Nasal spray for preventing COVID-19 infection and its manufacture and use

Abstract

This disclosure relates to a fully human-derived ACE2-Fc fusion protein, pharmaceutical compositions, formulations, and kits containing the same (for example, a nasal spray containing the fusion protein). Furthermore, this disclosure relates to the application of the fusion protein in the widespread prevention of coronavirus infections, for example, SARS-CoV-2 coronavirus and its known and unknown variants (including, but not limited to, prototype Hu-1, alpha, beta, gamma, delta, mu, omicron, JN.1, and / or other potentially emerging variants). This disclosure also relates to a method for producing the fusion protein and a method for preventing and / or treating infection with SARS-CoV-2 coronavirus and its known and unknown variants using the fusion protein. Furthermore, this disclosure relates to the fusion protein and its pharmaceutical compositions and formulations for preventing the transmission of SARS-CoV-2 coronavirus and its known and unknown variants in previously infected subjects.

Description

Technical Field

[0001] The present disclosure belongs to the field of biopharmaceuticals, and specifically relates to a fully human-derived ACE2-Fc fusion protein, a pharmaceutical composition, a formulation, and a kit (for example, a nasal spray containing the fusion protein) for the prevention and treatment of coronavirus infection and the prevention of the spread of coronavirus. The present disclosure further relates to a method for producing the fusion protein for preventing and / or treating infection by coronavirus SARS-CoV-2 and its known and unknown variants, and for preventing the spread of coronavirus SARS-CoV-2 and its known and unknown variants in infected subjects.

Background Art

[0002] Coronaviruses infect mammals including various birds and humans. Coronaviruses can spread annually in the human population and usually cause mild respiratory diseases, but tend to cause severe diseases in infants, the elderly, and people with weakened immune functions. However, some coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have high pathogenicity. Furthermore, the novel coronavirus has a high mutation rate, and since the end of 2020, multiple mutant SARS-CoV-2 strains (VOCs) with mutations that may lead to immune escape have begun to emerge. However, due to the long cycle from the research and development of ordinary vaccines to their market launch, it is completely insufficient to cope with the threats posed by the novel coronavirus to work, health, and global infection prevention. Therefore, emergency response facilities with broad protective effects play an important role in reducing people's infection risks, alleviating the pressure of infection prevention measures, and revitalizing social life. The present disclosure provides methods, applications, and articles of manufacture that meet the above and other requirements.

[0003] This application claims priority to Chinese Patent Application No. 202310217327.3, filed on March 4, 2023, all disclosures thereof are incorporated herein by reference. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Amid a global surge in COVID-19 cases, several new SARS-CoV-2 variants (VOCs) have emerged since the end of 2020. These VOCs may be associated with mutations in the spike (S) protein, which could lead to increased viral transmission and / or evasion of immunity from first-generation COVID-19 vaccination based on the SARS-CoV-2 Hu-1 strain (also known as the SARS-CoV-2 prototype or SARS-CoV-2 wild-type). The B.1.1.7 variant was classified as a VOC after it emerged and spread in the UK, the B.1.351 variant in South Africa, and the P.1 variant in Brazil. All of these VOCs contain the N501Y mutation in the receptor-binding domain (RBD) of the spike protein, and this mutation has been reported to increase transmissibility by 40-70%. Furthermore, the B.1.351 and P.1 mutant strains also possess two additional RBD mutations, E484K and K417, which may enable immune evasion from Hu-1 vaccines or antibodies induced by natural infection.

[0005] Randomized controlled trials of COVID-19 vaccines have shown decreased vaccine efficacy against VOCs compared to the SARS-CoV-2 Hu-1 strain. The adjuvant protein-based COVID-19 vaccine NVX-CoV2373 had an 89% efficacy rate in the UK (B.1.1.7 is dominant), but this dropped to only 49% in South Africa (B.1.351 is dominant). The adenovirus-carrier COVID-19 vaccine ChAdOx1 had a mere 10% efficacy rate against the B.1.351 variant. The efficacy rate against the B.1.351 variant was 75% in recipients of the Pfizer vaccine, compared to 95% against Hu-1. No neutralizing antibody titers against the P.1 variant were detected in subjects who received the Hu-1 strain-based inactivated vaccine Coronavac.

[0006] While there is promising evidence that the Hu-1 COVID-19 vaccine may prevent severe illness or death from VOCs, the low effectiveness of the vaccine against COVID-19 infections with increased transmissibility may make achieving herd immunity particularly difficult. If effective measures are not taken, the rapid global spread of SARS-CoV-2 VOCs could lead to the continued emergence of new target variants or new escape mutations in VOCs, such as the Indian variant (B.1.617). The Indian variant (B.1.617) emerged concurrently with the large surge in COVID-19 cases in the spring of 2021 and was recognized as a new VOC by the World Health Organization. The B.1.617.2 variant, classified under the B.1.617 lineage, was named Delta. Furthermore, the B.1.1.529 variant, first identified in South Africa, was named Omicron.

[0007] Under these circumstances, it is necessary to rapidly evaluate other infection prevention methods that can broadly address VOCs. [Means for solving the problem]

[0008] This disclosure provides a fusion protein comprising a plurality of recombinant polypeptides. The fusion protein comprises a human ACE2 protein or a fragment thereof and a human IgG Fc protein or a functional variant thereof. The fusion protein specifically binds to wild-type, mutant, or mutant human ACE2 receptors via one or more binding sites.

[0009] In some embodiments, the fusion protein is a water-soluble protein.

[0010] By utilizing fully human-derived antibody Fc fusion protein technology, the expression of soluble proteins under serum-free culture conditions can be significantly improved, resulting in advantages such as easier affinity purification and enhanced in vitro / in vivo stability of the ACE2-Fc protein. Since ACE2-Fc is a fully human-derived fusion protein and preferably achieves passive immunization via intranasal administration, high safety is expected.

[0011] In some embodiments, the human ACE2 protein or a fragment thereof includes the human ACE2 extracellular domain or a fragment thereof.

[0012] In some embodiments, the human ACE2 protein or fragment thereof comprises an amino acid sequence shown in any of SEQ ID NO: 5, 7, 11, 12, 13, and 14, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0013] In some embodiments, the human ACE2 protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0014] In some embodiments, the human IgG Fc protein or its functional variant comprises one or more Fc region sequences or functional variants and fragments selected from human IgG1 Fc, IgG2 Fc, IgG3 Fc, and IgG4 Fc.

[0015] In some embodiments, the human IgG Fc protein or its functional variant is selected from human IgG1 Fc region sequences or their functional variants and fragments.

[0016] In some embodiments, the human IgG Fc protein or its functional variant comprises an amino acid sequence shown in SEQ ID NO: 6 or 8, or a fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

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

[0018] In some embodiments, the fusion protein optionally includes a signal peptide.

[0019] In some embodiments, the fusion protein optionally includes a peptide linker, where the human ACE2 protein or a fragment thereof is directly linked to the human IgG Fc protein or a functional variant thereof, or linked via the peptide linker. For example, the peptide linker is selected from arginine-serine peptide linkers (-RS-), valine-serine linkers (-VS-), and glycine-serine linkers (-GS-). The peptide linker may be a common peptide linker known in the art for linking different functional polypeptide moieties in a fusion protein.

[0020] In some embodiments, the fusion protein optionally includes a mutant sequence. The mutant sequence is used for C-terminal modification to facilitate tracking and detection.

[0021] In some embodiments, the fusion protein includes an amino acid sequence shown in any of SEQ ID NO: 1-18, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a combination thereof, for example, an amino acid sequence shown in any of SEQ ID NO: 1-4 or 15-18, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0022] In some embodiments, the fusion protein further includes a detectable tag.

[0023] On the other hand, the present disclosure provides a pharmaceutical composition comprising the fusion protein of the present disclosure and an optional pharmaceutically acceptable carrier.

[0024] In some embodiments, the pharmaceutical composition contains the fusion protein according to any one of claims 1 to 15 at about 0.1 mg / ml to about 100 mg / ml, for example, at about 0.50 mg / ml to about 20.00 mg / ml, about 1.25 mg / ml, about 2.50 mg / ml, or about 5.00 mg / ml.

[0025] In some embodiments, the pharmaceutical composition is in a dosage form applicable to intramuscular, intradermal, subcutaneous, intravenous, intra - arterial, intra - articular, intra - peritoneal, nasal, sublingual, tonsillar, oral - pharyngeal, or other parenteral or mucosal administration routes, preferably a dosage form suitable for nasal administration such as a nasal spray.

[0026] On the one hand, the present disclosure discloses the fusion protein or pharmaceutical composition of the present disclosure for the prevention and treatment of infections caused by the coronavirus SARS - CoV - 2 and its variants, and / or for preventing the transmission of the coronavirus SARS - CoV - 2 and its variants in already - infected subjects.

[0027] On the one hand, the present disclosure discloses the application of the fusion protein or pharmaceutical composition of the present disclosure in the manufacture of a medicament for the prevention and treatment of infections caused by the coronavirus SARS - CoV - 2 and its variants, and / or for preventing the transmission of the coronavirus SARS - CoV - 2 and its variants in already - infected subjects.

[0028] On the one hand, the present disclosure provides a method for the prevention and treatment of infections caused by the coronavirus SARS - CoV - 2 and its variants, and / or for preventing the transmission of the coronavirus SARS - CoV - 2 and its variants in already - infected subjects, the method comprising administering to a subject a therapeutically effective amount of the fusion protein or pharmaceutical composition of the present disclosure.

[0029] In some embodiments, the administration is nasal administration.

[0030] On the one hand, this disclosure provides a kit for the prevention and treatment of infection with coronavirus SARS-CoV-2 and its variants, and / or for preventing the transmission of coronavirus SARS-CoV-2 and its variants in a previously infected subject, the kit comprising the fusion protein or pharmaceutical composition of this disclosure, Container and Optionally, include package inserts or labels indicating prevention and / or treatment.

[0031] In this disclosure, the coronavirus SARS-CoV-2 and its variants are not limited to SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, mu, omicron, and JN.1, but also include variants of unknown coronavirus SARS-CoV-2.

[0032] On the one hand, the Disclosure provides nucleic acids for encoding the fusion protein or a fragment thereof, a carrier containing the nucleic acid, and a host cell containing the nucleic acid or the carrier.

[0033] This disclosure provides a fully human-derived ACE2-Fc fusion protein for the prevention and treatment of coronavirus infection and for preventing the transmission of coronavirus, as well as pharmaceutical compositions, formulations, and kits containing the same, and methods for manufacturing and using the same. In some embodiments, nasal sprays containing ACE2-Fc fusion protein have shown broad-spectrum coronavirus infection prevention effects. Specifically, by applying the ACE2-Fc fusion protein receptor to the entire nasal mucosa, the binding of the virus to ACE2 on the surface of nasal mucosal epithelial cells can be efficiently and competitively inhibited, effectively suppressing viral entry. This is particularly effective for reducing the risk of infection in high-risk groups (healthcare workers, immigration personnel, etc.) and in crowded, enclosed, and poorly ventilated environments (airplanes, high-speed trains, and other public transportation). Furthermore, the ACE2-Fc fusion protein can prevent transmission of the virus through exsanguination, such as sneezing or saliva, by binding to the virus within an infected individual, particularly preventing transmission within the household. Moreover, the ACE2-Fc fusion protein exerts its therapeutic effect by binding to the virus within the infected individual and reducing the number of viruses in the body. This therapeutic effect can be achieved through both intranasal and systemic administration.

[0034] Currently, the number of COVID-19 cases worldwide exceeds 500 million, and the number of deaths exceeds 6 million. Further concern is the emergence of even more infectious variants, such as the Omicron strain, since the end of 2021, following infections caused by the Delta strain in India and variants in South Africa and Brazil. These variants not only increase infection rates but also acquire immunity evasion against commercially available vaccines and neutralizing antibodies, forcing countries worldwide to implement various countermeasures. However, with the mutation of Omicron BA.1, even more infectious variants such as BA.2, BA.2.12.1, BA.4 and BA.5, XBB, EG.5, and JN.1 are continuously appearing. Therefore, there is an urgent need for new, effective, and safe strategies globally to address the rapid mutation and high immunity evasion of the coronavirus. Given that vaccine research and development for COVID-19 variants is currently unable to keep pace with the mutations, it is essential to develop non-vaccine-based means to prevent infection by any COVID-19 variant.

[0035] The ACE2-Fc fusion protein disclosed herein is proposed as a powerful complementary agent to address shortcomings in existing vaccine development. It is expected to effectively mitigate the current global spread of COVID-19 by solving problems such as the rapid mutation, high immune evasion, and strong infectivity of the virus.

[0036] As a technology to solve the problem, the first important question is how the ACE2-Fc fusion protein, as a receptor capable of binding to all SARS-CoV-2 variants, can be applied to defense against SARS-CoV-2 variants. ACE2 is an essential receptor for SARS-CoV-2 to enter host cells, and due to the virus's constant mutations and evasion of vaccines and neutralizing antibodies, the protective effects of vaccines and therapeutic monoclonal antibodies are gradually weakening. However, the receptor through which the virus enters host cells does not constantly change, and research shows that the stronger the infectivity, the higher the affinity of the strain for the ACE2 receptor. Therefore, by using the ACE2-Fc fusion protein as a soluble receptor to block viral entry, it is possible to effectively deal with all current and future SARS-CoV-2 variants. This is determined by the fundamental mechanism of SARS-CoV-2 infection. On the other hand, existing monoclonal antibodies may lose their protective power against current and future variants. Next, we need to consider the practicality of using the ACE2-Fc fusion protein as a nasal spray for daily use, specifically application issues related to storage and usage temperatures.

[0037] The nasal cavity is a major entry point for the novel coronavirus, as it can enter the human body and cause infection via the nasal mucosa of the upper respiratory tract. By applying ACE2-Fc soluble receptors, which efficiently and competitively inhibit the binding of the virus to ACE2 on the surface of nasal mucosal epithelial cells, to the entire nasal mucosa via intranasal administration, viral entry can be effectively suppressed. This approach of passive immunity with ACE2-Fc soluble receptors via intranasal administration provides flexible, rapid, and effective protection against viral entry, especially when existing vaccines have limited protective effects against new variants. It is particularly effective in reducing the risk of infection in high-risk groups (healthcare workers, immigration personnel, etc.) and in crowded, enclosed, and poorly ventilated environments (airplanes, high-speed trains, and other public transportation). [Brief explanation of the drawing]

