dosage form for blocking viral invasion that can bind to ACE2 receptors in the upper respiratory tract and nasal cavity of a subject. The unit dosage form comprises a suitable single preselected dose for administration to a subject, or a suitably labeled or measured multiple of two or more preselected unit doses, and / or a metering mechanism for administering a unit dose or multiples thereof. In other embodiments, the composition further comprises one or more adjuvants.

[0450] IV. Receptor-Blocking Methods for Viral Invasion

[0451] In some embodiments, the present disclosure provides a method for preventing a subject from being infected with a coronavirus, comprising administering to the subject an effective amount of a composition comprising a recombinant fusion protein selected from the group consisting of SEQ ID NOs: 1-18. In some embodiments, the present disclosure provides a method for preventing a subject from being infected with a coronavirus, wherein the recombinant fusion protein comprises an ACE2 protein or a fragment thereof, and the method comprises administering to the subject an effective amount of a composition comprising a recombinant fusion protein selected from the group consisting of SEQ ID NOs: 1-18.

[0452] The disclosed fusion proteins (e.g., ACE2-Fc fusion proteins, e.g., human ACE2-Fc fusion proteins disclosed herein, nucleic acid molecules (e.g., RNA molecules) or vectors encoding human ACE2-Fc fusion proteins disclosed herein, or protein nanoparticles or virus-like particles comprising the disclosed human ACE2-Fc fusion proteins) can be administered to a subject to block infection by viruses that infect the human body through the ACE2 pathway (i.e., prevent viral infection). In a specific example, the subject is a human. The blockade can inhibit the subsequent binding of the corresponding coronavirus to the subject's ACE2, and can be used to prevent, treat, or inhibit infections and diseases associated with the corresponding coronavirus.

[0453] The fusion protein or pharmaceutical composition of the present disclosure can be administered via intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intranasal, sublingual, tonsil, oropharyngeal or other parenteral and mucosal routes, preferably intranasally.

[0454] A subject can be selected for protection having or being at risk of infection with a coronavirus, for example because of exposure or potential exposure to a coronavirus.

[0455] Typical subjects that are intended to be prevented with the therapy and methods of the present invention include humans and non-human primates and other animals. In order to identify the subject for prevention or treatment according to the method of the present invention, an acceptable screening method is adopted to determine the risk factors relevant to the target or suspected disease or condition, or to determine the state of the existing disease or condition in the subject. These screening methods include, for example, routine examinations, to determine the environment, family, occupation and other such risk factors that may be relevant to the target or suspected disease or condition, and diagnostic methods, such as various ELISAs and other immunoassays for detecting and / or characterizing coronavirus infection. These and other conventional methods allow clinicians to use the method and pharmaceutical composition of the present invention to select subjects who need to prevent or treat diseases. According to these methods and principles, compositions can be applied according to the teachings of the present disclosure or other conventional methods, as an independent prevention or treatment regimen, or as a follow-up, auxiliary or coordinated treatment regimen for other treatments.

[0456] Administration of the disclosed fusion proteins (e.g., ACE2-Fc fusion proteins, e.g., human ACE2-Fc fusion proteins described herein, disclosed nucleic acid molecules (e.g., RNA molecules) or vectors encoding human ACE2-Fc fusion proteins, or protein nanoparticles or virus-like particles comprising disclosed human ACE2-Fc fusion proteins) can be used for prophylactic or therapeutic purposes. When provided prophylactically, the disclosed therapeutic agents are provided before any symptoms occur, e.g., before infection occurs. Prophylactic administration of the disclosed therapeutic agents is used to prevent or ameliorate any subsequent infection. When provided therapeutically, the disclosed therapeutic agents are provided at or after the onset of disease or infection symptoms, e.g., after the development of symptoms of a coronavirus infection corresponding to a coronavirus S antigen, or after diagnosis of a coronavirus infection. Thus, a therapeutic agent can be provided before anticipated exposure to a coronavirus in order to attenuate the anticipated severity, duration, or extent of infection and / or related disease symptoms following exposure or suspected exposure to the virus, or after actual onset of infection.

[0457] The fusion protein and fusion protein composition thereof disclosed herein can effectively help subjects (preferably humans) prevent infection against coronavirus. The actual dosage of the disclosed fusion protein will vary according to factors such as the subject's disease signs and specific state (e.g., subject's age, size, health status, symptom degree, susceptibility factors, etc.), administration time and route, other drugs or treatments administered simultaneously, and the specific pharmacology of the composition to stimulate the desired activity or biological response in the subject. The dosage regimen can be adjusted to provide optimal prevention or therapeutic response.

[0458] The fusion proteins and fusion protein compositions described herein can be used together or sequentially with one or more immunogenic compositions of the disclosed immunogens.

[0459] In some embodiments, an effective amount of the ACE2-Fc fusion protein composition of the present disclosure is administered in a single dose or in a series of doses separated by one or more intervals. The intervals can be measured in hours, days, or weeks. In some embodiments, the composition containing an effective amount of the ACE2-Fc fusion protein of the present disclosure can be administered at intervals of 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours. In some embodiments, the composition can be administered 1, 2, or 3 times a day for multiple consecutive days, for example, once a day or twice a day for multiple consecutive days, for example, 28 days, or twice a day for one, two, three, four, or more consecutive days. In some embodiments, the composition can be administered once every two or three days for multiple consecutive days.

[0460] The suitability of the selected fusion protein parameters, such as formulation, dosage, regimen, etc., can be determined by taking aliquots of serum from the subject and measuring the antibody titer during administration. In addition, the clinical condition of the subject can be monitored to obtain the desired effect, such as preventing infection or improving the disease state (e.g., reducing viral load). If such monitoring shows that the ACE2-Fc fusion protein is suboptimal, the subject can be strengthened with an additional dose of the ACE2-Fc fusion protein composition, and the ACE2-Fc fusion protein composition usage parameters can be improved in a manner that is expected to enhance infection blocking.

[0461] Typically, each single human dose will include 0.01-20 mg of ACE2-Fc fusion protein, for example, from about 0.05 mg to about 10 mg, for example, from about 0.1 mg to about 8 mg, for example, about 0.50 mg, about 1.00 mg, 1.50 mg, about 2.00 mg, about 2.50, about 3.00 mg, about 3.50 mg, about 4.00 mg, about 4.50 mg, about 5.00 mg, about 5.50 mg, about 6.00 mg, about 6.50 mg, about 7.00 mg, about 7.50 mg, about 8.00 mg of ACE2-Fc fusion protein.

[0462] For protein therapy, generally, each single human dose can include about 0.01 to 3 mg of ACE2-Fc fusion protein. In some embodiments, a single dose of an ACE2-Fc fusion protein composition can include about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.39, 0.40 to 3 mg of ACE2-Fc fusion protein. In some embodiments, a single dose of an ACE2-Fc fusion protein composition may include about 0.05 to 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.2, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mg of an ACE2-Fc fusion protein. In some embodiments, a single dose of an ACE2-Fc fusion protein composition may include about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65 0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, or 3.00 mg of ACE2-Fc fusion protein. The single dose disclosed herein is a dose administered once, which can be one puff or multiple puffs per nostril, such as two puffs or three puffs.

[0463] The amount used in the fusion protein composition is selected based on the subject population (e.g., infants or the elderly). The optimal dosage of a particular component can be determined by standard studies involving observation of antibody titers and other responses in subjects. It should be understood that a therapeutically effective amount of the disclosed fusion protein can include an amount that is ineffective in blocking viral infection by a single dose but is effective in multiple doses.

[0464] After administering the fusion protein of the present invention, the viral load in the subject, such as the lung viral load, is lower (relative to the control group or before administration) or reduced, and this reaction means that an effective dose of fusion protein has been delivered to the subject. In some embodiments, the antibody response of the subject will be determined in the case of an effective dose / immunization regimen. In most cases, it is sufficient to assess the antibody titer in the serum or plasma obtained from the subject. The decision on whether to change the amount of the therapeutic agent administered to an individual can be based at least in part on the antibody titer level. The antibody titer level can be based on, for example, an immune binding assay that measures the concentration of antibodies to binding antigens (including, for example, recombinant coronavirus S antigens, such as S-trimers) in serum.

[0465] The method can be effective without completely eliminating, reducing or preventing coronavirus infection. For example, the use of the disclosed fusion protein can reduce or inhibit the coronavirus infection of the desired amount, such as at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (eliminating or preventing detectable infected cells), compared to coronavirus infection in the absence of fusion protein administration.

[0466] The fusion proteins disclosed in the present disclosure can also be administered via nucleic acid (Hoecke and Kenny Roose, J Transl Med (2019) 17:54, https: / / doi.org / 10.1186 / s12967-019-1804-8, incorporated by reference in its entirety for all purposes). In some embodiments, the fusion proteins disclosed in the present disclosure are administered using DNA.

[0467] In some embodiments, administering a therapeutically effective amount of one or more of the disclosed fusion proteins to a subject can block viral infection in the subject. To assess the effect of blocking viral infection, serum can be collected from the subject at an appropriate time point after administration, frozen, and stored for neutralization testing. Methods for determining neutralization activity are known to those of ordinary skill in the art and are further described herein, including but not limited to plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, a panel of coronavirus pseudoviruses can be used to determine serum neutralization activity.

[0468] V. Products or Kits

[0469] The present disclosure also provides products or kits containing the provided recombinant polypeptides and protein compositions. The products may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, test tubes, IV solution bags, etc. The container can be formed from various materials (e.g., glass or plastic). In some embodiments, the container has a sterile access port. Exemplary containers include intravenous solution bags and vials, including containers with stoppers that can be pierced by injection needles. The product or kit may further include a package insert indicating that the product can be used to prevent or treat specific conditions, such as the conditions described in the present disclosure (e.g., coronavirus infection). Alternatively, or additionally, the product or kit may further include another or the same container containing a pharmaceutically acceptable buffer or other excipients or adjuvants. It may further include other materials, such as other buffers, diluents, filters, needles, and / or syringes.

[0470] The label or package insert may indicate that the composition is used to prevent or treat a coronavirus infection in a subject. The label or package insert on or associated with the container may indicate instructions for reconstitution and / or use of the formulation. The label or package insert may further indicate that the formulation is used or intended for administration as a nasal spray to prevent a coronavirus infection in a subject.

[0471] In some embodiments, the container holds an ACE2-Fc fusion protein or a composition thereof, which is alone or in combination with another composition effective for treating, preventing and / or diagnosing a condition. An article of manufacture or kit may include (a) a first container having a composition contained therein (i.e., a first agent), wherein the composition includes an immune ACE2-Fc fusion protein or recombinant polypeptide or a composition thereof; and (b) a second container having a composition contained therein (i.e., a second agent), wherein the composition includes another agent, such as an adjuvant or other therapeutic agent, and the article or kit further includes instructions on a label or package insert for treating a subject with the second agent in an effective amount.

[0472] VI. Terminology

[0473] Unless otherwise defined, all special terms, symbols and other technical and scientific terms or terminology used in this disclosure are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, for clarity and / or ease of reference, this disclosure defines terms with commonly understood meanings, and the inclusion of such definitions in this disclosure does not necessarily represent a substantial difference from what is generally understood in the art.

[0474] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides (including provided receptors and other polypeptides, such as linkers or peptides) may include amino acid residues, including natural and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the polypeptide may include modifications to the native or natural sequence, as long as the protein retains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as through mutations in the host producing the protein or errors resulting from PCR amplification.

[0475] As used in this disclosure, a "subject" is a mammal, such as a human or other animal, and is typically a human. In some embodiments, the subject (e.g., patient) to which one or more agents, cells, cell populations, or compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject can be male or female and can be of any suitable age, including infants, teenagers, adolescents, adults, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0476] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to the complete or partial improvement or reduction of a disease, condition, or disorder, or a symptom, adverse reaction, or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or relieving the disease state, and alleviating or improving prognosis. The term does not imply a complete cure of the disease or the complete elimination of any symptom or the effect on all symptoms or outcomes.

[0477] As used herein, "delaying the progression of a disease" means delaying, hindering, slowing, slowing, stabilizing, inhibiting, and / or postponing the progression of a disease (e.g., cancer). The length of time for delay may vary depending on the history of the disease and / or the individual being treated. In some embodiments, sufficient or significant delay may actually encompass prevention, in that the individual will not develop the disease. For example, the development of advanced cancer, such as metastasis, may be delayed.

[0478] As used in this disclosure, "prevention" includes providing protection against the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease. In some embodiments, provided cells and compositions are used to delay the development of a disease or slow the progression of a disease.

[0479] As used herein, "inhibiting" a function or activity means reducing the function or activity when compared to other identical conditions except for the condition or parameter of interest or when compared to another condition. For example, cells that inhibit tumor growth will reduce the growth rate of a tumor compared to the growth rate of the tumor in the absence of the cells.

[0480] In the context of administration, an "effective amount" of an agent (eg, a pharmaceutical preparation, cell, or composition) refers to an amount effective at the dosage / amount and for the period of time necessary to achieve the desired effect (eg, a therapeutic or prophylactic effect).

[0481] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical formulation or cell) refers to an effective amount at the dosage and time period required to achieve the desired therapeutic effect (e.g., for treating a disease, condition, or disorder) and / or the pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the subject, as well as the cell population being administered. In some embodiments, provided methods involve administering cells and / or compositions in an effective amount (e.g., a therapeutically effective amount).