[0038] [Figure 1]This shows the mechanism by which the SARS-CoV-2 virus infects the host via the ACE2 receptor. Figures 2A to 2H show the affinity measurements of the ACE2-Fc fusion protein (SCB-719) with spike proteins from various different strains. [Figure 2A] The results of affinity measurements with the wild-type (Hu-1) spike protein are shown. [Figure 2B] The affinity measurement results for the Beta mutant spike protein are shown. [Figure 2C] The affinity measurement results for the Delta mutant spike protein are shown. [Figure 2D] The affinity measurement results for the Omicron mutant (BA.1) spike protein are shown. [Figure 2E] The affinity measurement results for the Omicron mutant (BA 4 / 5) spike protein are shown. [Figure 2F] The affinity measurement results for the Omicron mutant (XBB1.5) spike protein are shown. [Figure 2G] The affinity measurement results for the Omicron mutant (EG5.1) spike protein are shown. [Figure 2H] The affinity measurement results for the JN.1 mutant spike protein are shown. Figures 3A to 3D illustrate the principle of preventing SARS-CoV-2 virus infection using a nasal spray with an ACE2-Fc fusion protein. [Figure 3A] This shows how a nasal spray containing an ACE2-Fc fusion protein is sprayed into the nasal cavity. [Figure 3B] This shows how viruses or virus-containing droplets transmitted through the air enter the nasal cavity. [Figure 3C] This demonstration shows how a soluble ACE2-Fc fusion protein nasal spray, developed using Fc fusion protein technology, covers the upper respiratory tract mucosa, primarily the nasal cavity, when sprayed into the nasal cavity, forming a first line of defense against viral invasion as an "invisible mask with biological function." [Figure 3D]This illustrates the process by which the SARS-CoV-2 virus enters a cell and the process by which the ACE2-Fc fusion protein prevents the virus from entering the cell. When transmissible viruses or virus-containing droplets are present in the air, the virus primarily infects upper respiratory tract cells. In this case, the ACE2-Fc fusion protein distributed in the nasal cavity and upper respiratory tract competitively binds to any mutant strain of the SARS-CoV-2 virus, blocking the site where the virus's spike protein binds to host cells, thereby preventing the virus from entering host cells via the spike protein and infecting the human body. Figures 4A to 4C show the purification and characterization of the ACE2-Fc fusion protein according to the examples of this disclosure. [Figure 4A] This figure shows the detection of ACE2-Fc fusion protein expression levels in CHO. [Figure 4B] This figure shows the affinity purification of ACE2-Fc fusion proteins. [Figure 4C] This figure shows the detection of the purity of the ACE2-Fc fusion protein. Figures 5A to 5C exemplify the detection of the neutralizing activity of the ACE2-Fc fusion protein according to the examples of this disclosure against pseudoviruses of the original and mutant strains of the novel coronavirus. [Figure 5A] The mutant strains being detected include alpha, beta, gamma, delta, and omicron strains. [Figure 5B] The detected variants 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 neutralizing activity (neutralizing activity of the mutant against pseudovirus / neutralizing activity of the original strain against pseudovirus) is shown. As shown in Figures 5A-5C, the ACE2-Fc fusion protein disclosed herein has neutralizing activity against all currently known mutant strains of pseudovirus, and exhibits higher neutralizing activity against mutant strains than against the original strain. Figures 6A-6B show the design of the challenge trial. [Figure 6A]This report describes a delta challenge test using genetically modified mice expressing human ACE2. In this test, 40 mice were divided into four groups of 10 mice each. Weight measurement, lung viral load measurement, lung pathological analysis, and scoring were performed on 3 days (5 mice) and 10 days (5 mice) after infection. [Figure 6B] Group 1 was the control group, in which mice were administered physiological saline / excipient via nasal spray. Group 2 was in which mice were administered 0.1 ml of ACE2-Fc fusion protein at a dose of 5 mg / kg via nasal spray. Group 3 was in which mice were administered 0.1 ml of ACE2-Fc fusion protein at a dose of 50 mg / kg via nasal spray. Group 4 was in which mice were administered 0.2 ml of ACE2-Fc fusion protein at a dose of 50 mg / kg via intraperitoneal injection. Figures 7A and 7B exemplify the results of challenge tests (pharmacodynamic tests) using genetically modified mice with nasal and systemic administration. "in" indicates intranasal administration, and "ip" indicates systemic administration. [Figure 7A] The results of the live viral load test in the lungs are shown. [Figure 7B] The results of tests on the amount of viral (RNA) in the lungs are shown. [Figure 8A] Exemplary images (n=3,2) of the organ distribution of test substance B1, solvent B2, and test substance S4 at different time points are shown. Here, A: liver, B: spleen, C: kidney, D: heart, E: lung, F: brain, G: blood, H: nasal cavity. [Figure 8B] The fluorescence signal intensity in the nasal cavity of the test substance S4 at each time point ((p / s / cm2 / sr) / (μW / cm2), Mean±SEM, n=3) is shown. [Figure 9] The ACE2-Fc fusion protein (IV / IP) PK and in vitro efficacy curves are shown as examples. [Modes for carrying out the invention]

[0039] Figure 1 illustrates the principle by which the ACE2-Fc fusion protein solves problems such as the rapid mutation rate of the novel coronavirus, the high transmissibility of mutant strains, and the inability of vaccines to keep up with the mutations. ACE2 has been identified as a functional host receptor for severe acute respiratory syndrome coronavirus (SARS-CoV-2), and the mechanism by which the SARS-CoV-2 virus infects the host via the ACE2 receptor is shown in Figure 1. It is clear that the receptor through which the virus enters host cells remains constant, regardless of how the virus mutates or how immune evasion from vaccines or neutralizing antibodies occurs. Furthermore, studies have demonstrated that the stronger the infectivity, the higher the affinity of the strain for the receptor. For this reason, the ACE2 receptor can serve as a basis for inhibiting the infection of host cells by any SARS-CoV-2 mutant virus.

[0040] Figures 3A-3D exemplify the principle by which ACE2-Fc inhibits SARS-CoV-2 virus infection. By developing a soluble human ACE2-Fc fusion protein using Fc fusion protein technology and formulating it into a nasal spray, the ACE2-Fc fusion protein is administered intranasally to the upper respiratory tract, primarily the nasal cavity, forming a first line of defense against viral invasion as an "invisible mask with biological function." When transmissible viruses or virus-containing droplets are present in the air, the virus primarily infects through upper respiratory tract cells. In this case, ACE2-Fc fusion proteins distributed in the nasal cavity and upper respiratory tract competitively bind to any variant of the SARS-CoV-2 virus, blocking the site where the virus's spike protein binds to host cells, thereby preventing the virus from entering host cells via the spike protein and infecting the human body.

[0041] This disclosure provides a fusion protein comprising multiple recombinant polypeptides. The fusion protein comprises a human ACE2 protein or a fragment thereof and a human IgG Fc protein or a functional variant thereof. The fusion protein specifically binds to wild-type, mutant, or mutant human ACE2 receptors via one or more binding sites. It can also be used for the treatment of coronavirus infection (e.g., prophylactic or therapeutic). Methods of manufacturing and using the fusion protein are further disclosed in this disclosure. A nasal spray containing the ACE2-Fc fusion protein showed broad-spectrum effectiveness against coronavirus infection. Specifically, by applying the soluble ACE2-Fc fusion protein receptor to the entire nasal mucosa, the binding of the virus to ACE2 on the surface of nasal mucosal epithelial cells can be efficiently and competitively inhibited, effectively suppressing viral entry. Figures 2A-2H show that the ACE2-Fc fusion protein of this disclosure also has high affinity for various mutant spike proteins.

[0042] In some embodiments, the fusion protein described herein is a soluble protein comprising a human ACE2 protein or a fragment thereof and a human IgG Fc protein or a functional variant thereof.

[0043] The fusion protein specifically binds to wild-type, mutant, or mutant human ACE2 receptors via one or more binding sites.

[0044] In some embodiments, the fusion protein described herein includes a human ACE2 protein or a fragment thereof, which comprises a human ACE2 extracellular domain or a fragment thereof.

[0045] In some embodiments, the human ACE2 protein or fragment contained in the fusion protein described herein may be the wild-type human ACE2 extracellular domain or fragment thereof, or a human ACE2 extracellular domain variant or mutant or fragment thereof. Herein, the human ACE2 extracellular domain variant or mutant or fragment thereof only needs to retain the ability to bind to the wild-type, mutant, or mutant human ACE2 receptor.

[0046] In some embodiments, the human ACE2 protein or fragment contained in the fusion protein described herein includes an amino acid sequence shown in any of SEQ ID NO: 5, 7, 11, 12, 13, or 14, or an amino acid sequence or fragment having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0047] In some embodiments, the human ACE2 protein or fragment contained in the fusion protein described herein includes the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence or fragment having at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity thereto.

[0048] In some embodiments, the human IgG Fc protein or its functional variants contained in the fusion protein described herein include one or more Fc region sequences or functional variants and fragments selected from human IgG1 Fc, IgG2 Fc, IgG3 Fc, and IgG4 Fc.

[0049] In some embodiments, the human IgG Fc protein or a functional variant thereof contained in the fusion protein described herein is selected from human IgG1 Fc region sequences or their functional variants and fragments.

[0050] In some embodiments, the human IgG Fc protein or a functional variant thereof contained in the fusion protein described herein includes the amino acid sequence shown in SEQ ID NO: 6 or 8, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0051] In some embodiments, the human IgG Fc protein or a functional variant thereof contained in the fusion protein described herein includes the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0052] In some embodiments, the fusion protein optionally includes a signal peptide (e.g., the signal peptide shown in SEQ ID NO. 9).

[0053] In some embodiments, the fusion protein optionally includes a peptide linker, where the human ACE2 protein or a fragment thereof is directly bound to the human IgG Fc protein or a functional variant thereof, or is bound via the peptide linker.

[0054] In some embodiments, the fusion protein may optionally include a mutant sequence (e.g., the mutant sequence shown in SEQ ID NO. 10).

[0055] In some embodiments, the fusion protein described herein includes an amino acid sequence shown in any of SEQ ID NO: 1-18, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith, or a combination thereof, for example, an amino acid sequence shown in any of SEQ ID NO: 1-4 or 15-18, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[0056] In some embodiments, the fusion protein described herein further includes a detectable tag.

[0057] Furthermore, this disclosure provides a pharmaceutical composition comprising the fusion protein described herein and any pharmaceutically acceptable carrier.

[0058] In some embodiments, the pharmaceutical compositions described herein contain the fusion protein described in any one of claims 1 to 15 in an amount of about 0.1 mg / ml to about 100 mg / ml, for example, about 0.50 mg / ml to about 20.00 mg / ml, about 1.25 mg / ml, about 2.50 mg / ml, or about 5.00 mg / ml.

[0059] In some embodiments, the pharmaceutically acceptable carrier included in the pharmaceutical composition described herein is selected from one or more combinations of buffers and osmotic regulators.

[0060] In some embodiments, the buffering agent included in the pharmaceutical composition described herein is selected from sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dihydrate, or a combination thereof.

[0061] In some embodiments, the osmotic pressure modifier included in the pharmaceutical composition described herein is one or more combinations selected from the group consisting of sodium chloride, potassium chloride, glycerin, glucose, sorbitol, sucrose, xylitol, or mannitol.

[0062] In some embodiments, the osmotic pressure modifier included in the pharmaceutical composition described herein is sodium chloride and / or sucrose.

[0063] In some embodiments, the pharmaceutically acceptable carriers described herein optionally include a stabilizer, which is one or more combinations selected from the group consisting of proteins, peptides or their hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), and amino acids (e.g., monosodium glutamate), for example, sucrose.

[0064] In some embodiments, the pharmaceutically acceptable carrier described herein optionally comprises a bacteriostatic agent, which comprises one or more combinations selected from the group consisting of benzoic acid, sorbic acid, thimerosal, and phenethyl alcohol, for example, phenethyl alcohol and / or thimerosal.

[0065] In some embodiments, the pharmaceutical compositions described herein are in dosage forms applicable to intramuscular, intradermal, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, transnasal, sublingual, tonsillar, oral / pharyngeal, or other extraintestinal or mucosal administration routes, preferably in dosage forms suitable for transnasal administration, such as transnasal sprays.

[0066] On the one hand, this disclosure discloses a fusion protein or pharmaceutical composition for the prevention and treatment of infection with coronavirus SARS-CoV-2 and its variants, and / or for preventing the transmission of coronavirus SARS-CoV-2 and its variants to a previously infected subject.

[0067] In some embodiments, the fusion proteins or pharmaceutical compositions of the present disclosure are administered intranasally for the prevention and treatment of infection with coronavirus SARS-CoV-2 and its variants, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants to a previously infected subject.

[0068] On the one hand, this disclosure discloses the application of the fusion protein or pharmaceutical composition of this disclosure in the manufacture of pharmaceuticals for the prevention and treatment of infection with coronavirus SARS-CoV-2 and its variants, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants to subjects who have already been infected.

[0069] On the one hand, the present disclosure provides a method for the prevention and treatment of infection with coronavirus SARS-CoV-2 and its variants, and / or for preventing the transmission of coronavirus SARS-CoV-2 and its variants in a previously infected subject, the method comprising administering to a subject a therapeutically effective dose of the fusion protein or pharmaceutical composition of the present disclosure.

[0070] In some embodiments, the administration is performed via nasal infusion.

[0071] On the one hand, this disclosure provides a kit for the prevention and treatment of infection with coronavirus SARS-CoV-2 and its variants, and / or for preventing the transmission of coronavirus SARS-CoV-2 and its variants in a previously infected subject, the kit comprising the fusion protein or pharmaceutical composition of this disclosure, Container and Optionally, include package inserts or labels indicating prevention and / or treatment.

[0072] In this disclosure, the coronavirus SARS-CoV-2 and its variants are not limited to SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, mu, omicron, and JN.1, but also include variants of unknown coronavirus SARS-CoV-2.

[0073] On the one hand, the Disclosure provides nucleic acids for encoding the fusion protein or a fragment thereof, a carrier containing the nucleic acid, and a host cell containing the nucleic acid or the carrier.

[0074] In some embodiments, the carrier is phFC(IM).

[0075] In some embodiments, the host cell is a CHO cell (e.g., GH-CHO).

[0076] On the other hand, this disclosure states that (a) Culture recombinant cells containing a carrier comprising a nucleic acid molecule encoding a fusion protein of the present disclosure in a suspension culture medium, (b) A method for producing the fusion protein described herein is disclosed, comprising separating the fusion protein from the suspension culture.

[0077] In some embodiments, the disclosure describes a process after one or more steps of cell proliferation. (a) Culture recombinant cells containing a carrier comprising a nucleic acid molecule encoding a fusion protein of the present disclosure in a suspension culture medium, (b) A method for producing the fusion protein described herein is disclosed, comprising separating the fusion protein from the suspension culture.

[0078] In some embodiments, this disclosure is, (1) Rapid capture of the sample or concentration of the fusion protein, (2) One-step or multi-step purification of the fusion protein, (3) A method for producing the fusion protein described herein is disclosed, including obtaining the fusion protein stock solution by concentration and buffer exchange.

[0079] Like other enveloped RNA viruses (e.g., HIV, RSV, influenza virus), coronaviruses, including SARS-CoV-2, have trimer surface antigens on their viral envelope that allow them to enter different host cells via specific cell surface receptors during infection. Similar to SARS-CoV-1, SARS-CoV-2 also uses its trimer viral surface antigen spike protein to bind to its specific cell surface receptor ACE2 and enter host cells of the mammalian respiratory system. The fusion proteins provided in this disclosure can inhibit the binding of the virus to the human ACE2 receptor by binding to the human ACE2 receptor, thereby inhibiting and / or eliminating viral infection. Compositions containing the ACE2-Fc fusion protein provided in this disclosure (e.g., nasal sprays) can broadly and effectively suppress viral infections that enter the human body via the human ACE2 receptor. On the other hand, the compositions containing the ACE2-Fc fusion protein can broadly and effectively inhibit the entry of SARS-CoV-2 viral variants and mutants.

[0080] In some embodiments, the Disclosure discloses a method for treating a subject in need of treatment, comprising administering a therapeutically effective amount of a fusion protein to the subject. The fusion protein has 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 with SEQ ID NO:1 or SEQ ID NO:2.

[0081] This disclosure further discloses pharmaceutical compositions comprising the fusion protein provided herein, methods for producing the fusion protein provided herein, methods for treating a subject using the fusion protein and / or pharmaceutical composition provided herein, and related kits.

[0082] All publications, including patent documents, academic articles, and databases, referenced herein are incorporated herein by reference in their entirety for all purposes, to the same extent as if each individual publication were cited separately. In the event of any conflict or inconsistency between the definitions contained herein and those in patents, applications, published applications, and other publications incorporated herein, the definitions contained herein shall prevail over those incorporated herein. Chapter headings used herein are for organizational purposes only and shall not be construed as limiting the subject matter described herein.

[0083] 1. ACE2-Fc fusion protein resistant to coronavirus infection

[0084] This disclosure discloses an ACE2-Fc fusion protein and compositions comprising the same for preventing coronavirus infection. In some embodiments, the composition comprising the ACE2-Fc fusion protein may be a nasal spray. The nasal spray can cover the nasal cavity and upper respiratory tract. The ACE2-Fc fusion protein competitively binds to SARS-CoV-2 and any mutant (SARS-CoV-2) virus, thereby blocking the site where the viral spike protein binds to host cells, preventing the virus from entering host cells and infecting the human body via the spike protein. Those skilled in the art will readily understand that, based on the mechanism by which SARS-CoV-2 enters host cells and causes infection, the ACE2-Fc fusion protein can competitively bind to SARS-CoV-2 and any mutant (SARS-CoV-2) virus. Any mutants referred to herein include known SARS-CoV-2 mutants as well as unknown SARS-CoV-2 mutants. The aforementioned unknown SARS-CoV-2 mutant strains may be SARS-CoV-2 mutant strains that already exist but have not been discovered or identified by humankind, or they may be novel SARS-CoV-2 mutant strains that do not currently exist but may arise from future viral mutations.

[0085] Coronaviruses belong to the family Positive-Stranded Single-Stranded RNA Viruses, which are known to cause severe respiratory illnesses. They have the largest genome (26-32 kb) of any known RNA virus and are phylogenetically classified into four genera (α, β, γ, δ), of which β-coronaviruses are further subdivided into four lineages (A, B, C, D). Currently, among the Coronaviridae family, viruses that have been confirmed to infect humans originate from the α-coronavirus and β-coronavirus genera. Furthermore, the γ-coronavirus and δ-coronavirus genera are also thought to have the potential to infect humans in the future. Non-specific 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), Mouse Hepatitis Virus (MHV-CoV), Bat SARS-like Coronavirus WIV1 (WIV1-CoV), and Human Coronavirus HKU9 (HKU9-CoV). Non-specific examples of α-coronaviruses include Human Coronavirus 229E (229E-CoV), Human Coronavirus NL63 (NL63-CoV), Porcine Epidemic Diarrhea Virus (PEDV), and Infectious Gastroenteritis Coronavirus (TGEV). Non-specific examples of δ-coronaviruses include Porcine δ-coronavirus (SDCV).