[0482] A "prophylactically effective amount" refers to an effective amount for the dosage and time period required to achieve the desired preventive effect. Typically, but not necessarily, because a prophylactic dose is used for subjects before or in the early stages of the disease, the prophylactic effective amount will be less than the therapeutically effective amount. In cases where the tumor burden is low, the prophylactic effective amount in some aspects will be higher than the therapeutically effective amount. An effective amount of a vaccine or other agent is sufficient to produce the desired response, such as reducing or eliminating the signs or symptoms of a condition or disease, such as pneumonia. For example, this may be the amount necessary to inhibit viral replication or measurably change the external symptoms of a viral infection. Generally speaking, this amount will be sufficient to measurably inhibit viral (e.g., SARS-CoV-2) replication or infectivity. When administered to a subject, the dose typically used will achieve a target tissue concentration that has been shown to inhibit viral replication in vitro. In some embodiments, an "effective amount" is an amount that treats (including prevents) one or more symptoms and / or potential causes of any condition or disease, such as for the treatment of coronavirus infection. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is an amount that prevents the development of one or more signs or symptoms of a particular disease or condition, such as one or more signs or symptoms associated with coronavirus infection.

[0483] As used in this disclosure, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, typically a protein, that is capable of inducing an immune response in a subject. The term also refers to an immunologically active protein, i.e., one that, upon administration to a subject (directly or by administering a nucleotide sequence or vector encoding the protein to the subject), is capable of eliciting a humoral and / or cellular immune response against the protein. Unless otherwise indicated, the term "vaccine immunogen" is used interchangeably with "protein antigen" or "immunogenic polypeptide."

[0484] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to specific nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. With respect to polypeptide sequences, "conservatively modified variants" refer to variants having conservative amino acid substitutions, in which amino acid residues are replaced by other amino acid residues having side chains with similar charges. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0485] Epitopes are antigenic determinants. These are specific chemical groups or peptide sequences on an antigenic molecule that trigger a specific immune response. For example, epitopes are regions of an antigen that B cells and / or T cells respond to. Epitopes can be formed by either contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of a protein.

[0486] Unless otherwise indicated, the fusion protein of the present disclosure is a recombinant protein containing the amino acid sequences of at least two unrelated proteins, which are linked together by peptide bonds to form a single protein. Therefore, it does not contain naturally occurring coronavirus surface antigens. Unrelated amino acid sequences can be directly linked to each other, or they can be connected using a linker sequence. As used in the present disclosure, if the amino acid sequence of a protein is not usually linked together by peptide bonds in its natural environment (e.g., within a cell), the protein is unrelated. For example, the amino acid sequences of ACE2 and Fc are not usually linked together by peptide bonds.

[0487] An immunogen is a protein or portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected with or at risk of infection by a pathogen. Administration of an immunogen can result in protective immunity and / or active immunity against the target pathogen.

[0488] An immunogenic composition refers to a composition comprising an immunogenic polypeptide that induces a measurable CTL response against a virus expressing the immunogenic polypeptide, or induces a measurable B cell response (eg, production of antibodies) against the immunogenic polypeptide.

[0489] The sequence identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequence. When compared and aligned to obtain maximum correspondence by a comparison window or using one of the following sequence comparison algorithms or by manually comparing and visually inspecting the measured designated region, if the two sequences have the same amino acid residue or nucleotide of a specified percentage, the two sequences are "substantially identical" (i.e., on a specified region, or in the case of unspecified, on the entire sequence, 60% sequence identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity). Alternatively, identity is present in a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably in a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0490] Vaccine refers to a pharmaceutical composition that induces a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, vaccines induce an antigen-specific immune response against an antigen of a pathogen (e.g., a viral pathogen) or a cellular component associated with a pathological condition. Vaccines may include polynucleotides (e.g., nucleic acids encoding disclosed antigens), peptides or polypeptides (e.g., disclosed antigens), viruses, cells, or one or more cellular components. In some embodiments, a vaccine or vaccine immunogen or vaccine composition is expressed from a fusion construct and self-assembles into nanoparticles that display an immunogenic polypeptide or protein on the surface.

[0491] Virus-like particles (VLPs) refer to non-replicable viral shells, derived from any of several viruses. VLPs are generally composed of one or more viral proteins, such as, but not limited to, proteins called capsids, coats, shells, surfaces, and / or envelope proteins, or particle-forming polypeptides derived from these proteins. After recombinant expression of proteins in an appropriate expression system, VLPs can form spontaneously. Methods for producing specific VLPs are known in the art. The presence of VLPs after recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, etc. See, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Virol. 68: 4503-4505, which are incorporated by reference in their entirety for all purposes. For example, VLPs can be separated and / or identified by characteristic density bands by density gradient centrifugation. Alternatively, cryo-electron microscopy can be performed on a vitrified aqueous sample prepared from the VLP in question, and images are recorded under appropriate exposure conditions.

[0492] The term "about" used in this disclosure refers to the normal error range of each value that is readily known to those skilled in the art. References to "about" values ​​or parameters in this disclosure include (and describe) embodiments for the value or parameter itself.

[0493] As used in this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more."

[0494] In this disclosure, various aspects of the claimed subject matter are presented in range format. It should be understood that descriptions in range format are merely for convenience and brevity and should not be construed as inflexible limitations on the scope of the claimed subject matter. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual values ​​within that range. For example, where a range of values ​​is provided, it should be understood that each intervening value between the upper and lower limits of that range and any other stated or intervening value within that range is included in the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the claimed subject matter, subject to any explicitly excluded limitations within the stated ranges. If the stated range includes one or both limits, then ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies to any width of the range.

[0495] As used in this disclosure, a composition refers to any mixture of two or more products, substances or compounds (including cells). It can be a solution, a suspension, a liquid, a powder, a paste, aqueous, non-aqueous or any combination thereof.

[0496] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term encompasses vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0497] VII. Exemplary Protocols

[0498] Scheme 1. A fusion protein comprising multiple recombinant polypeptides, wherein the fusion protein comprises human ACE2 protein or a fragment thereof and human IgG Fc protein or a functional variant thereof.

[0499] Option 2. The fusion protein according to Option 1, wherein the human ACE2 protein or a fragment thereof comprises the human ACE2 extracellular domain or a fragment thereof.

[0500] Option 3. The fusion protein according to Option 1 or 2, wherein the human ACE2 protein or a fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 5, 7, 11, 12, 13 and 14, or an amino acid sequence or a fragment thereof having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0501] Option 4. The fusion protein according to any one of Options 1 to 3, wherein the human ACE2 protein or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence or a fragment thereof having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0502] Option 5. The fusion protein according to any one of Options 1 to 4, wherein the human IgG Fc protein or a functional variant thereof comprises any one or more Fc region sequences selected from human IgG1Fc, IgG2Fc, IgG3Fc and IgG4Fc, or functional variants and fragments thereof.

[0503] Option 6. The fusion protein according to any one of Options 1 to 5, wherein the human IgG Fc protein or a functional variant thereof is selected from a human IgG1 Fc region sequence or a functional variant and fragment thereof.

[0504] Option 7. The fusion protein according to any one of Options 1 to 6, wherein the human IgG Fc protein or a functional variant thereof comprises the amino acid sequence of SEQ ID NO: 6 or 8, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a fragment thereof.

[0505] Option 8. The fusion protein according to any one of Options 1 to 7, wherein the human IgG Fc protein or a functional variant thereof comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a fragment thereof.

[0506] Option 9. The fusion protein according to any one of Option 1 to Option 8, which optionally comprises a signal peptide.

[0507] Scheme 10. The fusion protein according to any one of Schemes 1 to 9, which optionally comprises a peptide linker, wherein the human ACE2 protein or a fragment thereof and the human IgG Fc protein or a functional variant thereof are directly connected or connected through a peptide linker.

[0508] Scheme 11. The fusion protein according to Scheme 10, wherein the peptide linker is selected from the group consisting of an arginine-serine peptide linker (-RS-), a valine-serine linker (-VS-), and a glycine-serine linker (-GS-).

[0509] Option 12. The fusion protein according to any one of Option 1 to Option 11, which optionally includes a mutant sequence.

[0510] Option 13. The fusion protein according to Option 1, comprising the amino acid sequence of any one of SEQ ID NOs: 1-18, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a fragment thereof, or a combination thereof.

[0511] Option 14. The fusion protein according to Option 1, comprising the amino acid sequence of any one of SEQ ID NOs: 1-4 and 15-18, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a fragment thereof, for example, comprising the amino acid sequence of any one of SEQ ID NOs: 1-4 or 15-18, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a fragment thereof.

[0512] Option 15. The fusion protein according to any one of Option 1 to Option 14, further comprising a detectable tag.

[0513] Embodiment 16. A pharmaceutical composition comprising the fusion protein according to any one of Embodiments 1 to 15 and an optional pharmaceutically acceptable carrier.

[0514] 17. The pharmaceutical composition of 16, comprising about 0.1 mg / ml to about 100 mg / ml of the fusion protein of any one of 1 to 15, such as about 0.50 mg / ml to about 20.00 mg / ml, such as about 1.25 mg / ml, about 2.50 mg / ml, or about 5.00 mg / ml.

[0515] Embodiment 18. The pharmaceutical composition according to embodiment 16 or 17, wherein the pharmaceutically acceptable carrier comprises a combination of one or more of a buffer and an osmotic pressure regulator.

[0516] Embodiment 19. The pharmaceutical composition of embodiment 18, wherein the buffer comprises sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, or a combination thereof.

[0517] Embodiment 20. The pharmaceutical composition according to embodiment 18 or 19, wherein the osmotic pressure regulating agent comprises a combination of one or more selected from the group consisting of sodium chloride, potassium chloride, glycerol, glucose, sorbitol, sucrose, xylitol, and mannitol.

[0518] 21. The pharmaceutical composition of any one of 18 to 20, wherein the osmotic pressure regulating agent comprises sodium chloride and / or sucrose.

[0519] Option 22. The pharmaceutical composition according to any one of Option 18 to Option 21, wherein the pharmaceutically acceptable carrier optionally comprises a stabilizer, wherein the stabilizer comprises one or more combinations selected from the group consisting of proteins, peptides or hydrolysates such as albumin, gelatin; sugars such as sucrose, lactose, sorbitol; and amino acids such as sodium glutamate, for example, the stabilizer comprises sucrose.

[0520] 23. The pharmaceutical composition according to any one of claims 18 to 22, wherein the pharmaceutically acceptable carrier optionally comprises a bacteriostatic agent, wherein the bacteriostatic agent comprises a combination of one or more selected from the group consisting of benzoic acid, sorbic acid, thimerosal and phenylethanol, for example, the bacteriostatic agent comprises phenylethanol and / or thimerosal.

[0521] Embodiment 24. The pharmaceutical composition according to any one of embodiments 18 to 23, which is in a dosage form suitable for administration by intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intranasal, sublingual, tonsil, oropharyngeal or other parenteral and mucosal routes, preferably in a dosage form suitable for intranasal administration, such as a nasal spray.

[0522] Scheme 25. The fusion protein according to any one of Schemes 1 to 15 or the pharmaceutical composition according to any one of Schemes 16 to 24 is used to prevent or treat infection with coronavirus SARS-CoV-2 and its variants and / or prevent the spread of coronavirus SARS-CoV-2 and its variants in infected subjects.

[0523] Scheme 26. A fusion protein according to any one of Schemes 1 to 15 or a pharmaceutical composition according to any one of Schemes 16 to 24 for preventing, treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects according to Scheme 25, wherein the coronavirus SARS-CoV-2 and its variants include but are not limited to SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Mu, Omicron, JN.1; and unknown coronavirus SARS-CoV-2 variants.

[0524] Scheme 27. A fusion protein according to any one of Schemes 1 to 15 or a pharmaceutical composition according to any one of Schemes 16 to 24 for preventing or treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects according to Scheme 25 or 26, wherein the fusion protein or pharmaceutical composition is administered intramuscularly, intradermally, subcutaneously, intravenously, intraarterially, intraarticularly, intraperitoneally, intranasally, sublingually, tonsilally, oropharyngeally or other parenteral and mucosal routes, preferably intranasally.

[0525] Option 28. A fusion protein according to any one of Options 1 to 15 or a pharmaceutical composition according to any one of Options 16 to 24 for use in preventing, treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects according to any one of Options 25 to 27, wherein the fusion protein or pharmaceutical composition is administered intranasally as a single dose or a series of doses separated by one or more intervals, the intervals being measured in hours, days or weeks.

[0526] Scheme 29. A fusion protein according to any one of Schemes 1 to 15 or a pharmaceutical composition according to any one of Schemes 16 to 24 for preventing or treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects according to Scheme 28, wherein the interval is 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours.

[0527] Scheme 30. The fusion protein according to any one of Schemes 1 to 15 or the pharmaceutical composition according to any one of Schemes 16 to 24 for preventing or treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects according to any one of Schemes 25 to 29, wherein the fusion protein or pharmaceutical composition is administered once or more times / day, for example, once / day or twice / day.

[0528] Scheme 31. Use of the fusion protein according to any one of Schemes 1 to 15 or the pharmaceutical composition according to any one of Schemes 16 to 24 in the preparation of a medicament for preventing or treating infection by coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects.