[0086] This disclosure provides a list of coronaviruses associated with severe acute respiratory syndrome. Bat coronavirus Cp / Yunnan2011 Bat coronavirus RaTG13 Bat coronavirus Rp / Shaanxi2011 Bat SARS-CoV-3 Bat SARS coronavirus HKU3-1 Bat SARS coronavirus HKU3-10 Bat SARS coronavirus HKU3-11 Bat SARS coronavirus HKU3-12 Bat SARS coronavirus HKU3-13 Bat SARS coronavirus HKU3-2 Bat SARS coronavirus HKU3-3 Bat SARS coronavirus HKU3-4 Bat SARS coronavirus HKU3-5 Bat SARS coronavirus HKU3-6 Bat SARS coronavirus HKU3-7 Bat SARS coronavirus HKU3-8 Bat SARS coronavirus HKU3-9 Bat SARS-CoV-1 Bat SARS-CoV-2 Bat SARS-CoV Rf1 / 2004 Bat CoV 273 / 2005 Bat SARS-CoV Rm1 / 2004 Bat CoV 279 / 2005 Bat SARS-CoV Rp3 / 2004 Bat-like SARS-like coronavirus Bat-like SARS-like coronavirus Rs3367 Bat-like SARS-like coronavirus RsSHC014 Bat-like SARS-like coronavirus WIV1 Bat SARS-like coronavirus YNLF_31C Bat SARS-like coronavirus YNLF_34C BtRf-BetaCoV / HeB2013 BtRf-BetaCoV / JL2012 BtRf-BetaCoV / SX2013 BtRs-BetaCoV / GX2013 BtRs-BetaCoV / HuB2013 BtRs-BetaCoV / YN2013 Civet SARS-CoV 007 / 2004 Civet SARS-CoV SZ16 / 2003 Civet SARS-CoV SZ3 / 2003 Recombinant SARSr-CoV SARS-CoV-1 ExoN1 SARS coronavirus MA15 SARS-CoV-15 ExoN1 SARS-CoV-14 Nakagi horseshoe bat coronavirus SARS bat coronavirus SARS coronavirus A001 SARS-CoV-13 SARS-CoV-21 SARS-CoV-22 SARS-CoV-2030 SARS coronavirus A031 SARS coronavirus AS SARS-CoV-12 SARS-CoV-24 SARS-CoV-29 SARS-CoV-13 SARS-CoV-19 SARS-CoV-140 SARS coronavirus BJ01 SARS-CoV-2 SARS-CoV-2 (BJ03) SARS-CoV-24 SARS-CoV-2 (BJ162) SARS-CoV-2 (BJ182-12) SARS-CoV-2 (BJ182-4) SARS-CoV-2 (BJ182-8) SARS coronavirus BJ182a SARS coronavirus BJ182b SARS-CoV-202 SARS coronavirus BJ2232 SARS-CoV-2 (BJ302) SARS-CoV-13 SARS-CoV-14 SARS-CoV-17 SARS-CoV-18 SARS-CoV-19 SARS-CoV-25 SARS-CoV-28 SARS-CoV-29 SARS coronavirus CDC#200301157 SARS coronavirus civet010 SARS coronavirus civet014 SARS coronavirus civet019 SARS coronavirus civet020 SARS-CoV-21 SARS-CoV-24 SARS-CoV-2 CUHK-AG01 SARS-CoV-2 CUHK-AG02 SARS-CoV-2 CUHK-AG03 SARS-CoV-2 SARS-CoV-2 CUHK-SU10 SARS-CoV-2 CUHK-W1 SARS coronavirus cw037 SARS coronavirus cw049 SARS-CoV-291 SARS-CoV-260 SARS-CoV-2 (FRA) SARS coronavirus Frankfurt 1 SARS coronavirus Frankfurt1-v01 SARS-CoV-2 (GD01) SARS coronavirus GD03T0013 SARS-CoV-2 (GD322) SARS-CoV-29 SARS-CoV-2 (GDH-BJH01) SARS-CoV-2 (GZ-A) SARS-CoV-2 (GZ-B) SARS-CoV-2 (GZ-C) SARS-CoV-2 (GZ-D) SARS-CoV-2 SARS coronavirus GZ0401 SARS-CoV-2 (GZ0402) SARS coronavirus GZ0403 SARS-CoV-2GZ43 SARS coronavirus GZ50 SARS coronavirus GZ60 SARS-CoV-19 SARS coronavirus HC / SZ / 61 / 03 SARS-CoV-2 HGZ8L1-A SARS coronavirus HGZ8L1-B SARS coronavirus HGZ8L2 SARS-CoV-2004 SARS coronavirus HKU-36871 SARS coronavirus HKU-39849 SARS coronavirus HKU-65806 SARS coronavirus HKU-66078 SARS coronavirus Hong Kong / 03 / 2003 SARS-CoV-2003 SARS-CoV-2 HSR-1 SARS-CoV-2 HSZ-A SARS-CoV-2 (HSZ-Bb) SARS-CoV-2 (HSZ-Bc) SARS-CoV-2 (HSZ-Cb) SARS-CoV-2 (HSZ-Cc) SARS-CoV-2 HSZ2-A SARS coronavirus HZS2-Bb SARS-CoV-2 (HZS2-C) SARS-CoV-2 (HZS2-D) SARS-CoV-2 (HZS2-E) SARS coronavirus HZS2-Fb SARS coronavirus HZS2-Fc SARS-CoV-2 (JMD) SARS-CoV-1 SARS-CoV-2 SARS-CoV-13 SARS-CoV-4 SARS-CoV-15 SARS-CoV-2004 SARS-CoV-2 NS-1 SARS-CoV-2 SARS coronavirus PC4-115 SARS coronavirus PC4-127 SARS coronavirus PC4-13 SARS-CoV-2 (PC4-136) SARS coronavirus PC4-137 SARS coronavirus PC4-145 SARS coronavirus PC4-199 SARS-CoV-205 SARS coronavirus PC4-227 SARS-CoV-241 SARS-CoV-1 PUMC01 SARS-CoV-2 (PUMC02) SARS-CoV-23 SARS coronavirus Rs_672 / 2006 SARS coronavirus SF098 SARS coronavirus SF099 SARS coronavirus ShanghaiQXC1 SARS coronavirus ShanghaiQXC2 SARS coronavirus Shanghai LY SARS coronavirus Sin0409 SARS coronavirus Sin2500 SARS coronavirus Sin2677 SARS coronavirus Sin2679 SARS coronavirus Sin2748 SARS coronavirus Sin2774 SARS coronavirus Sin3408 SARS coronavirus Sin3408L SARS coronavirus Sin3725V SARS coronavirus Sin3765V SARS coronavirus Sin842 SARS coronavirus Sin845 SARS coronavirus Sin846 SARS coronavirus Sin847 SARS coronavirus Sin848 SARS coronavirus Sin849 SARS coronavirus Sin850 SARS coronavirus Sin852 SARS coronavirus Sin_WNV SARS coronavirus Sino1-11 SARS coronavirus Sino3-11 SARS coronavirus SinP1 SARS coronavirus SinP2 SARS coronavirus SinP3 SARS coronavirus SinP4 SARS coronavirus SinP5 SARS-CoV-21 SARS coronavirus SZ1 SARS coronavirus SZ13 SARS coronavirus Taiwan SARS-CoV-2003 (Taiwan JC-2003) SARS coronavirus Taiwan TC1 SARS coronavirus Taiwan TC2 SARS coronavirus Taiwan TC3 SARS-CoV-2 TJ01 SARS-CoV-21 SARS-CoV-2 SARS-CoV-2 SARS-CoV-2 TW-GD1 SARS-CoV-2 (TW-GD2) SARS-CoV-2 (TW-GD3) SARS-CoV-2 (TW-GD4) SARS-CoV-2 (TW-GD5) SARS-CoV-2 TW-HP1 SARS-CoV-2 (TW-HP2) SARS-CoV-2 (TW-HP3) SARS-CoV-2 (TW-HP4) SARS-CoV-2 TW-JC2 SARS-CoV-2 TW-KC1 SARS-CoV-2 TW-KC3 SARS-CoV-2 TW-PH1 SARS-CoV-2 TW-PH2 SARS-CoV-2 TWYM1 SARS-CoV-2 (TWYM2) SARS-CoV-2 TWYM3 SARS-CoV-2 TWYM4 SARS-CoV-21 SARS-CoV-20 SARS-CoV-21 SARS-CoV-2 SARS-CoV-23 SARS-CoV-24 SARS-CoV-25 SARS-CoV-2 TW6 SARS coronavirus TW7 SARS-CoV-28 SARS-CoV-29 SARS-CoV-2019 SARS-CoV-2 SARS-CoV-3 SARS-CoV-21 SARS-CoV-2 (TWJ) SARS-CoV-2019 SARS-CoV-2015 SARS coronavirus TWY SARS coronavirus (Urbania) SARS coronavirus Vietnam SARS coronavirus WF188 SARS-CoV-20 SARS-CoV-20 SARS coronavirus xw002 SARS coronavirus ZJ01 SARS-CoV-2 SARS coronavirus ZJ0301 SARS-CoV-2 (ZMY-1) SARS-CoV-2 (ZS-A) SARS-CoV-2 (ZS-B) SARS-CoV-2 (ZS-C) SARS-related bat coronavirus RsSHC014 SARS-related β-coronavirus Rp3 / 2004 Severe Acute Respiratory Syndrome Coronavirus 2

[0087] The following are representative SARS-CoV-2 strains.

[0088] [Table 1]

[0089] The coronavirus genome is capped, polyadenylated, and covered by a nucleocapsid protein. Coronavirus particles contain a viral envelope that includes a type I fusion glycoprotein, which is the spike (S) protein. Most coronaviruses have a common genomic structure in which the replicase gene is located in the 5′ region of the genome and the structural gene is located in the 3′ region of the genome.

[0090] The coronavirus spike (S) protein is a type I fusion glycoprotein initially synthesized as a precursor protein. A single precursor spike polypeptide forms a homotrimer, undergoing glycosylation in the Golgi apparatus, which removes the signal peptide. Further cleavage by cellular proteases into separate S1 and S2 polypeptide chains is then performed. The homotrimer is a heterodimeric trimer, as the S1 / S2 protomers remain bound in the homotrimer. The S1 subunit is distal to the viral membrane and contains a receptor-binding domain (RBD) that mediates attachment to the host receptor. The S2 subunit contains the fusion peptide, two heptadrepeat sequences (HR1 and HR2) characteristic of fusion glycoproteins, and fusion protein mechanisms including a central helix, a transmembrane domain, and a cytosolic tail domain.

[0091] ACE2 acts as a receptor for SARS-CoV-2, with its extracellular domain binding to the spike protein on the SARS-CoV-2 envelope. During this process, a transmembrane serine protease (TMPRSS2) present on the cell membrane surface cleaves the spike protein into S1 and S2 subunits, exposing the fusion peptide. This achieves fusion with the cell membrane, releasing genetic material and initiating replication.

[0092] Viral antigens or immunogens contain protease cleavage sites, which are, as appropriate, furin protease (furin), trypsin, factor Xa, or cathepsin L. During viral assembly, the furin protease cleaves the spike protein into different subunits (S1 and S2), which are released to infect other cells. SARS-CoV lacks a furin protease recognition site, and due to differences in the spike protein cleavage and assembly mechanism, SARS-CoV-2 has a stronger membrane fusion ability and a higher efficiency of entry into cells. Studies have shown that SARS-CoV-2's ability to bind to ACE2 is 10 to 20 times greater than that of SARS, which contributes to its high transmissibility.

[0093] In some embodiments, the ACE2-Fc fusion protein comprises an ACE2 protein and a polypeptide. In some embodiments, the ACE2 protein comprises an ACE2 protein derived from a primate (e.g., human ACE2 protein or a fragment thereof). In some embodiments, the human ACE2 protein or a fragment thereof is the full-length extracellular domain protein or a fragment protein of human ACE2. In some embodiments, the non-dimerized Fc domain is an IgG, IgM, IgD, IgA, or IgE Fc region, or a variant, mutant, or fragment thereof. In some embodiments, the non-dimerized 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 (e.g., human IgG Fc protein or a functional variant thereof). In some embodiments, human IgG Fc or a fragment thereof is the full-length extracellular domain protein or a fragment protein of human IgG Fc. 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 extracellular domain protein / polypeptide or a fragment of the human ACE2 protein / polypeptide, and human IgG Fc or a fragment of the human IgG protein / polypeptide.

[0094] In some embodiments, the ACE2-Fc fusion protein includes the sequence or fragment thereof shown in SEQ ID NO:1. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:1 below. In some embodiments, the ACE2-Fc fusion protein includes an amino acid fragment RS for linking the ACE2 and Fc sequences. MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPRSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDSNLWN(SEQ ID NO:1)

[0095] In some embodiments, the ACE2-Fc fusion protein comprises the sequence or fragment thereof shown in SEQ ID NO:2. In some embodiments, the ACE2-Fc fusion protein comprises an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:2 below. QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPRSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:2)

[0096] In some embodiments, the ACE2-Fc fusion protein comprises the sequence or fragment thereof shown in SEQ ID NO:3. In some embodiments, the ACE2-Fc fusion protein comprises an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:3 below. MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPRSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3)

[0097] In some embodiments, the ACE2-Fc fusion protein includes the sequence or fragment thereof shown in SEQ ID NO:4. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:4 below. QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPRSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDSNLWN(SEQ ID NO:4)

[0098] In some embodiments, the ACE2-Fc fusion protein includes the sequence or fragment thereof shown in SEQ ID NO:15. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:15 below. In some embodiments, the ACE2-Fc fusion protein includes an amino acid fragment VS for linking the ACE2 and Fc sequences. MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDSNLWN(SEQ ID NO:15)

[0099] In some embodiments, the ACE2-Fc fusion protein includes the sequence or fragment thereof shown in SEQ ID NO:16. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:16 below. QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:16)

[0100] In some embodiments, the ACE2-Fc fusion protein comprises the sequence or fragment thereof shown in SEQ ID NO:17. In some embodiments, the ACE2-Fc fusion protein comprises an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:17 below. MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:17)

[0101] In some embodiments, the ACE2-Fc fusion protein includes the sequence or fragment thereof shown in SEQ ID NO:18. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:18 below. QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDSNLWN(SEQ ID NO:18)

[0102] In some embodiments, the ACE2-Fc fusion protein includes the ACE2 extracellular domain sequence or a fragment thereof, as shown in SEQ ID NO:5. 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 with the sequence shown in SEQ ID NO:5 below. (SEQ ID NO:5)

[0103] In some embodiments, the ACE2-Fc fusion protein includes the ACE2 extracellular domain sequence or a fragment thereof, as shown in SEQ ID NO:11. 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 with the sequence shown in SEQ ID NO:11 below. (SEQ ID NO:11)

[0104] In some embodiments, the ACE2-Fc fusion protein comprises the ACE2 extracellular domain sequence or a fragment thereof, as shown in SEQ ID NO:12. 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 with the sequence shown in SEQ ID NO:12 below. (SEQ ID NO:12)

[0105] In some embodiments, the ACE2-Fc fusion protein comprises the ACE2 extracellular domain sequence or a fragment thereof, as shown in SEQ ID NO:13. 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 with the sequence shown in SEQ ID NO:13 below. (SEQ ID NO:13)

[0106] In some embodiments, the ACE2-Fc fusion protein comprises the ACE2 extracellular domain sequence or a fragment thereof, as shown in SEQ ID NO:14. 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 with the sequence shown in SEQ ID NO:14 below. (SEQ ID NO:14)

[0107] In some embodiments, the ACE2-Fc fusion protein comprises the ACE2 extracellular domain sequence or a fragment thereof, as shown in SEQ ID NO:7. 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 with the sequence shown in SEQ ID NO:7 below. (SEQ ID NO:7)

[0108] In some embodiments, the ACE2-Fc fusion protein includes an Fc sequence or fragment thereof shown in SEQ ID NO:6. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:6 below. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:6)

[0109] In some embodiments, the ACE2-Fc fusion protein includes an Fc tag sequence or fragment thereof, as shown in SEQ ID NO:8. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:8 below. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDSNLWN(SEQ ID NO:8)

[0110] In some embodiments, the fusion protein of the present disclosure further comprises a signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence or fragment thereof shown in SEQ ID NO:9. In some embodiments, the signal peptide comprises an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:9.