[0529] Scheme 32. The use according to Scheme 31, wherein the coronavirus SARS-CoV-2 and its variants include but are not limited to SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Miu, Omicron, JN.1; and unknown variants of the coronavirus SARS-CoV-2.

[0530] Scheme 33. A method for preventing, treating infection by coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in an infected subject, comprising administering to the subject a therapeutically effective amount of the fusion protein according to any one of Schemes 1 to 15 or the pharmaceutical composition according to any one of Schemes 16 to 24.

[0531] Option 34. A method according to Option 33, wherein the coronavirus SARS-CoV-2 and its variants include but are not limited to SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Mu, Omicron, JN.1; and unknown variants of the coronavirus SARS-CoV-2.

[0532] Scheme 35. The method according to Scheme 33 or 34, wherein the fusion protein or pharmaceutical composition is administered intramuscularly, intradermally, subcutaneously, intravenously, intraarterially, intraarticularly, intraperitoneally, intranasally, sublingually, tonsilally, oropharyngeally or other parenteral and mucosal routes, preferably intranasally.

[0533] 36. The method of any one of claims 33 to 35, wherein the fusion protein of any one of claims 1 to 15 or the pharmaceutical composition of any one of claims 16 to 24 is administered intranasally as a single dose or a series of doses separated by one or more intervals measured in hours, days, or weeks.

[0534] Option 37. The method of Option 36, wherein the interval period is 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours.

[0535] Scheme 38. The scheme according to any one of schemes 33 to 37, wherein the fusion protein or pharmaceutical composition is administered once or more times per day, for example, once per day or twice per day.

[0536] Scheme 39. A kit for preventing or treating infection by coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in an infected subject, comprising the fusion protein according to any one of Schemes 1 to 15 or the pharmaceutical composition according to any one of Schemes 16 to 24;

[0537] Container; and

[0538] Optional package insert or label indicating prophylaxis and / or treatment.

[0539] Option 40. A kit according to Option 39, wherein the coronavirus SARS-CoV-2 and its variants include but are not limited to SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Mu, Omicron, JN.1; and unknown variants of the coronavirus SARS-CoV-2.

[0540] A nucleic acid encoding the fusion protein or a fragment thereof according to any one of claims 1 to 15.

[0541] Option 42. A vector comprising the nucleic acid according to Option 41.

[0542] Scheme 43. The vector according to Scheme 33, which is phFC(IM).

[0543] Option 44. A host cell comprising the nucleic acid according to Option 41 or the vector according to Option 42.

[0544] Option 45. The host cell according to Option 44, which is a CHO cell, such as a GH-CHO.

[0545] Example

[0546] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.

[0547] method

[0548] Example 1: Production and purification of ACE2-Fc fusion protein and preparation of nasal spray

[0549] In this example, an ACE2-Fc fusion protein with the amino acid sequence set forth in SEQ ID NO:4 (SCB-719) was produced and purified and used in the biological assays performed in the following examples. SEQ ID NO:4 synthesized in the examples has a C-terminal modification, which facilitates tracking and detection without affecting the functional effects of the ACE2-Fc fusion protein. Those skilled in the art will readily appreciate that ACE2-Fc fusion proteins with or without this C-terminal modification, such as SEQ ID NOs:1-3 and 15-18, can achieve the same or comparable biological effects.

[0550] To rapidly express ACE2-Fc fusion protein (Liu et al., Scientific Reports, 7(1):8953, 2017, incorporated by reference in its entirety for all purposes), the cDNA encoding ACE2-Fc fusion protein was subcloned into the expression vector phFC(IM) and propagated in the Chinese hamster ovary cell line GH-CHO.

[0551] Taking the 200L cell culture process as an example, the 200L cell culture process is divided into the seed expansion stage and the fed-batch culture stage. The seed expansion stage: Using CD118 medium, a working cell bank (WCB) cell is revived in a 250ml shake flask. After 3-5 days of culture, it is subcultured and expanded. After N-4 and N-3 shake flask subculture, it is inoculated into a WAVE reactor for N-2 and N-1 expansion. The fed-batch culture stage: When the N-1 level culture reaches day 3, it is inoculated into a 200L reactor. From day 3 to day 15, 2% CB7a and 0.2% CB7b of the initial culture volume are added daily. On day 4, the culture temperature is lowered from 37°C to 32°C. The glucose concentration is maintained at 2-10g / L during the culture period. The cells are harvested on day 14-16 or when the cell viability is less than 90%. The harvest is deep filtered using Merck's D0SP and A1HC secondary membrane packages. After sterile filtration, the cell harvest is transferred to downstream purification.

[0552] In order to obtain a fusion protein in a high-purity form for drug research, the sample was first quickly captured from the cell culture harvest using Cytiva's affinity chromatography filler Mabselect PrismA, and then a flow-through cation exchange chromatography was performed. The filler was the cation exchange chromatography filler Monomix HC 60SP from Suzhou Saifen Technology Co., Ltd. The main purpose of this step was to remove the target product polymers. A final concentration of 1% Tween80 and 0.3% TNBP were added to the cation exchange chromatography collection liquid to inactivate the S / D virus. The sample was then purified in a binding elution mode using Merck's anion exchange chromatography filler Eshmuno Q to further improve the sample purity and remove impurities such as HCP and DNA. The virus was then removed by nano-membrane filtration using Planova 20N from Asahi Kasei Corporation. Finally, a 50kDa ultrafiltration membrane package made of PES material from Hangzhou Kebet Filter Equipment Co., Ltd. was used for concentration and liquid exchange to 20mM PB, 50mM The sample concentration was ≥10 g / L in a 5% NaCl sucrose solution, and the stock solution was sterile filtered through 0.2 μm. The ACE2-Fc fusion protein purification platform yielded a high-purity target protein with a purity >98%. The detection profile is shown in Figure 4C. Stability analysis of the purified ACE2-Fc fusion protein demonstrated that the ACE2-Fc fusion protein remained stable at 25°C.

[0553] The ACE2-Fc fusion protein stock solution is thawed, the semi-finished product is prepared, sterilized and filtered, aseptically filled, capped, labeled, and packaged to prepare the ACE2-Fc fusion protein nasal spray.

[0554] The formulation development test for the ACE2-Fc fusion protein nasal spray was divided into four phases: pH screening, single-factor additive testing, DoE testing, and formulation determination. The product formulation was determined using the purity (SEC-HPLC) trend under accelerated conditions (25±2°C) and high-temperature conditions (40±2°C) as the primary indicator, and the osmotic pressure of the drug solution as the secondary indicator. The formulation is shown in Tables 2a-2e.

[0555] Table 2a. Nasal spray prescription

[0556] Table 2b. Nasal spray prescription

[0557] Table 2c. Nasal spray prescription

[0558] Table 2d. Nasal spray prescription

[0559] Table 2e. Nasal spray prescription Note: The formula contains water.

[0560] The nasal spray preparation disclosed in the present disclosure can be placed under long-term conditions (2-8°C) for 6 months and accelerated conditions (25±2°C) for 4 months. The purity shows no obvious change trend compared with the 0 point and meets the acceptable standard; after being placed at high temperature (40±2°C) for 1 month, the purity shows a significant downward trend, but is still within the acceptable standard range.

[0561] The nasal spray disclosed herein is a multi-dose sterile nasal spray (e.g., 100 μl per nostril, 2.8 ml per bottle). It is manufactured using sterile (sterile) packaging and aseptic filling processes, ensuring the sterility of the released product. The unique sealing technology of the antibacterial pump at the spray hole and the separate airway filter design ensure the sterility of the product during multiple uses.

[0562] Example 2: Construction and production of pseudoviruses

[0563] The spike protein (S) gene of the SARS-CoV-2 high-concern variant was optimized using mammalian codons, synthesized by GenScript, and then cloned into the pcDNA3.1(+) eukaryotic expression vector. Plasmids encoding the S glycoproteins of SARS-CoV-2 variants, including Hu-1, Alpha (α), Beta (β), Gamma (γ), Delta (δ), Miu (μ), Omicron (ο), and JN.1, were constructed. The lentiviral packaging plasmid psPAX2 and the pLVX-AcGFP-N1-Fluc lentiviral reporter plasmid expressing GFP and luciferase were from HonorGene (HonorGene, China). Pseudoviruses were generated by co-transfecting psPAX2, pLVX-AcGFP-N1-Fluc, and plasmids encoding various S genes into HEK 293T cells using Lipofectamine 3000 (Invitrogen, L3000-015). The supernatant was harvested 24 ± 2 hours after transfection, centrifuged at 1500 rpm for 5 minutes to remove cell debris, and then stored at -80 ° C. The pseudovirus reservoir was titrated by infecting 293T-ACE2 cells, and the luciferase activity was determined using a microplate reader (TECAN, Spark) after a 44 to 48-hour incubation period at 37 ° C and 5% CO2 by adding the Bright-Glo luciferase detection system (Promega, E2650). The TCID of the pseudovirus was then calculated according to the Reed-Muench method (Quantification of SARS-CoV-2neutralizing antibody by a pseudotyped virus based assay. Nie J. et al. DOI: 10.21203 / rs.3.pex-941 / v11) 50 .

[0564] Example 3: Neutralization test

[0565] Aliquots of the test serum samples were first heat inactivated at 56°C for 30 minutes and then clarified by centrifugation at 10,000 rcf for 5 minutes. The samples were serially diluted (3-fold) with the assay medium (100 ml) and diluted with 650 TCID 50The pseudovirus (50 ml) was incubated at 37 ° C for 1 hour, along with virus-infected untreated controls (virus alone) and cells alone (background control). Fresh trypsinized 293T-ACE2 cells were then added to each well at 20,000 cells / well in 100 mcL. After incubation at 37 ° C in a 5% CO2 incubator for 44 to 48 hours, the cells were lysed according to the manufacturer's protocol and luciferase activity was determined by the Bright-Glo luciferase assay system (Promega). The IC of a given serum sample was 24.5 mmol / l. 50 Neutralizing antibody titers were defined as the serum dilution at which the sample showed a 50% reduction in relative light units (RLU) compared to virus-infected control wells. The detailed method was based on the Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay reported by Nie J. et al. DOI: 10.21203 / rs.3.pex-941 / v11.

[0566] result

[0567] The test results show that the ACE2-Fc fusion protein disclosed in this disclosure has neutralizing activity against all current new coronavirus mutant strains, and its neutralizing activity against mutant strains is higher than that against the wild-type original strain.

[0568] Example 4: Neutralization activity detection of ACE2-Fc fusion protein with currently available SARS-CoV-2 mutants and original strain pseudovirus

[0569] The test results of Figures 5A-5C show that the ACE2-Fc fusion protein has neutralizing activity against all current novel coronavirus mutants, and the neutralizing activity against mutants is higher than that against the original strain. The experimental results confirm that the selection of ACE2-Fc fusion protein as a biological protective barrier against novel coronavirus infection can cope with any novel coronavirus mutant strain. Regardless of the mutation site of the novel coronavirus mutant strain, its mechanism of invading the human body has not changed fundamentally. The ACE2-Fc fusion protein has high neutralizing activity against any novel coronavirus mutant strain, including known and unknown novel coronavirus mutant strains. This also shows that the ACE2-FC fusion protein disclosed herein may be used to prevent the infection and spread of the novel coronavirus, and can also be used for therapeutic purposes.

[0570] Example 5: Nasal administration of the drug in transgenic mice (pharmacological experiment)

[0571] As shown in Figures 6A-6B, a delta challenge test was conducted in transgenic mice carrying human ACE2. The 40 mice participating in the experiment were divided into 4 groups, with 10 mice in each group. On the third day after the challenge, samples were taken to measure body weight, lung viral load, and lung pathology analysis and scoring. The first group was a control group, in which normal saline / vehicle was administered to mice via nasal spray; the second group was administered 50 μl / nostril ACE2-Fc fusion protein (SCB-719) via nasal spray at a dose of 50 μg / nostril; the third group was administered 50 μl / nostril ACE2-Fc fusion protein (SCB-719) via nasal spray at a dose of 500 μg / nostril; and the fourth group was administered 0.2 ml ACE2-Fc fusion protein (SCB-719) via intraperitoneal injection at a dose of 1000 μg / mouse.

[0572] The test results showed that the experimental group using nasal administration had a lower lung viral load than the intraperitoneal administration group. The lung viral load of both experimental groups given ACE2-Fc fusion protein was significantly lower than that of the non-administered group. Among them, the live lung viral load of the 50μg administration group was below the detection limit. Specific test results are shown in Figures 7A-7B. The experimental results show that nasal administration can effectively block the invasion of the new coronavirus into the body and is superior to systemic administration (intraperitoneal injection, i.p.), proving the feasibility of practical application of ACE2-Fc fusion protein.

[0573] Example 6: Evaluation of distribution in mice after nasal administration

[0574] Thirty-two all-female experimental animals were divided into vehicle B2 group, test substance B1 group, and test substance S4 group according to body weight. Among them, the protein (SCB-719) administered to the animals in groups B2 (2 animals), B1 (3 animals), and S4 (27 animals) was labeled with AF750 fluorescein at a protein content of 6 mg / ml.

[0575] Specific dosing information is shown in Table 3.

[0576] Table 3. Dosage information Note: The first digit of the animal number represents the group (1, 2, and 3 represent vehicle B2 group, test substance B1 group, and test substance S4 group, respectively). The second letter represents the sex (F represents female). The third and fourth digits represent the cage number within the group. The fifth and sixth digits represent the animal number within the cage within the group.