[0111] In some embodiments, the fusion protein of the Disclosure further includes a mutant sequence for C-terminal modification to facilitate traceability detection. The mutant sequence included in the ACE2-Fc fusion protein of the Disclosure includes the sequence or fragment thereof shown in SEQ ID NO:10. In some embodiments, the ACE2-Fc fusion protein includes an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:10.

[0112] In some embodiments, the fusion protein of the Disclosure comprises a non-dimerized Fc domain having one or more cysteine ​​substitutions compared to human IgGl Fc, optionally selected from C5S, C11S, and C14S, with the substitution site relative to SEQ ID NO:6, and optionally the cysteine ​​being replaced with serine. In some embodiments, the fusion protein of the Disclosure comprises a non-dimerized Fc domain 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 with the sequence shown in SEQ ID NO:5.

[0113] In some embodiments, the ACE2 protein peptide is produced from a codon-optimized nucleic acid sequence. In some embodiments, the ACE2 protein peptide is produced from a non-codon-optimized nucleic acid sequence.

[0114] The ACE2 provided in this disclosure is expected to be able to form a fusion protein with Fc, and also to bind (e.g., ligate) with other proteins or peptides to form a recombinant polypeptide containing the fusion peptide. In some embodiments, the ACE2-Fc fusion protein comprises the ACE2 and Fc proteins and polypeptides. In some embodiments, the ACE2 is primate-derived ACE2 (e.g., human ACE2 protein or a fragment thereof). In some embodiments, the human ACE2 protein or a fragment thereof is the full-length extracellular domain protein or a fragment protein of human ACE2. In some embodiments, the Fc is primate Fc (e.g., human IgG Fc protein or a functional variant thereof). In some embodiments, the human IgG Fc or a fragment thereof is the full-length extracellular domain protein or a fragment protein of human IgG Fc. In some embodiments, the ACE2-Fc fusion protein comprises the human ACE2 protein or a fragment thereof, and the human IgG Fc protein or a functional variant thereof. In some embodiments, the ACE2-Fc fusion protein comprises the full-length extracellular domain protein / polypeptide or a fragment of the human ACE2 protein / polypeptide and the human IgG Fc protein or a functional variant protein / polypeptide. In some embodiments, the ACE2 protein / polypeptide is directly linked to the Fc protein / polypeptide. In some embodiments, the ACE2 and Fc proteins and polypeptides are linked via one or more amino acids.

[0115] In some embodiments, one or more peptide linkers (e.g., glycine-serine linkers, e.g., arginine-serine peptide linkers) are used to link ACE2 to Fc. In some embodiments, the linker comprises a non-dimerized Fc domain or fragment thereof capable of binding to an Fc receptor (e.g., FcRn), thereby extending the half-life (e.g., half-life in human serum) of the fusion protein and its complex. In some embodiments, recombinant ACE2-Fc may be dimerized or multimerized (e.g., trimer, tetramer, pentamer, hexamer) as long as it retains the desired properties (e.g., pre-fusion conformation), and may contain any (or a combination thereof) of the stabilizing mutations provided in this disclosure. In some embodiments, the non-dimerized Fc domain or fragment thereof in the trimer complex maintains a monomeric state. That is, the Fc domain or fragment thereof does not form interchain covalent bonds (such as disulfide bonds) and does not form oligomers through direct non-covalent Fc interactions between domains. In some embodiments, the recombinant polypeptide or fusion protein has a first sequence indicated by SEQ ID NO: 5, 7, 11, 12, 13, or 14, the first sequence being linked to a second sequence indicated by SEQ ID NO: 6 or 8. In some embodiments, the recombinant polypeptide or fusion protein has a first sequence indicated by SEQ ID NO: 11, 13, the first sequence being linked to a second sequence indicated by SEQ ID NO: 6 or 8. In some embodiments, the first and second sequences are linked via a linker, the linker being linked by one or more amino acids (e.g., two amino acids: -RS- or -VS-). In some embodiments, the linker includes a sequence containing a glycine-XY repeat sequence.

[0116] Papain digestion of the antibody produces two identical antigen-binding fragments (called Fab fragments) and one residual Fc fragment (a name reflecting its crystallization ability). Each Fab fragment consists of a complete light chain, a variable region of the heavy chain, and the first constant region (CH1) of the heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. The Fc fragment contains the carboxyl group terminals of two heavy chains linked by disulfide bonds. The effector function of the antibody is determined by the sequence of the Fc region, which is also recognized by Fc receptors (FcRs) on specific cells.

[0117] The Fc domain of Fc fusion proteins, by binding to Fc receptors on the surface of immune cells, exerts diverse biological functions such as mediating passage through the placenta and mucosal barrier, inflammatory responses, antibody-dependent cell phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cell-mediated cytotoxicity (CDC). It can also promote dendritic cell (DC) maturation, regulate cytokine secretion, and regulate B cell proliferation and differentiation.

[0118] [Table 2]

[0119] Antibodies can extend their serum half-life by interacting with the neonatal Fc receptor (FcRn) via a pH-dependent Fc (fragment, crystallizable) domain. In some embodiments, this disclosure provides Fc fusion molecules having a modified Fc domain that can extend the serum half-life of a therapeutic protein or peptide. Because antibody Fc is homodimerized, naturally occurring IgG antibodies and modified Fc fusion molecules containing naturally occurring IgG sequences typically exhibit bivalent and monospecificity. In the specific therapeutic applications disclosed herein, modified Fc polypeptides and their fusion polypeptides can achieve flexibility and specificity while retaining the beneficial properties of monomeric and non-dimerized Fc.

[0120] The most abundant immunoglobulin category in human serum is IgG. IgG structure consists of two light chains and two heavy chains, each light chain containing two domains and each heavy chain containing four domains. The antigen-binding site is located in the Fab region (antigen-binding fragment), which includes the variable light chain (VL) and variable heavy chain (VH) domains, as well as the constant light chain (CL) and constant heavy chain (CH1) domains. The Fc region contains the CH2 and CH3 domain regions of the heavy chains, and the two heavy chains are linked by a disulfide bond (-SS-) at the hinge region.

[0121] Because the FcRn (neonatal Fc receptor) binding site of IgG is located in the Fc region of the antibody, the extended serum half-life property of the antibody is retained in the Fc fragment. A single Fc fragment can be considered a homodimer of the heavy chain containing CH2 and CH3 domains. In some embodiments, the monomeric non-dimerized Fc domain of this disclosure contains 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, the monomeric non-dimerized Fc domain contains three or more mutations in the Fc amino acid sequence compared to the corresponding wild-type Fc domain. In some embodiments, the monomeric non-dimerized Fc domain contains two mutations in the Fc amino acid sequence compared to the corresponding wild-type Fc domain.

[0122] In some embodiments, the Fc domain or fusion polypeptide of the Disclosure comprises an immunoglobulin moiety (e.g., a hinge, CH2 and / or CH3 domain), and any or all cysteine ​​residues present in the hinge domain located in the immunoglobulin moiety are mutated to prevent the formation of disulfide bonds between one or more of these cysteine ​​residues. For example, one or more of the cysteines at positions 220, 226, and 229 of the human IgG1 Fc domain (corresponding to positions 233, 239, and 242 in Kabat numbering, respectively) may be replaced with other amino acid residues such as serine, according to the Kabat EU index number. In some embodiments, the Fc domain or fusion polypeptide of the Disclosure comprises a cysteine ​​at position 220, which is replaced with, for example, serine. In some embodiments, the Fc domain or fusion polypeptide of the Disclosure comprises a cysteine ​​at position 226, which is replaced with, for example, serine. In some embodiments, the Fc domain or fusion polypeptide of the Disclosure comprises a cysteine ​​at position 229, wherein the cysteine ​​is substituted with, for example, serine. In some embodiments, the Fc domain or fusion polypeptide of the Disclosure further comprises one or more other amino acid substitutions, insertions and / or deletions. In some embodiments, the Fc domain or fusion polypeptide of the Disclosure further comprises a proline at position 238 (corresponding to the proline at position 251 in Kabat numbering), which is substituted with, for example, serine.

[0123] In some embodiments, the Fc domain or its fusion polypeptide of the Disclosure exhibits increased affinity for FcRn in a first pH range compared to the corresponding human IgG1 Fc domain or its fusion polypeptide. In some embodiments, the Fc domain or its fusion polypeptide of the Disclosure exhibits rapid dissociation of the corresponding human IgG1 Fc domain or its fusion polypeptide from FcRn in 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 less than 7.0 and the second pH range is greater than 7.0. In some embodiments, the first pH range is between approximately 5.5 and approximately 6.5 and the second pH range is between approximately 7.0 and approximately 8.0. In some embodiments, the first pH range is approximately 6.0 and the second pH range is approximately 7.4.

[0124] In some embodiments, the Fc domain or its fusion polypeptide of the Disclosure is located 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, and 409, and according to the Kabat EU Index, positions 410, 413, 424, 438 and 442 in the human IgG1Fc domain are These 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, each following the Kabat numbering system. In some embodiments, the Fc domain or its fusion polypeptide of the Disclosure is P217R, P228K, F243V, V262I, V273L, K274V, K288V, K288D, K288I, K288F, K290L, S298N, V305I, L309E, H310S, C321V, K326G, R344T, P353K, P353D, D356P, V363N, S3 It contains one or more amino acid substitutions selected from the group consisting of 64N, L368W, L368G, S375Y, E388G, N389V, N390L, V397L, L398W, D399K, D401G, F405E, F405K, F405Q, F405R, F405V, Y407S, K409N, K409D, L410N, D413R, S424G, Q438S, and S442K.

[0125] In some embodiments, the Fc domain or fusion polypeptide of the Disclosure is located at positions 221, 234, 297, 306, 312, 315, 306, 312, and 315, respectively, and according to the Kabat EU index, positions 325, 343, 356, 401, 406, and 421 in the human IgG1 Fc domain correspond to positions 234, 247, 314, 325, 331, 334, 344, 364, 377, 430, 437, and 452, respectively, according to the Kabat numbering system. In some embodiments, the Fc domain or fusion polypeptide of the Disclosure includes one or more amino acid substitutions selected from the group N421H.

[0126] In some embodiments, the Fc domain or fusion polypeptide of the Disclosure is located at positions 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. Located in [location], and according to the European Union Kabat index, it corresponds to 234th, 237th, 283rd, 284th, 286th, 307th, 311th, 324th, 334th, 338th, 341st, 364th, 370th, 378th, 387th, 391st, 419th, 436th, 440th, 455th, 457th, 466th, 468th, 469th, 472nd, and 478th, respectively, following the Kabat numbering system. In some embodiments, the Fc domain or its fusion polypeptide of the 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.

[0127] In some embodiments, the Fc domain or its fusion polypeptide of the Disclosure includes one or more amino acid substitutions, insertions, and / or deletions at positions 252, 254, 256, 428, and 434. According to the Kabat EU index, the human IgG1 Fc domain corresponds to positions 265, 267, 269, 459, and 465, respectively, according to the Kabat numbering system. In some embodiments, the Fc domain or its fusion polypeptide of the Disclosure includes one or more amino acid substitutions selected from M252Y, S254T, T256E, M428L, and N434S.

[0128] In some embodiments, the fusion proteins described herein may contain monomers or fragments of an immunoglobulin Fc domain to extend the serum half-life of the polypeptide. In some embodiments, the fusion proteins described herein cannot form dimers (e.g., homodimers or heterodimers) through interactions between any two Fc domain monomers in the non-covalent complex disclosed herein. Conversely, the fusion proteins described herein may trimerize (independently of their Fc domains) to form a trimer complex comprising three Fc domain monomers having identical or different sequences.

[0129] In some embodiments, the Fc domain is mutable to lack effector function and is typically an "inactivated" Fc domain that does not bind to Fc receptors such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and / or FcγRIV. For example, the Fc domain may include certain amino acid substitutions known to minimize interaction between the Fc domain and the Fcγ receptor. In some embodiments, the Fc domain is derived from an IgGl antibody and includes amino acid substitutions L234A, L235A, and G237A. In some embodiments, the Fc domain is derived from an IgGl antibody and includes amino acid substitutions D265A, K322A, and N434A. The above amino acid positions are based on Kabat's definition (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). By comparing homologous regions of an antibody sequence with a "standard" Kabat number sequence, the Kabat number of an amino acid residue in a given antibody can be determined. Furthermore, in some embodiments, the Fc domain does not induce immune system-related responses. For example, the Fc domain in a polypeptide dimer can be modified to reduce its interaction with or binding to the Fcγ receptor.

[0130] In some cases, the fusion protein described herein comprises the Fc region of human immunoglobulin or a fragment or variant thereof. In some embodiments, the Fc domain of the fusion protein is the Fc region of human IgG1 or a fragment or variant thereof. In some embodiments, the Fc domain of the fusion protein is shortened at the N-terminus compared to the native human immunoglobulin Fc region. In some embodiments, the Fc domain of the fusion protein does not contain the CH1 domain of the native human immunoglobulin Fc region.

[0131] In some embodiments, the Fc domain of the fusion protein contains one or more amino acid substitutions, deletions, or insertions compared to the natural human IgGl sequence. In some embodiments, the Fc domain of the fusion protein contains substitutions at one or more amino acid positions within the CH2 domain. In some embodiments, the Fc domain of the fusion protein contains substitutions at one or more amino acid positions within the DE turn. In some embodiments, the Fc domain of the fusion protein contains a naturally occurring amino acid substitution at position 297, where the substitution detectably reduces and / or removes glycosylation at position 297. In certain embodiments, the Fc domain of the fusion protein contains a substitution of asparagine at position 297 of the antibody heavy chain with cysteine. 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 contains an N297Q substitution. In each case, the constant region numbering system is the EU index numbering system as defined by Kabat.

[0132] In some embodiments, amino acid substitutions, deletions, or insertions may result in improvements to the expression, purification, or stability profile of the fusion protein. In some embodiments, amino acid substitutions, deletions, or insertions may result in improvements to the binding affinity and specificity spectrum of the fusion protein. In some cases, the Fc domain of the fusion protein contains no cysteine ​​molecules capable of forming interchain disulfide bonds.

[0133] In some embodiments, the Fc domain of the fusion protein or complex includes the sequence shown in SEQ ID NO:5. In some embodiments, the Fc domain of the fusion protein or complex includes an amino acid sequence or fragment thereof having at least or about 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:5.

[0134] In some embodiments, the recombinant fusion protein comprises the ACE2 protein or a fragment thereof as described in Section I. In some embodiments, the recombinant fusion protein comprises the ACE2 as described in Section I of the Disclosure, ligated to the Fc protein or a functional variant thereof as described in the Disclosure.

[0135] In some embodiments, the Fc protein is produced by a codon-optimized nucleic acid sequence. In some embodiments, the Fc protein is produced by a non-codon-optimized nucleic acid sequence.

[0136] In some embodiments, the ACE2-Fc fusion protein provided in this disclosure is further linked to a conjugate portion, which is selected from the group consisting of detectable markers, pharmaceuticals, toxins, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, coat proteins, or VLPs, or a combination thereof. In some embodiments, the ACE2-Fc fusion protein is linked to the conjugate portion via a conjugate linker.

[0137] 2. Polynucleotides and carriers

[0138] The disclosure further provides polynucleotides (nucleic acid molecules) encoding the ACE2-Fc fusion protein provided herein, and carriers for cells genetically modified to express the ACE2-Fc fusion protein.

[0139] In some embodiments, the present disclosure provides polynucleotides encoding fusion proteins. On the other hand, the polynucleotide comprises a single nucleic acid sequence, for example, the nucleic acid sequence of a polypeptide encoding an ACE2-Fc fusion protein. In other embodiments, the polynucleotide comprises a recombinant polypeptide, particularly a second nucleic acid sequence encoding a first nucleic acid of ACE2 and a recombinant polypeptide of Fc.

[0140] In some embodiments, the polynucleotide encoding the fusion protein comprises at least one promoter, the promoter being functionally linked to control the expression of the recombinant polypeptide. In some embodiments, the polynucleotide comprises two, three, or more promoters, the promoters being functionally linked to control the expression of the recombinant polypeptide.