[0577] All animals in each group were tested at the time points of 0 + 5 min, 1 ± 0.1, ± 0.1, 4 ± 0.1, 6 ± 0.1, 8 ± 0.1, 12 ± 0.1, 16 ± 0.1 and 24 ± 0.1 h after administration. The specific testing time points are shown in Table 3.

[0578] Detection methods and indicators: After anesthesia, the animals in each group were anesthetized and the distribution and attenuation of the test substance in the body of the animals at different time points before and after administration were evaluated using a live imaging system. The p / s / cm 2 After each time point, the luminescence signal intensity of the main organs (heart, liver, spleen, lungs, kidneys, brain, nasal cavity), and blood (2 μL) were measured immediately after the test. The maximum excitation wavelength of AF750 fluorescein is 753 nm, and the maximum emission wavelength is 782 nm. Each mouse was tested three times in total.

[0579] After a single intranasal administration, no fluorescent signal was observed for the unlabeled ACE2-Fc fusion protein (test group B1) and the solvent B2. AF750 fluorescein-labeled ACE2-Fc fusion protein (test group S4) was enriched in the nose, and a fluorescent signal was clearly observed 0-4 hours after administration. Figure 8A shows that fluorescent signals of ACE2-Fc fusion protein were observed in the nasal cavity at different time points, while there were no fluorescent signals in other organs, indicating that ACE2-Fc fusion protein was enriched in the nasal cavity. After a single intranasal administration, a fluorescent signal was clearly detected in the 0-6 hours, the fluorescent signal was significantly weakened in the 6-8 hours but the signal could be detected, and the fluorescent signal could still be detected in the 12 hours. The imaging of each organ is shown in Figure 8A, and the trend of changes in the intensity of the nasal fluorescence signal is shown in Figure 8B.

[0580] In summary, ACE2-Fc fusion protein can accumulate in the mouse nasal cavity for more than 12 hours. This result demonstrates that ACE2-Fc fusion protein can remain at the administration site for a long time, proving that the tissue half-life of ACE2-Fc fusion protein supports its nasal administration for protection against the new coronavirus.

[0581] Figure 9 exemplifies the ACE2-Fc fusion protein (IV / IP) PK and in vitro efficacy curves. ACE2-Fc fusion protein was administered to female BALB / c mice aged 6-8 weeks by intravenous or intraperitoneal injection at a dose of 50 mg / kg in a volume of 0.2 mL / mouse. There were 3 mice in each group. After a single dose, about 50 μL of whole blood was collected from the mice at specific time points (5 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 120 h). After standing at room temperature for 1 h, serum was separated for blood drug concentration detection. Drug concentrations in serum were determined using an enzyme-linked immunosorbent assay (ELISA). Mouse anti-human Fc protein was immobilized in a 96-well microplate. ACE2-Fc fusion protein in the test serum was captured by the mouse anti-human Fc. Biotin-labeled ACE2-Fc fusion protein (SCB-2019-biotin) was then added to bind to the ACE2-Fc fusion protein. Finally, HRP-labeled strepavidin was added to bind to the biotin. HRP catalyzed a color reaction with TMB substrate. The reaction was terminated with 1M sulfuric acid solution, and the readings were taken on a microplate reader at a wavelength of 450 nm. The theoretical concentration of ACE2-Fc fusion protein was plotted as the horizontal axis, and the measured OD value as the vertical axis. The ACE2-Fc fusion protein content in the test samples was calculated using a four-parameter regression model to fit the relevant parameters of the standard curve. The quantitative range of this method was 3-100 ng / mL. The figure shows the changes in blood drug concentration over time. The mean drug concentration (mean) and standard error (SEM) of three mice are shown at each time point.

[0582] The results showed that at a dose of 50 mg / kg, the half-life was 38.0 hours and the AUC was 10454 h*μg / mL when administered via intravenous injection, while the half-life was 24.6 hours and the AUC was 10127 h*μg / mL when administered via intraperitoneal injection. The drug exposure and half-life were comparable for both routes of administration. This also suggests that the ACE2-Fc fusion protein disclosed herein may be used to prevent infection and transmission of the novel coronavirus, as well as for therapeutic purposes.

[0583] The present invention is not intended to be limited in scope to specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the present invention. Through the description and teachings of the present disclosure, various modifications of the compositions and methods will become apparent. These modifications may be implemented without departing from the true scope and spirit of the present invention, and are intended to fall within the scope of the present invention.

Claims

1. A fusion protein comprising multiple recombinant polypeptides, wherein the fusion protein comprises human ACE2 protein or a fragment thereof and human IgG Fc protein or a functional variant thereof.

2. The fusion protein according to claim 1, wherein the human ACE2 protein or a fragment thereof comprises a human ACE2 extracellular domain or a fragment thereof.

3. The fusion protein according to claim 1 or 2, wherein the human ACE2 protein or a fragment thereof comprises an amino acid sequence as described in any one of SEQ ID NOs: 5, 7, 11, 12, 13 and 14, or an amino acid sequence or a fragment thereof having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

4. The fusion protein according to any one of claims 1 to 3, wherein the human ACE2 protein or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence or a fragment thereof having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The fusion protein according to any one of claims 1 to 4, wherein the human IgG Fc protein or a functional variant thereof comprises any one or more Fc region sequences selected from human IgG1 Fc, IgG2 Fc, IgG3 Fc and IgG4 Fc, or functional variants and fragments thereof. The fusion protein according to any one of claims 1 to 5, wherein the human IgG Fc protein or a functional variant thereof is selected from a human IgG1 Fc region sequence or a functional variant and fragment thereof.

7. The fusion protein according to any one of claims 1 to 6, wherein the human IgG Fc protein or a functional variant thereof comprises the amino acid sequence of SEQ ID NO: 6 or 8 or an amino acid sequence or a fragment thereof having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

8. The fusion protein according to any one of claims 1 to 7, wherein the human IgG Fc protein or a functional variant thereof comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto or a fragment thereof.

9. The fusion protein according to any one of claims 1 to 8, optionally comprising a signal peptide.

10. The fusion protein according to any one of claims 1 to 9, optionally comprising a peptide linker, wherein The human ACE2 protein or its fragment and the human IgG Fc protein or its functional variant are directly connected or connected through a peptide linker. The fusion protein according to claim 10 , wherein the peptide linker is selected from the group consisting of an arginine-serine peptide linker (-RS-), a valine-serine linker (-VS-), and a glycine-serine linker (-GS-).

12. The fusion protein according to any one of claims 1 to 11, optionally comprising a mutant sequence.

13. The fusion protein of claim 1, comprising an amino acid sequence of any one of SEQ ID NOs: 1-18, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a fragment thereof, or a combination thereof.