[0141] In some embodiments, for example, if the polynucleotide comprises two or more nucleic acid coding sequences (e.g., a first nucleic acid sequence containing ACE2 and a second nucleic acid sequence encoding an Fc recombinant polypeptide), at least one promoter is functionally linked to control the expression of these two or more nucleic acid sequences. In some embodiments, the polynucleotide comprises two, three, or more promoters, the promoters being functionally linked to control the expression of the recombinant polypeptide.

[0142] In some embodiments, the expression of recombinant polypeptides is either induced or conditional. Therefore, in some cases, the polynucleotide encoding the recombinant polypeptide includes a conditional promoter, enhancer, or transactivator. In some such embodiments, the conditional promoter, enhancer, or transactivator is either an inducible or repressive promoter, enhancer, or transactivator. For example, in some embodiments, an inducible or conditional promoter can be used to restrict 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 factor (e.g., heat, radiation, or drug).

[0143] If a polynucleotide contains nucleic acid sequences encoding one or more recombinant polypeptides, the polynucleotide may further contain nucleic acid sequences for encoding peptides between the nucleic acid sequences. In some cases, the nucleic acid located between the nucleic acid sequences encodes a peptide that separates the translation product of the nucleic acid sequence either during or after translation. In some embodiments, the peptide includes peptides that induce ribosome skipping, such as internal ribosome entry sites (IRESs), self-cleaving peptides, or T2A peptides.

[0144] In some embodiments, polynucleotides encoding recombinant polypeptides are introduced into a composition containing cultured cells (e.g., host cells) by, for example, retroviral transduction, transfection, or transformation. In some embodiments, this enables the expression (e.g., production) of the recombinant polypeptide. In some embodiments, the expressed recombinant polypeptide is purified.

[0145] In some embodiments, the polynucleotides (nucleic acid molecules) provided by this disclosure encode the nucleic acid sequence of the ACE2-Fc fusion protein described in this disclosure. In some embodiments, the polynucleotides (nucleic acid molecules) provided by this disclosure encode the nucleic acid sequence of the ACE2-Fc fusion protein described in Disclosure I. In some embodiments, the polynucleotides (nucleic acid molecules) provided by this disclosure encode a recombinant polypeptide containing ACE2 described in this disclosure. In some embodiments, the polynucleotides (nucleic acid molecules) provided by this disclosure encode a recombinant polypeptide containing Fc described in this disclosure. Further, carriers or constructs comprising the nucleic acid molecules described in this disclosure are provided. In some embodiments, the carrier or construct comprises one or more promoters, the promoters functionally linked to a nucleic acid molecule encoding a recombinant polypeptide, and driving its expression. In some embodiments, the promoters functionally linked to one or more nucleic acid molecules, for example, a nucleic acid molecule encoding a polypeptide containing ACE2.

[0146] In some embodiments, the carrier is a viral carrier. In some embodiments, the viral carrier is a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the retroviral vector is a γ-retroviral vector.

[0147] In some embodiments, the carrier or construct includes a single promoter that drives the expression of one or more nucleic acid molecules within a polynucleotide. In some embodiments, such a promoter may be polycistronic (bisicronic or tricistronic, see, e.g., U.S. Patent No. 6,060,273). For example, in some embodiments, the transcription unit can be fabricated as a bicistronic unit containing an IRES (internal ribosome entry site), thereby enabling co-expression of gene products (e.g., encoding different recombinant polypeptides) with a message derived from a single promoter. In some embodiments, the carrier provided by the Disclosure is bicistronic, enabling the carrier to contain and express two nucleic acid sequences. In some embodiments, the carrier provided by the Disclosure is tricistronic, enabling the carrier to contain and express three nucleic acid sequences.

[0148] In some embodiments, a single promoter directs RNA expression, and the RNA contains two or three genes (e.g., one encoding a chimeric signaling receptor and the other a recombinant receptor) in a single open reading frame (ORF), which are separated from each other by encoding a self-cleaving peptide sequence (e.g., a 2A sequence) or a protease recognition site (e.g., a furin protease). Thus, the ORF encodes a single polypeptide. During translation (in the case of 2A) or post-translation, the polypeptide is processed into a single protein. In some cases, the peptide (e.g., T2A) causes the ribosome to skip the synthesis of the C-terminal peptide bond of the 2A element (ribosome skipping), resulting in separation between the end of the 2A sequence and the next downstream peptide (see, e.g., de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004), which are incorporated in full for all purposes). Many 2A elements are known in the art. Examples of 2A sequences usable in the methods and nucleic acids of this disclosure include, but are not limited to, 2A sequences derived from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine teschovirus-1 (P2A) (including those described in U.S. Patent Publication No. 20070116690).

[0149] 3. ACE2-Fc fusion protein composition and formulation

[0150] In some embodiments, the Disclosure provides ACE2-Fc fusion protein compositions, i.e., compositions (pharmaceutical compositions) comprising an ACE2-Fc fusion protein, wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 1-18, or any combination thereof. In some embodiments, the Disclosure provides compositions comprising a recombinant fusion protein having the sequence shown in SEQ ID NO: 1, or a fragment, variant, or mutant thereof. The fusion protein compositions referred to in the Disclosure comprise a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 5-8, or a fragment, variant, or mutant thereof, and can be used in formulations.

[0151] In some embodiments, a single dose of the ACE2-Fc fusion protein composition contains about 0.1 mg to about 100 mg of ACE2-Fc fusion protein, preferably about 0.25 mg to about 35 mg of ACE2-Fc fusion protein, more preferably about 0.25 mg to about 15 mg of ACE2-Fc fusion protein. In some embodiments, the single dose is 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, 1.6 The formulation contains 0, 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 of ACE2-Fc fusion protein. In some embodiments, a single dose contains 0.5 mg of ACE2-Fc fusion protein. In other embodiments, a single dose contains 1 mg of ACE2-Fc fusion protein. In yet another embodiment, the dose contains 2.0 mg of ACE2-Fc fusion protein.

[0152] In some embodiments, the ACE2-Fc fusion protein composition provided herein may contain about 0.1 mg / ml to about 100 mg / ml of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain about 0.1 mg / ml to about 95 mg / ml of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain about 0.1 mg / ml to about 85 mg / ml of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain about 0.1 mg / ml to about 75 mg / ml of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain about 0.1 mg / ml to about 65, 55, 45, 35, 25, or 15 mg / ml of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain about 0.1 mg / ml to about 5, 6, 7, 8, 9, or 10 mg / ml of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain 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 of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain 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 of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition contains 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 of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition contains 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 of ACE2-Fc fusion protein.Preferably, in some embodiments, the ACE2-Fc fusion protein composition contains 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 of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition contains about 1 mg / ml to about 5 mg / ml of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain 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 of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain about 1.00 mg / ml of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain about 1.25 mg / ml of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain about 2.50 mg / ml of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain about 5.00 mg / ml of ACE2-Fc fusion protein. Preferably, in some embodiments, the ACE2-Fc fusion protein composition may contain about 10.00 mg / ml of ACE2-Fc fusion protein.

[0153] In some cases, the disclosed fusion protein may need to be used in combination with other agents (e.g., vaccines) or other pharmaceutical products that induce a protective response. For example, a nasal spray of a quadrivalent influenza vaccine is expected to provide protection against influenza A(H1N1), influenza A(H3N2), and two types of influenza B viruses. Formulations containing the fusion protein described herein can be administered concurrently or sequentially with vaccines or other pharmaceutical products against other viruses that infect humans via the oral cavity, nasal cavity, or upper respiratory tract ACE2 pathway, or against influenza.

[0154] The aforementioned combination therapy provides protection against multiple pathogens. In some cases, the combination therapy provides protection against multiple strains of the same pathogen. The combination therapy is crucial in minimizing the number of vaccinations required to provide protection against multiple pathogens or pathogenic strains, thereby reducing management costs and improving vaccination rates. This advantage can be particularly evident in the vaccination of infants and children.

[0155] The Disclosure also provides compositions comprising the ACE2-Fc fusion protein and a pharmaceutically acceptable carrier. In some embodiments, the compositions of the Disclosure comprise the ACE2-Fc fusion protein provided in the Disclosure and any pharmaceutically acceptable carrier such as water, buffer, or saline. In some embodiments, the compositions of the Disclosure comprise the ACE2-Fc fusion protein provided in the Disclosure and PBS buffer or saline. In some embodiments, the compositions of the Disclosure comprise the ACE2-Fc fusion protein provided in the Disclosure and PB buffer or saline. In some embodiments, the compositions of the Disclosure comprise the ACE2-Fc fusion protein provided in the Disclosure, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate monohydrate, sodium chloride, sucrose, phenethyl alcohol, and water, specifically comprising 0.1 to 15 mg / ml of ACE2-Fc fusion protein, 0.5 to 10 mg of disodium hydrogen phosphate dihydrate, 0.01 to 5 mg / ml of sodium dihydrogen phosphate monohydrate, 0.1 to 10 mg / ml of sodium chloride, 10 to 100 mg / ml of sucrose, 0.25 to 5 mg / ml of phenethyl alcohol, and an appropriate amount of water. In some embodiments, the composition of the present disclosure comprises 1 to 5 mg / ml of ACE2-Fc fusion protein, 2.13 mg of disodium hydrogen phosphate dihydrate, 0.82 mg / ml of sodium dihydrogen phosphate monohydrate, 2.92 mg / ml of sodium chloride, 50 mg / ml of sucrose, 2.50 mg / ml of phenethyl alcohol, and an appropriate amount of water. In some embodiments, the composition of the present disclosure comprises 1.25 mg / ml of ACE2-Fc fusion protein, 2.13 mg of disodium hydrogen phosphate dihydrate, 0.82 mg / ml of sodium dihydrogen phosphate monohydrate, 2.92 mg / ml of sodium chloride, 50 mg / ml of sucrose, and 2.50 mg / ml of phenethyl alcohol. In some embodiments, the compositions of the present disclosure contain 2.0 mg / ml of ACE2-Fc fusion protein, 2.13 mg of disodium hydrogen phosphate dihydrate, 0.82 mg / ml of sodium dihydrogen phosphate monohydrate, 2.92 mg / ml of sodium chloride, 50 mg / ml of sucrose, and 2.50 mg / ml of phenethyl alcohol.In some embodiments, the composition of the Disclosure comprises 2.5 mg / ml of ACE2-Fc fusion protein, and 2.13 mg of disodium hydrogen phosphate dihydrate, 0.82 mg / ml of sodium dihydrogen phosphate monohydrate, 2.92 mg / ml of sodium chloride, 50 mg / ml of sucrose, and 2.50 mg / ml of phenethyl alcohol. In some embodiments, the composition of the Disclosure comprises 5.0 mg / ml of ACE2-Fc fusion protein, and 2.13 mg of disodium hydrogen phosphate dihydrate, 0.82 mg / ml of sodium dihydrogen phosphate monohydrate, 2.92 mg / ml of sodium chloride, 50 mg / ml of sucrose, and 2.50 mg / ml of phenethyl alcohol. In some embodiments, the compositions of the Disclosure include the ACE2-Fc fusion protein provided in the Disclosure and disodium hydrogen phosphate (e.g., disodium hydrogen phosphate dihydrate), sodium dihydrogen phosphate (e.g., sodium dihydrogen phosphate monohydrate), sodium chloride, and Tween 80.

[0156] In some embodiments, the ACE2-Fc fusion protein composition comprises protein nanoparticles provided in this disclosure and optionally a pharmaceutically acceptable carrier. In some embodiments, the ACE2-Fc fusion protein composition comprises VLPs provided in this disclosure and optionally a pharmaceutically acceptable carrier. In some embodiments, the ACE2-Fc fusion protein composition comprises extracted nucleic acids provided in this disclosure and optionally a pharmaceutically acceptable carrier. In some embodiments, the ACE2-Fc fusion protein composition comprises a carrier provided in this disclosure and optionally a pharmaceutically acceptable carrier. In some embodiments, the ACE2-Fc fusion protein composition comprises a virus provided in this disclosure and optionally a pharmaceutically acceptable carrier. In some embodiments, the ACE2-Fc fusion protein composition comprises a pseudovirus provided in this disclosure and optionally a pharmaceutically acceptable carrier. In some embodiments, the ACE2-Fc fusion protein composition comprises cells provided in this disclosure and optionally a pharmaceutically acceptable carrier.

[0157] In some embodiments, ACE2-Fc fusion proteins are used prophylactically. In some embodiments, ACE2-Fc fusion proteins are used therapeutically. In some embodiments, ACE2-Fc fusion proteins are used prophylactically and therapeutically. Such pharmaceutical compositions can be administered to subjects via a variety of routes of administration known to those skilled in the art (e.g., intramuscular, intradermal, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, transnasal, sublingual, amygdala, oral / pharyngeal, and other extraintestinal and mucosal routes). Preferably, ACE2-Fc fusion protein compositions are administered intranasally. Practical methods for preparing administerable compositions are known or obvious to those skilled in the art and are described in detail in publications such as "Remingtons Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pa., 1995".

[0158] Therefore, the ACE2-Fc fusion proteins described herein may be formulated with pharmaceutically acceptable carriers to help maintain biological activity and promote improved stability during storage within an acceptable temperature range. Potential carriers include, but are not limited to, physiological equilibrium medium, phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, antifreeze additives or stabilizers (proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., monosodium glutamate) or their protective agents). The resulting aqueous solutions may be packaged and used as is or lyophilized. The lyophilized formulations are mixed with a sterile solution before single or multiple doses. In some embodiments, the phosphate-buffered saline contains sodium dihydrogen phosphate monohydrate or disodium hydrogen phosphate dihydrate.

[0159] In some embodiments, the ACE2-Fc fusion protein composition of the present invention comprises an aqueous support as a solvent. Suitable supports include, for example, sterile water, physiological saline, phosphate-buffered saline (PBS), and Ringer's solution. In some embodiments, the composition is isotonic. In some embodiments, the ACE2-Fc fusion protein composition of the present invention comprises phosphate-buffered saline (PBS) (e.g., NaCl, KCl, NaHPO₄, KH₂PO₄, or NaCl, NaHPO₄, NaH₂PO₄).

[0160] Formulations, particularly liquid formulations, may contain bacteriostatic agents that inhibit microbial growth to prevent or minimize degradation during storage. These include, but are not limited to, benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben at an effective concentration (usually 1% w / v). The bacteriostatic agent may comprise one or more of essential oils, high-concentration salts, high-concentration sugars, high-acid agents, and anaerobic environment-forming agents. In some embodiments, the bacteriostatic agent described herein comprises one or more of benzoic acid, sorbic acid, thimerosal, and phenethyl alcohol. In some embodiments, the bacteriostatic agent comprises thimerosal and / or phenethyl alcohol. Because the use of bacteriostatic agents may be prohibited for some patients, lyophilized formulations are reconstituted in solutions containing or not containing these components.

[0161] The composition of the present invention may contain pharmaceutically acceptable carrier substances necessary to approximate physiological conditions, such as pH adjusters and buffers, tension adjusters, isotonic agents, and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate, in order to approximate physiological conditions. The isotonic agent is one or more selected from glucose, dextrorotatory glucose, sucrose, glycerin, mannitol, sodium chloride, and potassium chloride, and is preferably dextrorotatory glucose.

[0162] In some embodiments, the ACE2-Fc fusion protein composition includes pharmaceutically acceptable excipients such as solvents, fillers, buffers, tension modifiers, and preservatives (Pramanick et al., Pharma Times, 45:65-77, 2013, which is incorporated herein by reference in its entirety for all purposes). In some embodiments, the fusion protein composition of the present disclosure may include excipients that function as one or more of solvents, fillers, buffers, and tension modifiers (for example, sodium chloride in physiological saline may function simultaneously as both an aqueous carrier and a tension modifier).

[0163] The ACE2-Fc fusion protein compositions described herein may contain buffers. Buffers control pH and inhibit the degradation of the active agent during handling, storage, and optionally during reconstitution. Suitable buffers include, for example, salts containing acetates, citrates, phosphates, or sulfates. Other suitable buffers include, for example, amino acids such as arginine, glycine, histidine, and lysine. Buffers may further contain hydrochloric acid or sodium hydroxide. In some embodiments, the buffer maintains the pH of the composition in the range of 6 to 9. In some embodiments, the pH is greater than 6, 7, or 8 (as a lower limit). In some embodiments, the pH is less than 9, 8, or 7 (as an upper limit). That is, the pH is in the range of approximately 6 to 9, with the lower limit less than the upper limit. In some embodiments, the pH value is between approximately 5.8 and 6.8.

[0164] The ACE2-Fc fusion protein compositions described herein may contain tension modifiers. Suitable tension modifiers include, for example, glucose, glycerol, sodium chloride, sucrose, and mannitol.

[0165] The ACE2-Fc fusion protein compositions described herein may contain one or more osmotic modifiers selected from sodium chloride, potassium chloride, glycerin, glucose, sorbitol, sucrose, xylitol, and mannitol. For example, sodium chloride and / or sucrose. The ACE2-Fc fusion protein compositions described herein may contain a solvent. The solvent includes one or more selected from ethanol, propylene glycol, and pure water, for example, water.

[0166] The ACE2-Fc fusion protein compositions described herein may include fillers. Fillers are particularly useful when the pharmaceutical composition is freeze-dried before administration. In some embodiments, the fillers function as protective agents, contributing to stabilization and prevention of degradation during the freeze-drying or spray-drying period and / or storage period. Suitable fillers are sugars (monosaccharides, disaccharides, and polysaccharides) such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0167] The ACE2-Fc fusion protein compositions described herein may contain preservatives. 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, with potassium sorbate being preferred. The suitable antioxidants may be a combination of one or more selected from EDTA-2Na, butylhydroxyanisole, 2,6-di-tert-butyl-4-methylphenol, sodium metabisulfite, potassium metabisulfite, butylated hydroxytoluene, L-ascorbic acid palmitate, sodium thiosulfate, and vitamin E. However, in preferred embodiments, the fusion protein compositions of this disclosure are prepared under sterile conditions and filled into single-use containers, and therefore do not need to contain preservatives.

[0168] The ACE2-Fc fusion protein compositions described herein may contain antimicrobial agents. The antimicrobial agent is a substance or drug that kills or inhibits the activity of bacteria or microorganisms. Suitable antimicrobial agents and / or bacteriostatic agents include one or more β-lactams (penicillins, cephalosporins, carbapenems, β-lactams and monobactams including enzyme inhibitors, etc.), aminoglycosides, tetracyclines, fluoroquinolones, sulfamonomethoxine, chloramphenicol, glycopeptides (vancomycin and teicoplanin), macrolides, etc. The antimicrobial agent may also include one or more chemically synthesized drugs such as sulfonamides, furans, and quinolones. The ACE2-Fc fusion protein compositions described herein do not have to contain bacteriostatic agents / antimicrobial agents.

[0169] The ACE2-Fc fusion protein compositions described herein may contain pharmaceutically acceptable flavorings or other taste and odor modifiers.

[0170] In some embodiments, the composition may be provided as a sterile composition. The pharmaceutical composition typically contains an effective amount of the ACE2-Fc fusion protein of this disclosure and is prepared by the prior art. Generally, the amount of the ACE2-Fc fusion protein composition per dose is selected to function as a soluble receptor that inhibits viral entry without serious side effects. In some embodiments, the composition is provided in unit dosage forms and may be used to inhibit viral entry that can bind to ACE2 receptors in the upper respiratory tract and nasal cavity of a subject. The unit dosage forms include a single predetermined dose suitable for administration to a subject, or two or more appropriately labeled or measured multiples of predetermined unit doses, and / or a measuring mechanism for administering unit doses or multiples thereof. In other embodiments, the composition further includes one or more adjuvants.

[0171] 4. Methods for inhibiting viral entry via receptors

[0172] In some embodiments, the Disclosure provides a method for preventing coronavirus infection in a subject. The method comprises administering an effective amount of a composition to a subject, the composition comprising a recombinant fusion protein selected from the group consisting of SEQ ID NO: 1-18. In some embodiments, the Disclosure provides a method for preventing coronavirus infection in a subject. The recombinant fusion protein comprises an ACE2 protein or a fragment thereof, and the method comprises administering an effective amount of a composition comprising a recombinant fusion protein selected from the group consisting of those shown in SEQ ID NO: 1-18 to a subject.

[0173] By administering the fusion proteins of this disclosure (e.g., ACE2-Fc fusion proteins, e.g., human ACE2-Fc fusion proteins disclosed herein, nucleic acid molecules (e.g., RNA molecules) or carriers encoding the human ACE2-Fc fusion proteins disclosed herein, or protein nanoparticles or virus-like particles containing the human ACE2-Fc fusion proteins disclosed herein) to a subject, infection by viruses that infect humans via the ACE2 pathway can be inhibited (i.e., viral infection can be prevented). In certain examples, the subject is human. This inhibition can be used to prevent, treat or suppress infections and diseases associated with the corresponding coronavirus, as it can then suppress the binding of the corresponding coronavirus to the subject's ACE2.

[0174] The fusion proteins or pharmaceutical compositions of this disclosure can be administered via intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intra-articular, intraperitoneal, transnasal, sublingual, tonsillar, oral cavity / pharynx, or other extraintestinal and mucosal administration routes, preferably transnasal administration.

[0175] It is possible to select subjects who are in situations where there is a high probability of coronavirus infection, such as being exposed to or potentially exposed to the coronavirus, and to protect them from infection.

[0176] Typical subjects receiving prevention by the therapies or methods of the present invention include humans, non-human primates, and other animals. To identify subjects receiving prevention or treatment by the methods of the present invention, acceptable screening methods are employed to assess risk factors associated with the target or suspected disease or condition, or to determine the pre-existing disease or condition status in the subjects. These screening methods include, for example, routine tests to assess environmental, familial, occupational, and other risk factors associated with 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 methods and other conventional methods enable clinicians to select subjects requiring prevention or treatment of disease using the methods and pharmaceutical compositions of the present invention. Based on these methods and principles, the compositions may be administered as a standalone preventive or therapeutic regimen, or as a follow-up, adjunct, or modified therapeutic regimen to other treatments, in accordance with the instructions of this disclosure or other conventional methods.

[0177] The fusion proteins of this disclosure (e.g., ACE2-Fc fusion proteins, e.g., human ACE2-Fc fusion proteins disclosed herein, nucleic acid molecules (e.g., RNA molecules) or carriers encoding the human ACE2-Fc fusion proteins disclosed herein, or protein nanoparticles or virus-like particles containing the human ACE2-Fc fusion proteins disclosed herein) can be administered for prophylactic or therapeutic purposes. In the case of prophylactic administration, the disclosed therapeutic agent is administered before symptoms such as infection appear. Prophylactic administration of the therapeutic agent of this disclosure is used to prevent or mitigate any subsequent infection. In the case of therapeutic administration, the disclosed therapeutic agent is administered when symptoms of the disease or infection appear or thereafter, e.g., after the progression of coronavirus infection symptoms corresponding to the coronavirus S antigen, or after a diagnosis of coronavirus infection. Thus, the therapeutic agent may be administered after exposure to the virus or after suspected exposure, or after actual infection has started, or before exposure to coronavirus is expected, in order to reduce the expected severity, duration, or degree of infection and / or associated disease symptoms.

[0178] The fusion proteins and their fusion protein compositions described in the present disclosure can effectively assist in preventing the infection of coronavirus in a subject (preferably a human). The actual dosage of the fusion protein of the present disclosure may vary based on factors such as, for example, the signs and specific conditions of the subject's disease (such as the subject's age, physique, health status, symptom degree, susceptibility factors, etc.), the administration time and route, other medications or treatments used in combination, and the specific pharmacological properties of the composition that elicit the desired activity or biological response in the subject. The dosage regimen can be adjusted to obtain an optimal prophylactic or therapeutic response.

[0179] The fusion proteins and their fusion protein compositions described in the present disclosure can be used simultaneously with, or before or after, one or more of the disclosed immunogenic compositions.

[0180] In some embodiments, an effective amount of the ACE2-Fc fusion protein composition of the present disclosure can be administered as a single dose or a series of doses at one or more intervals. The intervals can be set in units of time, days, or weeks. In some embodiments, a composition containing an effective amount of the ACE2-Fc fusion protein described in the present disclosure can be administered at intervals of 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours. In some embodiments, administration can be performed once, twice, or three times a day for several consecutive days. For example, administration can be performed once a day or twice a day for several days (such as 28 days) in a row, and administration twice a day can be performed continuously for one week, two weeks, three weeks, four weeks, or more. In some embodiments, administration once every two or three days can be performed continuously for several days.

[0181] The suitability of the fusion protein parameters to be selected (e.g., formulation, dosage, administration schedule, etc.) can be determined by collecting equivalent sera from the subject and measuring the antibody titer during the administration period. Furthermore, the clinical condition of the subject can be monitored to obtain the expected effects such as prevention of infection or reduction of disease state (e.g., reduction of virus amount). If such monitoring indicates that the ACE2-Fc fusion protein is not optimal, the subject is enhanced by additional administration of the ACE2-Fc fusion protein composition, and the usage parameters of the ACE2-Fc fusion protein formulation are improved in a manner expected to enhance the infection-blocking effect.

[0182] Typically, a single dose for humans contains 0.01 to 20 mg of the ACE2-Fc fusion protein, such as about 0.05 mg to about 10 mg, or about 0.1 mg to about 8 mg, specifically about 0.50 mg, about 1.00 mg, 1.50 mg, about 2.00 mg, about 2.50 mg, 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 the ACE2-Fc fusion protein.

[0183] In protein therapy, a typical single dose for humans contains approximately 0.01–3 mg of ACE2-Fc fusion protein. In some embodiments, a single dose of ACE2-Fc fusion protein composition may contain approximately 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 ACE2-Fc fusion protein composition may contain about 0.05–0.06, 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 ACE2-Fc fusion protein.In some embodiments, a single dose of the ACE2-Fc fusion protein composition is approximately 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, 0.39, 0. It may contain 4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.55, 0.6, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.20, 1.25, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.00, 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. In this disclosure, “single dose” means the amount applied in a single administration, which may be one or more (e.g., two or three) sprays to each nostril.

[0184] In fusion protein compositions, the dose is selected based on the target population (e.g., infants or the elderly). Standard studies observing the antibody titers and other responses of subjects can determine the optimal dose of a particular component. It should be understood that the therapeutically effective dose of a disclosed fusion protein may include a dose that is insufficient to block viral infection with a single dose, but is effective with multiple doses.

[0185] A decrease or reduction in the viral load in a subject's body (e.g., viral load in the lungs) after administration of the fusion protein of this disclosure (compared to the control group or before administration) indicates that an effective dose of the fusion protein has been delivered to the subject. In some embodiments, the subject's antibody response is measured when evaluating the effective dose / immunization plan. In most cases, it is sufficient to assess the antibody titer in serum or plasma collected from the subject. The decision of whether to change the therapeutic dose to an individual can be made at least in part on the level of antibody titer. The level of antibody titer can be assessed, for example, by an immunobinding assay, which measures the concentration of antibodies in serum that bind to an antigen (including recombinant coronavirus S antigen, e.g., S-trimer).

[0186] The method is effective even without the need for complete elimination, reduction, or prevention of coronavirus infection. For example, the fusion protein of this disclosure can reduce or suppress a desired level of coronavirus infection compared to coronavirus infection without administration of the fusion protein. For example, suppression effects of 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%, and even up to 100% (complete elimination or prevention of detectable infected cells) can be achieved.

[0187] The fusion proteins disclosed herein 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; this document is cited herein as an incorporated reference for all purposes). In some embodiments, the fusion proteins disclosed herein are administered via DNA.

[0188] In some embodiments, administering one or more of the disclosed fusion proteins to a subject in a therapeutically effective dose can inhibit the subject's viral infection. To evaluate the viral infection inhibitory effect, serum is collected at an appropriate time after administration to the subject and cryopreserved for neutralization testing. Methods for measuring neutralizing activity are known to those skilled in the art and include, but are not limited to, plaque reduction neutralization assays (PRNT), microneutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, viral neutralizing activity in serum can be measured using a panel of coronavirus pseudoviruses.

[0189] 5. Product or kit

[0190] This disclosure further provides a product or kit comprising a recombinant polypeptide, protein composition, etc. The product further includes a container and labels or accompanying documents attached to or related to the container. Suitable containers include, for example, bottles, vials, syringes, test tubes, IV infusion bags, etc. Containers may be made of various materials (e.g., glass or plastic). In some embodiments, the container is equipped with a sterile inlet. Typical containers include intravenous infusion bags, vials, containers with stoppers that can be punctured with a needle, etc. The product or kit may further include accompanying documents indicating that the product can be used for the prevention or treatment of a specific disease (e.g., coronavirus infection as described in this disclosure). Alternatively, the product or kit may further include another container, or the same container, containing pharmaceutically acceptable buffers, other additives, or adjuvants. Furthermore, it may include other materials such as other buffers, diluents, filters, needles and / or syringes.

[0191] The label or accompanying information may indicate that the composition is used for the prevention or treatment of coronavirus infection in individuals. Labels or accompanying information attached to or associated with the container may provide instructions on the preparation or use of the formulation. The label or accompanying information may further indicate that the formulation can be used for intranasal administration for the purpose of preventing coronavirus infection in individuals.

[0192] In some embodiments, the container contains an ACE2-Fc fusion protein or a composition thereof. The ACE2-Fc fusion protein or a composition thereof may be used alone or in combination with other compositions that can effectively treat, prevent and / or diagnose a disease. The product or kit may include (a) a first container containing a composition comprising an immune ACE2-Fc fusion protein or recombinant polypeptide or a composition thereof (i.e., a first agent), and (b) a second container containing a composition comprising another agent such as an adjuvant or other therapeutic agent (i.e., a second agent). Furthermore, the product or kit may include a label or accompanying leaflet containing instructions for treating a subject with an effective amount of the second agent.

[0193] All terms, symbols, and other technical and specialized terms or terminology used in this disclosure have the same meaning as commonly understood by those skilled in the art in the field in which the subject matter of protection is claimed, unless otherwise specified. In some cases, for clarity and / or convenience of reference, terms that have a commonly understood meaning may be defined in this disclosure, and the definitions included in this disclosure do not necessarily represent a substantial difference from the commonly understood meaning in the art.

[0194] 6. Explanation of Terms

[0195] The terms “polypeptide” and “protein” are used interchangeably to refer to polymers of amino acid residues and are not limited to the minimum length. Polypeptides (the receptors and other polypeptides provided, e.g., linkers or peptides) may contain amino acid residues, including native and / or non-native amino acid residues. The term further includes post-translational modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, polypeptides may include modifications to the native or natural sequence, as long as the protein retains the desired activity. These modifications may be intentional alterations by site-directed mutagenesis or accidental alterations due to mutations in the protein-producing host or errors associated with PCR amplification.

[0196] As used in this disclosure, “subject” refers to a mammal, including humans or other animals, and usually means human. In some embodiments, the subject (e.g., patient) administered one or more drugs, cells, cell populations, or compositions is a mammal, usually a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject may be male or female and may be of any appropriate age group, such as an infant, child, adolescent, adult, or elderly. In some embodiments, the subject may be a non-primate mammal, such as a rodent.

[0197] As used in this disclosure, “treatment” (and grammatical variations such as “treat” and “treating”) means to completely or partially improve or reduce a disease, disorder, or condition, or any symptoms, adverse reactions, consequences, or phenotype associated therewith. Favorable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, improvement or mitigation of the disease state, and improvement of prognosis. The term does not necessarily imply a complete cure of the disease, complete disappearance of any symptoms, or an effect on all symptoms or consequences.

[0198] As used herein, "slowing the progression of a disease" means slowing, hindering, alleviating, decelerating, stabilizing, suppressing, and / or delaying the progression of a disease (e.g., cancer). The time to slow may vary depending on the medical history and / or the individual receiving the treatment. In some embodiments, "sufficient or substantial delay" may include "prevention" since the individual does not develop the disease. For example, the progression of advanced cancer such as metastasis can be slowed.

[0199] As used herein, "prevention" includes preventing the onset or recurrence of a disease in a subject who is susceptible to the disease but has not yet had a confirmed diagnosis. In some embodiments, the provided cells and compositions are used to delay the progression of a disease or reduce the rate of progression of a disease.

[0200] As used herein, "suppressing" a function or activity refers to reducing a function or activity as compared to another identical condition excluding the condition or parameter of interest, or as compared to another condition. For example, cells that suppress tumor growth reduce the rate of tumor growth as compared to the rate of tumor growth in the absence of cells.

[0201] When administered, an "effective amount" of an agent (e.g., a pharmaceutical formulation, cell or composition) refers to an amount effective to achieve a desired effect such as a therapeutic or prophylactic effect, for example, the dosage / administration and period necessary to achieve the desired effect.

[0202] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical formulation or cell) refers to an amount effective to achieve a desired therapeutic effect (such as the treatment of a disease, disorder or condition) and / or pharmacokinetic and pharmacodynamic effects, such as the dosage and period necessary to achieve the desired effect. The therapeutically effective amount may vary depending on factors such as the severity of the disease in the subject, age, gender, weight, and the characteristics of the cell population being administered. In some embodiments, the methods of the present disclosure include administering cells and / or compositions in an effective amount (e.g., a therapeutically effective amount).