14. The fusion protein of claim 1, comprising an amino acid sequence of any one of SEQ ID NOs: 1-4 and 15-18, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a fragment thereof, such as an amino acid sequence of any one of SEQ ID NOs: 1-4 or 15-18, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a fragment thereof.

15. The fusion protein according to any one of claims 1 to 14, further comprising a detectable label.

16. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 15 and an optional pharmaceutically acceptable carrier.

17. A pharmaceutical composition according to claim 16, comprising about 0.1 mg / ml to about 100 mg / ml of the fusion protein according to any one of claims 1 to 15, such as about 0.50 mg / ml to about 20.00 mg / ml, such as about 1.25 mg / ml, about 2.50 mg / ml, or about 5.00 mg / ml.

18. The pharmaceutical composition according to claim 16 or 17, wherein the pharmaceutically acceptable carrier comprises a combination of one or more of a buffer and an osmotic pressure regulator.

19. The pharmaceutical composition of claim 18, wherein the buffer comprises sodium phosphate monobasic monohydrate, sodium phosphate dibasic dihydrate, or a combination thereof.

20. The pharmaceutical composition according to claim 18 or 19, wherein the osmotic pressure regulator comprises a combination of one or more selected from the group consisting of sodium chloride, potassium chloride, glycerol, glucose, sorbitol, sucrose, xylitol and mannitol.

21. The pharmaceutical composition according to any one of claims 18 to 20, wherein the osmotic pressure regulator comprises sodium chloride and / or sucrose.

22. A pharmaceutical composition according to any one of claims 18 to 21, wherein the pharmaceutically acceptable carrier arbitrarily comprises a stabilizer, the stabilizer comprising a combination of one or more selected from the group consisting of proteins, peptides or hydrolysates such as albumin, gelatin; sugars such as sucrose, lactose, sorbitol; and amino acids such as sodium glutamate, for example, the stabilizer comprises sucrose.

23. A pharmaceutical composition according to any one of claims 18 to 22, wherein the pharmaceutically acceptable carrier optionally comprises an antibacterial agent, the antibacterial agent comprising a combination of one or more selected from the group consisting of benzoic acid, sorbic acid, thimerosal and phenylethyl alcohol, for example, the antibacterial agent comprises phenylethyl alcohol and / or thimerosal.

24. A pharmaceutical composition according to any one of claims 18 to 23, which is in a dosage form suitable for administration by intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intranasal, sublingual, tonsil, oropharyngeal or other parenteral and mucosal routes, preferably in a dosage form suitable for intranasal administration, such as a nasal spray.

25. The fusion protein according to any one of claims 1 to 15 or the pharmaceutical composition according to any one of claims 16 to 24 is used for preventing and treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects.

26. A fusion protein according to any one of claims 1 to 15 or a pharmaceutical composition according to any one of claims 16 to 24 for preventing, treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects according to claim 25, wherein the coronavirus SARS-CoV-2 and its variants include but are not limited to SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Mu, Omicron, JN.1; and unknown coronavirus SARS-CoV-2 variants.

27. A fusion protein according to any one of claims 1 to 15 or a pharmaceutical composition according to any one of claims 16 to 24 for preventing, treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects according to claim 25 or 26, wherein the fusion protein or pharmaceutical composition is administered intramuscularly, intradermally, subcutaneously, intravenously, intraarterially, intraarticularly, intraperitoneally, intranasally, sublingually, tonsilally, oropharyngeally or other parenteral and mucosal routes, preferably intranasally.

28. A fusion protein according to any one of claims 1 to 15 or a pharmaceutical composition according to any one of claims 16 to 24 for preventing, treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects according to any one of claims 25 to 27, wherein the fusion protein or pharmaceutical composition is administered intranasally in a single dose or a series of doses separated by one or more intervals, and the intervals are measured in hours, days or weeks.

29. A fusion protein according to any one of claims 1 to 15 or a pharmaceutical composition according to any one of claims 16 to 24 for preventing, treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects according to claim 28, wherein the interval is 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours.

30. A fusion protein according to any one of claims 1 to 15 or a pharmaceutical composition according to any one of claims 16 to 24 for preventing, treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects according to any one of claims 25 to 29, wherein the fusion protein or pharmaceutical composition is administered once or more per day, for example, once per day or twice per day.

31. Use of the fusion protein according to any one of claims 1 to 15 or the pharmaceutical composition according to any one of claims 16 to 24 in the preparation of a medicament for preventing, treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects.

32. The use according to claim 31, wherein the coronavirus SARS-CoV-2 and its variants include but are not limited to SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Miu, Omicron, JN.1; and unknown coronavirus SARS-CoV-2 variants.

33. A method for preventing and treating infection by coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in an infected subject, comprising administering to the subject a therapeutically effective amount of a fusion protein according to any one of claims 1 to 15 or a pharmaceutical composition according to any one of claims 16 to 24.

34. The method of claim 33, wherein the coronavirus SARS-CoV-2 and its variants include but are not limited to SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Mu, Omicron, JN.1; and unknown coronavirus SARS-CoV-2 variants.

35. The method of claim 33 or 34, wherein the fusion protein or pharmaceutical composition is administered via intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intranasal, sublingual, tonsil, oropharyngeal or other parenteral and mucosal routes, preferably intranasally.

36. The method according to any one of claims 33 to 35, wherein the fusion protein according to any one of claims 1 to 15 or the pharmaceutical composition according to any one of claims 16 to 24 is administered intranasally in a single dose or a series of doses separated by one or more intervals, the intervals being measured in hours, days or weeks.

37. The method of claim 36, wherein the interval period is 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours.

38. The method according to any one of claims 33 to 37, wherein the fusion protein or pharmaceutical composition is administered once or more times per day, for example once or twice per day.

39. A kit for preventing and treating infection with coronavirus SARS-CoV-2 and its variants and / or preventing the spread of coronavirus SARS-CoV-2 and its variants in infected subjects, comprising a fusion protein according to any one of claims 1 to 15 or a pharmaceutical composition according to any one of claims 16 to 24; Container; and Optional package insert or label indicating prevention and / or treatment.

40. A kit according to claim 39, wherein the coronavirus SARS-CoV-2 and its variants include but are not limited to SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Mu, Omicron, JN.1; and unknown variants of the coronavirus SARS-CoV-2.

41. A nucleic acid encoding the fusion protein or fragment thereof according to any one of claims 1 to 15.

42. A vector comprising the nucleic acid according to claim 41.

43. The vector according to claim 33, which is phFC(IM).

44. A host cell comprising the nucleic acid of claim 41 or the vector of claim 42.

45. The host cell according to claim 44, which is a CHO cell, such as GH-CHO.