[0203] The “preventive effective dose” refers to the effective dose and duration required to achieve the desired preventive effect. In most cases (but not all), prophylactic administration is given to subjects who are asymptomatic or in the early stages, so the preventive effective dose is less than the therapeutic effective dose. In situations with low tumor burden, the preventive effective dose may exceed the therapeutic effective dose in some embodiments. The effective dose of a vaccine or other drug is sufficient to elicit a desired response, such as reducing or eliminating the signs or symptoms of a disease or illness, such as pneumonia. For example, this may be the amount necessary to suppress viral replication or to alter the external symptoms of a viral infection to a measurable degree. Generally, this amount is sufficient to suppress the replication or infectivity of a virus (e.g., SARS-CoV-2) to a measurable degree. When administered to a subject, a dose is usually used that reaches the target tissue concentration in which suppression of viral replication has been demonstrated in vitro. In some embodiments, the “effective dose” refers to the amount necessary for the treatment (including prevention) of one or more symptoms and / or potential causes of any disease or illness, such as coronavirus infection. In some embodiments, the effective dose is the therapeutic effective dose. In some embodiments, an effective dose is an amount sufficient to prevent the progression of signs or symptoms of a particular disease or condition, such as one or more signs or symptoms associated with coronavirus infection.

[0204] As used in this disclosure, the terms “antigen” or “immunogen” are synonymous when referring to a substance (usually a protein) that can induce an immune response in a subject. The terms further refer to immunoactive proteins, i.e., substances that, when administered to a subject (directly or indirectly via a nucleotide sequence or vector encoding such protein), can evoke a humoral and / or cellular immune response to such protein. Unless otherwise specified, the term “vaccine immunogen” is synonymous with “protein antigen” or “immunogenic polypeptide.”

[0205] The term "conservative modified variant" applies to amino acid sequences and nucleic acid sequences. For a given nucleic acid sequence, a conservative modified variant refers to a nucleic acid that codes for the same or essentially the same amino acid sequence, or, in the case of nucleic acids that do not code for an amino acid sequence, an essentially identical sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For polypeptide sequences, a "conservative modified variant" refers to a variant having a conservative amino acid substitution, meaning that an amino acid residue is replaced by another amino acid residue with a side chain having similar charge properties. Families of amino acid residues with side chains having similar charge properties have already been defined in the art. These families include 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), β-branched chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0206] An epitope refers to an antigenic determinant. These are specific chemical groups or peptide sequences on an antigen molecule that induce a specific immune response. For example, an epitope functions as an antigenic region that induces a B cell and / or T cell response. Epitopes can be formed by consecutive amino acids or by discontinuous amino acids arranged in parallel through the triple folding of a protein.

[0207] Unless otherwise specified, the fusion proteins of this disclosure are recombinant proteins, which comprise the amino acid sequences of at least two non-homologous proteins linked together via peptide bonds to form a single protein. Therefore, they do not contain naturally occurring coronavirus surface antigens. Non-homologous amino acid sequences may be linked directly to one another or indirectly via linker sequences. As used in this disclosure, proteins are non-homologous if their amino acid sequences do not normally link via peptide bonds in their natural environment (e.g., intracellularly). For example, the amino acid sequences of ACE2 and Fc are not normally linked via peptide bonds.

[0208] Immunogens are proteins or parts thereof that can induce an immune response in mammals infected with or at risk of infection by a pathogen. Administration of immunogens can induce protective and / or active immunity against a target pathogen.

[0209] An immunogenic composition refers to a composition comprising an immunogenic polypeptide, the immunogenic polypeptide inducing a measurable CTL response to a virus expressing the immunogenic polypeptide, or a measurable B-cell response (e.g., antibody production) to the immunogenic polypeptide.

[0210] Sequence identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is indicated by the identity or similarity between the sequences. Sequence identity is expressed as percentage identity, with higher percentages indicating higher sequence identity. When comparison and alignment are performed on a specified region measured through a comparison window to obtain the maximum correspondence, or using one of the following sequence comparison algorithms, or by manual comparison and visual inspection, the two sequences are "basically identical" (i.e., 60% sequence identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity in the specified region or, if not specified, in the entire sequence). Where necessary, identity resides in a region of at least about 50 nucleotides (or 10 amino acids) in length, more preferably in a region of 100-500 or 1000 or more nucleotides (or 20, 50, or 200 or more amino acids) in length.

[0211] A 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 to a pathogen antigen (e.g., a viral pathogen) or cellular components associated with a pathological situation. A vaccine may contain a polynucleotide (e.g., a nucleic acid encoding the disclosed antigen), a peptide or polypeptide (e.g., the disclosed antigen), a virus, a cell, or one or more cellular components. In some embodiments, the vaccine or vaccine immunogen, or vaccine composition, is expressed from a fusion construct and self-associates to form nanoparticles having immunogenic polypeptides or proteins on their surface.

[0212] Virus-like particles (VLPs) refer to non-replicating viral shells derived from any one virus selected from several species. VLPs typically consist of one or more viral proteins (including, but not limited to, proteins called capsid, coating, shell, surface, and / or envelope proteins, or polypeptides formed by particles derived from these proteins). VLPs formed spontaneously by the expression of recombinant proteins in a suitable expression system. Methods for producing specific VLPs are known in the field. Following recombinant expression of viral proteins, the presence of VLPs makes them detectable by techniques known in the field (e.g., electron microscopy, biophysical assessment). See Baker et al. (1991) Biophys. J. 60:1445-1456 and Hagensee et al. (1994) J. Virol. 68:4503-4505, whose full texts are incorporated herein by reference for all purposes. For example, VLPs can be separated by density gradient centrifugation and / or identified by characteristic density bands. Alternatively, cryo-electron microscopy can be performed on vitrified, hydrated samples prepared from the VLP in question, and images can be recorded under appropriate exposure conditions.

[0213] As used in this disclosure, the term “approximately” refers to a general range of error for each numerical value that is readily apparent to those skilled in the art. In this disclosure, “approximately” descriptions relating to numerical values ​​or parameters include embodiments that include such value or parameter itself.

[0214] As used in this disclosure, the singular forms “a / an” and “the” refer to multiple objects unless the context explicitly indicates otherwise. For example, “a / an” means “at least one,” “one or more,” or “one or more.”

[0215] In this disclosure, various aspects of the subject matter for which protection is claimed are presented in range form. It should be understood that this range form is adopted for convenience and brevity and should not be interpreted as a rigid limitation on the scope of the claimed subject matter. Therefore, the range description should be considered as specifically disclosing all possible sub-ranges and individual numerical values ​​within those ranges. For example, if a range of values ​​is provided, each intermediate value between the upper and lower limits of that range, and any other described intermediate value within that range, should be understood to be included in the subject matter of the claims. The upper and lower limits of these smaller ranges are independently included within the smaller ranges and are included in the subject matter of the claims, but subject to the limitations explicitly excluded within the ranges. If the range includes one or two limit values, the range that does not include either or both of those limit values ​​is also included in the subject matter of the claims. This applies to the width of any range.

[0216] As used in this disclosure, a composition means two or more products, substances, or compounds (including cells), or any mixture thereof. This may be a solution, suspension, liquid, powder, paste, or aqueous, non-aqueous, or any combination thereof.

[0217] As used in this disclosure, the term “carrier” refers to a nucleic acid molecule capable of propagating another nucleic acid bound to it. The term includes both carriers as self-replicating nucleic acid structures and carriers integrated into the genome of a host cell into which they are introduced. Certain carriers can induce the expression of a nucleic acid functionally linked to them. Such carriers are referred to in this disclosure as “expression carriers.”

[0218] 7. Exemplary Embodiments

[0219] Embodiment 1. A fusion protein comprising a plurality of recombinant polypeptides, wherein the fusion protein comprises a human ACE2 protein or a fragment thereof, or a human IgG Fc protein or a functional variant thereof.

[0220] Embodiment 2. The fusion protein according to Embodiment 1, wherein the human ACE2 protein or a fragment thereof includes the human ACE2 extracellular domain or a fragment thereof.

[0221] Embodiment 3. A fusion protein according to Embodiment 1 or 2, wherein the human ACE2 protein or a fragment thereof comprises an amino acid sequence shown in any of SEQ ID NO: 5, 7, 11, 12, 13, or 14, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0222] Embodiment 4. A fusion protein according to any one of Embodiments 1 to 3, wherein the human ACE2 protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0223] Embodiment 5. A fusion protein according to any one of Embodiments 1 to 4, wherein the human IgG Fc protein or its functional variant comprises one or more Fc region sequences selected from human IgG1 Fc, IgG2 Fc, IgG3 Fc, and IgG4 Fc, or functional variants and fragments thereof.

[0224] Embodiment 6. A fusion protein according to any one of Embodiments 1 to 5, wherein the human IgG Fc protein or its functional variant is selected from human IgG1 Fc region sequences or their functional variants and fragments.

[0225] Embodiment 7. A fusion protein according to any one of Embodiments 1 to 6, wherein the human IgG Fc protein or its functional variant comprises an amino acid sequence shown in SEQ ID NO: 6 or 8, or a fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0226] Embodiment 8. A fusion protein according to any one of Embodiments 1 to 7, wherein the human IgG Fc protein or its functional variant comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0227] Embodiment 9. A fusion protein according to any one of Embodiments 1 to 8, which optionally includes a signal peptide.

[0228] Embodiment 10. A fusion protein according to any one of Embodiments 1 to 9, which optionally includes a peptide linker, wherein the human ACE2 protein or a fragment thereof and the human IgG Fc protein or a functional variant thereof are either directly bound or bound via the peptide linker.

[0229] Embodiment 11. A fusion protein according to Embodiment 10, wherein the peptide linker is selected from an arginine-serine peptide linker (-RS-), a valine-serine linker (-VS-), and a glycine-serine linker (-GS-).

[0230] Embodiment 12. A fusion protein according to any one of Embodiments 1 to 11, which optionally includes a mutant sequence.

[0231] Embodiment 13. A fusion protein according to Embodiment 1, comprising an amino acid sequence shown in any of SEQ ID NO: 1-18, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith, or a combination thereof.

[0232] Embodiment 14. A fusion protein according to Embodiment 1, comprising an amino acid sequence shown in any of SEQ ID NO: 1-4 and 15-18, or an amino acid sequence or fragment having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith, for example, an amino acid sequence shown in any of SEQ ID NO: 1-4 or 15-18, or an amino acid sequence or fragment having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[0233] Embodiment 15. A fusion protein according to any one of Embodiments 1 to 14, further comprising a detectable tag.

[0234] Embodiment 16. A pharmaceutical composition comprising a fusion protein described in any one of Embodiments 1 to 15 and any pharmaceutically acceptable carrier.

[0235] Embodiment 17. A pharmaceutical composition according to Embodiment 16, comprising the fusion protein described in any one of Embodiments 1 to 15 in an amount of about 0.1 mg / ml to about 100 mg / ml, for example, about 0.50 mg / ml to about 20.00 mg / ml, about 1.25 mg / ml, about 2.50 mg / ml, or about 5.00 mg / ml.

[0236] Embodiment 18. A pharmaceutical composition according to Embodiment 16 or 17, wherein the pharmaceutically acceptable carrier comprises one or more combinations of buffers and osmotic pressure modifiers.

[0237] Embodiment 19. The pharmaceutical composition according to Embodiment 18, wherein the buffering agent is selected from sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dihydrate, or a combination thereof.

[0238] Embodiment 20. A pharmaceutical composition according to Embodiment 18 or 19, wherein the osmotic pressure regulator comprises one or more combinations selected from the group consisting of sodium chloride, potassium chloride, glycerin, glucose, sorbitol, sucrose, xylitol, or mannitol.

[0239] Embodiment 21. A pharmaceutical composition according to any one of Embodiments 18 to 20, wherein the osmotic pressure adjusting agent comprises sodium chloride and / or sucrose.

[0240] Embodiment 22. A pharmaceutical composition according to any one of Embodiments 18 to 21, wherein the pharmaceutically acceptable carrier optionally includes one or more combinations selected from the group consisting of proteins, peptides or their hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), and amino acids (e.g., monosodium glutamate), for example, a stabilizer containing sucrose.

[0241] Embodiment 23. A pharmaceutical composition according to any one of Embodiments 18 to 22, wherein the pharmaceutically acceptable carrier optionally comprises one or more combinations selected from the group consisting of benzoic acid, sorbic acid, thimerosal, and phenethyl alcohol, for example, a bacteriostatic agent containing phenethyl alcohol and / or thimerosal.

[0242] Embodiment 24. A pharmaceutical composition according to any one of Embodiments 18 to 23, wherein the composition is in a dosage form applicable to intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intra-articular, intraperitoneal, transnasal, sublingual, tonsillar, oral cavity / pharynx, or other extraintestinal or mucosal administration routes, preferably a dosage form suitable for transnasal administration such as a nasal spray.

[0243] Embodiment 25. Application of a fusion protein according to any one of Embodiments 1 to 15, or a pharmaceutical composition according to any one of Embodiments 16 to 24, for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants to a previously infected subject.

[0244] Embodiment 26. A fusion protein according to any one of Embodiments 1 to 15, or a pharmaceutical composition according to any one of Embodiments 16 to 24, for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants as described in Embodiment 25, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants to a previously infected subject, wherein the coronavirus SARS-CoV-2 and its variants are not limited to SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, mu, omicron, and JN.1, but also include unknown coronavirus SARS-CoV-2 variants.

[0245] Embodiment 27. A fusion protein according to any one of Embodiments 1 to 15, or a pharmaceutical composition according to any one of Embodiments 16 to 24, for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants in a previously infected subject, wherein the fusion protein or pharmaceutical composition is administered via intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intra-articular, intraperitoneal, transnasal, sublingual, tonsillar, oral cavity / pharynx, or other extraintestinal and mucosal administration routes, preferably transnasally.

[0246] Embodiment 28. A fusion protein according to any one of Embodiments 1 to 15, or a pharmaceutical composition according to any one of Embodiments 16 to 24, for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants to a previously infected subject, wherein the fusion protein or pharmaceutical composition is administered intranasally as a single dose or as multiple doses at intervals of hours, days, or weeks.

[0247] Embodiment 29. A fusion protein according to any one of Embodiments 1 to 15, or a pharmaceutical composition according to any one of Embodiments 16 to 24, for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants as described in Embodiment 28, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants to a previously infected subject, wherein the interval is 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours.

[0248] Embodiment 30. A fusion protein according to any one of Embodiments 1 to 15, or a pharmaceutical composition according to any one of Embodiments 16 to 24, for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants to a previously infected subject, wherein the fusion protein or pharmaceutical composition is administered once or more times per day, for example, once or twice per day.

[0249] Embodiment 31. Application of a fusion protein according to any one of Embodiments 1 to 15, or a pharmaceutical composition according to any one of Embodiments 16 to 24, in the manufacture of a pharmaceutical for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants to a previously infected subject.

[0250] Embodiment 32. The use described in Embodiment 31, wherein the coronavirus SARS-CoV-2 and its variants are not limited to SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, mu, omicron, and JN.1, but also include variants of unknown coronavirus SARS-CoV-2.

[0251] Embodiment 33. A method for preventing and treating infection with coronavirus SARS-CoV-2 and its variants, and / or a method for preventing the transmission of coronavirus SARS-CoV-2 and its variants to a previously infected subject, comprising administering to the subject a therapeutically effective amount of the fusion protein described in any one of Embodiments 1 to 15 or the pharmaceutical composition described in any one of Embodiments 16 to 24.

[0252] Embodiment 34. The method according to Embodiment 33, wherein the coronavirus SARS-CoV-2 and its variants are not limited to SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, mu, omicron, and JN.1, but also include variants of unknown coronavirus SARS-CoV-2.

[0253] Embodiment 35. The method according to Embodiment 33 or 34, wherein the fusion protein or pharmaceutical composition is administered via intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intra-articular, intraperitoneal, transnasal, sublingual, amygdala, oral cavity / pharynx, or other extraintestinal and mucosal administration routes, preferably transnasal administration.

[0254] Embodiment 36. The method according to any one of Embodiments 33 to 35, wherein the fusion protein described in any one of Embodiments 1 to 15 or the pharmaceutical composition described in any one of Embodiments 16 to 24 is administered intranasally as a single dose or as multiple doses separated by hours, days, or weeks.

[0255] Embodiment 37. The method according to Embodiment 36, wherein the interval is 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours.

[0256] Embodiment 38. The method according to any one of Embodiments 33 to 37, wherein the fusion protein or pharmaceutical composition is administered once or more times a day, for example, once a day or twice a day.

[0257] Embodiment 39. A kit for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants to a previously infected subject, wherein the kit comprises a fusion protein according to any one of Embodiments 1 to 15, or a pharmaceutical composition according to any one of claims 16 to 24. Container and Optionally, include package inserts or labels indicating prevention and / or treatment.

[0258] Embodiment 40. The kit according to Embodiment 39, wherein the coronavirus SARS-CoV-2 and its variants are not limited to SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, mu, omicron, and JN.1, but also include variants of unknown coronavirus SARS-CoV-2.

[0259] Embodiment 41. A nucleic acid encoding a fusion protein or a fragment thereof as described in any one of Embodiments 1 to 15.

[0260] Embodiment 42. A carrier comprising the nucleic acid described in Embodiment 41.

[0261] Embodiment 43. The carrier described in Embodiment 33, wherein the carrier is phFC(IM).

[0262] Embodiment 44. A host cell comprising the nucleic acid described in Embodiment 41 or the carrier described in Embodiment 42.

[0263] Embodiment 45. The host cell described in Embodiment 44, wherein the host cell is a CHO cell (e.g., GH-CHO).

[0264] The following examples are provided for illustrative purposes only and do not limit the scope of the present invention. [Examples]

[0265] Production, purification, and manufacture of ACE2-Fc fusion protein and nasal spray.

[0266] In this example, an ACE2-Fc fusion protein having the amino acid sequence shown in SEQ ID NO:4 (SCB-719) was prepared and purified and used in the biological tests performed in the following examples. SEQ ID NO:4 synthesized in the example has a C-terminal modification, which allows for traceable detection without affecting the functional effect of the ACE2-Fc fusion protein. Those skilled in the art will fully understand that ACE2-Fc fusion proteins shown, for example, SEQ ID NO:1-3 and 15-18, can exhibit equivalent or comparable biological functional effects, regardless of the presence or absence of this C-terminal modification.

[0267] To efficiently express the ACE2-Fc fusion protein (see Liu et al., Scientific Reports, 7(1):8953, 2017, which is incorporated herein by reference in its entirety for all purposes), the cDNA encoding the ACE2-Fc fusion protein was subcloned into the expression carrier phFC(IM) and grown in the Chinese hamster ovarian cell line GH-CHO.

[0268] Taking the 200L cell culture process as an example, this process can be divided into a seed propagation stage and a fed-batch culture stage. Seed propagation stage: Using CD118 medium, one vial of cells is collected from the working cell bank (WCB), cultured in a 250ml shaking flask for 3-5 days for subculturing, and after subculturing in shaking flasks for N-4 and N-3 stages, the cells are inoculated into a WAVE bioreactor for N-2 and N-1 stages of growth. Fed-batch culture stage: On day 3 of the N-1 stage culture, the cells are inoculated into a 200L bioreactor. From day 3 to day 15, 2% CB7a and 0.2% CB7b are added daily relative to the initial culture volume. On day 4, the culture temperature is lowered from 37°C to 32°C. The glucose concentration is maintained at 2-10 g / L during the culture period. Cells are harvested on days 14-16 of culture, or when the cell viability is <90%. During harvesting, deep filtration is performed using a two-stage membrane of Merck's D0SP and A1HC. After sterilization filtration, the obtained cell harvest is transferred to the downstream purification process.

[0269] To obtain high-purity fusion proteins for pharmaceutical research, the following steps were performed. First, the sample was rapidly captured from the cell culture recovery using Cytiva's Mabselect PrismA affinity chromatography packing, followed by cation exchange chromatography in flow mode (packing: Monomix HC 60SP cation exchange chromatography packing manufactured by Suzhou Saifen Technology Co., Ltd.). This step was intended to remove the polymer of the target product. The cation exchange chromatography fraction was treated with S / D virus inactivation by adding 1% Tween 80 and 0.3% TNBP, and then purified using Merck's Eshmuno Q anion exchange chromatography packing in elution mode to further improve sample purity by removing impurities such as HCP and DNA. Next, the virus was removed by nanofiltration using Asahi Kasei Corporation's Planova 20N. Finally, the solution was concentrated and buffer-exchanged using a 50kDa ultrafiltration module made of PES material manufactured by Hangzhou KEBAITE Filtration Equipment Co., Ltd. The solution was then replaced with a 20mM PB, 50mM NaCl, and 5% sucrose solution to adjust the sample concentration to ≥10g / L. Finally, a 0.2μm sterilization filtration was performed to obtain the stock solution. Using the ACE2-Fc fusion protein purification platform, a high-purity target protein with a purity of >98% was obtained (see Figure 4C for the assay chromatogram). Stability analysis of the purified ACE2-Fc fusion protein confirmed that the ACE2-Fc fusion protein maintains its stability at 25°C.

[0270] The ACE2-Fc fusion protein concentrate underwent thawing, semi-finish preparation, sterilization filtration, aseptic filling, capping, labeling, and packaging processes to be formulated as an ACE2-Fc fusion protein nasal spray.

[0271] The formulation development study for the ACE2-Fc fusion protein nasal spray was conducted in four stages: pH screening, additive single-factor testing, DoE testing, and formulation determination testing. Under accelerated conditions (25±2℃) and high-temperature conditions (40±2℃), the trend in change in purity (SEC-HPLC) was set as the primary evaluation indicator, and the drug solution osmotic pressure was set as a secondary evaluation indicator to determine the formulation. The final formulations are shown in Tables 2a-2e.

[0272] [Table 3]

[0273] [Table 4]

[0274] [Table 5]

[0275] [Table 6]

[0276] [Table 7]

[0277] When the nasal spray disclosed in this disclosure was stored under long-term conditions (2-8°C) for 6 months and under accelerated conditions (25±2°C) for 4 months, its purity did not show any clear trend of change compared to time zero and met the acceptable standard. When stored under high-temperature conditions (40±2°C) for 1 month, a clear decrease in purity was observed, but it was still within the acceptable standard range.

[0278] The nasal spray disclosed herein is a sterile nasal spray for multiple doses (e.g., 100 μl per nostril, 2.8 ml per bottle). The manufacturing process employs sterile packaging materials and a sterile filling process to ensure the sterility of the shipped product. The antimicrobial pump used employs special sealing technology at the nozzle and an independent filter design in the flow path, maintaining the sterility of the product even after multiple uses. [Examples]

[0279] Construction and production of pseudoviruses

[0280] The spike protein (spike, S) gene of the SARS-CoV-2 high-concern mutant was synthesized by Genscript after mammalian codon optimization and cloned onto the pcDNA3.1(+) eukaryotic expression carrier. Plasmids encoding SARS-CoV-2 mutant S glycoproteins such as Hu-1, Alpha (α), Beta (β), Gamma (γ), Delta (δ), Miu (μ), Omicron (ο), and JN.1 were constructed. The lentivirus packaging plasmid psPAX2 and the pLVX-AcGFP-N1-Fluc lentivirus reporter plasmid expressing GFP and luciferase were procured from HonorGene (China). Pseudoviruses were produced by co-transfection of HEK 293T cells with plasmids encoding psPAX2, pLVX-AcGFP-N1-Fluc, and various S genes using Lipofectamine 3000 (Invitrogen, L3000-015). The supernatant was collected 24±2 hours after transfection, centrifuged at 1500 rpm for 5 minutes to remove cell residue, and then stored at -80°C. Titration of the pseudovirus reservoir was performed by infection of 293T-ACE2 cells. After adding the Bright-Glo luciferase assay system (Promega, E2650) and culturing at 37°C and 5% CO2 for 44-48 hours, luciferase activity was measured using a microplate reader (TECAN, Spark). Subsequently, the TCID of pseudoviruses was determined based on the Reed-Muench assay (Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus based assay. Nie J. et al. DOI:10.21203 / rs.3.pex-941 / v11). 50 The result was calculated. [Examples]

[0281] Neutralization test

[0282] Equal volumes of the test serum sample were first heat-inactivated at 56°C for 30 minutes, then clarified by centrifugation at 10,000 rcf for 5 minutes. The samples were successively diluted 3-fold with detection medium (100 ml) and 650 TCID. 50 Pseudovirus (50 ml) was cultured at 37°C for 1 hour, while simultaneously using untreated virus-infected controls (virus only) and cell-only controls (background controls). Fresh trypsin-treated 293T-ACE2 cells were added to each well at 100 mcL of 20,000 cells / well. After culturing at 37°C in a 5% CO2 incubator for 44–48 hours, cells were lysed according to the manufacturer's solution, and luciferase activity was measured using the Bright-Glo luciferase assay system (Promega). The IC50 neutralizing antibody titer of a given serum sample was defined as the serum dilution at which the relative luminescence (RLU) decreased by 50% compared to the virus-infected control well. Detailed methods followed the procedures described in "Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay. Nie J. et al. DOI:10.21203 / rs.3.pex-941 / v11".

[0283] The measurement results revealed that the ACE2-Fc fusion protein disclosed in this disclosure has neutralizing activity against all currently existing mutant strains of the novel coronavirus, and that its neutralizing activity against mutant strains is higher than that of the wild-type original strain. [Examples]

[0284] Evaluation of the neutralizing activity of ACE2-Fc fusion protein against existing SARS-CoV-2 mutant strains and primitive pseudovirus strains.

[0285] The measurement results in Figures 5A-5C clearly show that the ACE2-Fc fusion protein exhibits neutralizing activity against all currently identified COVID-19 mutants, and that its neutralizing activity against mutants is higher than that against the original strain. The test results demonstrate that selecting the ACE2-Fc fusion protein as a biological protective barrier against COVID-19 infection is effective against all mutants, and that regardless of the site of mutation in the COVID-19 mutant, there is no fundamental change in its mechanism of entry into the human body. The ACE2-Fc fusion protein is demonstrated to exhibit high neutralizing activity against all coronavirus mutants, both known and unknown. This suggests that the ACE2-Fc fusion protein disclosed herein can be applied for the prevention and treatment of COVID-19 infection and transmission. [Examples]

[0286] Challenge trials (pharmacodynamic studies) in genetically modified mice using nasal administration.

[0287] As shown in Figures 6A-6B, a delta virus challenge test was conducted using genetically modified mice expressing human ACE2. In the test, 40 mice were divided into four groups of 10 mice each. On the third day after infection (5 mice), body weight, lung viral load measurement, lung pathological analysis, and scoring were performed. Group 1 was the control group, and the mice were administered physiological saline / excipient via nasal spray. Group 2 was administered 50 μg / nostril of ACE2-Fc fusion protein (SCB-719) via nasal spray at a dose of 50 μg / nostril. Group 3 was administered 50 μg / nostril of ACE2-Fc fusion protein (SCB-719) via nasal spray at a dose of 500 μg / nostril. Group 4 was administered 0.2 ml of ACE2-Fc fusion protein (SCB-719) intraperitoneally at a dose of 1000 μg / mouse.

[0288] The measurement results showed that the group that received intranasal administration had lower pulmonary viral loads compared to the group that received intraperitoneal administration. In both experimental groups that received ACE2-Fc fusion protein, the pulmonary viral load was significantly lower than that of the untreated group, and in particular, the 50 μg dose group showed a reduction in the amount of live virus in the lungs to below the detection limit. Detailed experimental results are shown in Figures 7A-7B. These experimental results demonstrate that intranasal administration effectively prevents the entry of the novel coronavirus into the body and is superior to systemic administration methods (intraperitoneal injection, ip), confirming the practical applicability of ACE2-Fc fusion protein. [Examples]

[0289] Evaluation of distribution in mice after intranasal administration.

[0290] Thirty-two all-female experimental animals were randomly assigned to solvent group B2, test substance group B1, and test substance group S4 based on their body weight. In this study, the protein (SCB-719) administered to the animals in group B2 (2 animals), group B1 (3 animals), and group S4 (27 animals) was all labeled with AF750 fluorescence, and the protein content was 6 mg / mL.

[0291] Specific administration information is shown in Table 3.

[0292] [Table 8]

[0293] [Table 9]

[0294] Measurements were performed on all animals in all groups at the following time points: 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 measurement time points are shown in Table 3.

[0295] Measurement Method and Evaluation Indices: After anesthetizing the animals in each group, the distribution and decay of the test substance within the animal body were evaluated at different time points before and after administration using an in vivo imaging system, and the number of photons per square centimeter per second (p / s / / sr) was measured. Immediately after the completion of measurements at each time point, gross dissection was performed, and the emission signal intensity of the excised major organs (heart, liver, spleen, lungs, kidneys, brain, nasal cavity) and blood (2 μL) was measured. The maximum excitation wavelength of the AF750 fluorescent dye was 753 nm, and the maximum emission wavelength was 782 nm. A total of three measurements were performed for each mouse.

[0296] After a single intranasal administration, no fluorescence signal was observed for unlabeled ACE2-Fc fusion protein (test substance group B1) and solvent group B2. AF750-labeled ACE2-Fc fusion protein (test substance group S4) was concentrated in the nasal cavity, and a clear fluorescence signal was observed 0-4 hours after administration. Figure 8A shows that ACE2-Fc fusion protein showed fluorescence in the nasal cavity at different time points, and since no fluorescence signal was observed in other organs, this indicates that ACE2-Fc fusion protein is concentrated in the nasal cavity. After a single intranasal administration, a clear fluorescence signal was detected 0-6 hours, and although the signal clearly attenuated at 6-8 hours, it remained at a detectable level, and the signal was still detectable at 12 hours. Imaging results for each organ are shown in Figure 8A, and the time course of nasal cavity fluorescence signal intensity is shown in Figure 8B.

[0297] In summary, it was confirmed that the ACE2-Fc fusion protein was continuously concentrated in the mouse nasal cavity for more than 12 hours. This result demonstrates that the ACE2-Fc fusion protein can remain at the administration site for a long period of time, and that the tissue half-life of the ACE2-Fc fusion protein supports its application in defense against COVID-19 via nasal administration.

[0298] Figure 9 illustrates the PK and in vitro efficacy curves of ACE2-Fc fusion protein (IV / IP). Female BALB / c mice aged 6-8 weeks were administered ACE2-Fc fusion protein at a dose of 50 mg / kg and 0.2 mL / mouse by intravenous or intraperitoneal injection (3 mice per group). After a single dose, approximately 50 μL of whole blood from the mice was collected 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 hour, serum was separated and used for blood concentration measurement. Serum drug concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Specifically, mouse anti-human Fc protein was immobilized in a 96-well microplate, and ACE2-Fc fusion protein in the sample serum was captured. Then, biotin-labeled ACE2-Fc fusion protein (SCB-2019-biotin) was added and bound to it. Finally, HRP-labeled strepavidin was added and bound to biotin. After stopping the HRP-induced color reaction of the TMB substrate with 1M sulfuric acid solution, the absorbance was measured at a 450 nm wavelength using a microplate reader. The ACE2-Fc fusion protein concentration in the test sample was calculated by fitting the parameters of a standard curve using a 4-parameter logistic regression model, with the theoretical concentration of ACE2-Fc fusion protein on the x-axis and the measured OD value on the y-axis. The quantitative range of this method was 3 to 100 ng / mL. The figure shows the mean blood concentration (Mean) and standard error (SEM) of three mice at each time point.

[0299] The result was 50 mg / μg / μg / mL, and it was confirmed that the drug exposure and half-life were equivalent in both administration routes. This result suggests that the ACE2-Fc fusion protein disclosed herein may be applicable for the prevention and treatment of COVID-19 infection and transmission.

[0300] The present invention is not limited to any particular disclosed embodiment (for example, those provided to illustrate various aspects of the invention). Through the description and teachings of this disclosure, various modifications to the described compositions and methods will become apparent. Such modifications are feasible and within the scope of the invention without departing from the true scope and spirit of the invention.

[0301] [Table 10]

[0302] [Table 11]

[0303] [Table 12]

[0304] [Table 13]

[0305] [Table 14]

[0306] [Table 15]

[0307] [Table 16]

[0308] [Table 17]

[0309] [Table 18]

[0310] Table 19

[0311] Table 20

[0312] Table 21

[0313] Table 22

[0314] Table 23

[0315] Table 24

[0316] Table 25

Claims

1. A fusion protein comprising multiple recombinant polypeptides, comprising 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 fragment thereof comprises the human ACE2 extracellular domain or fragment thereof.

3. The fusion protein according to claim 1 or 2, wherein the human ACE2 protein or fragment thereof comprises an amino acid sequence shown in any of SEQ ID NO: 5, 7, 11, 12, 13, or 14, or an amino acid sequence or fragment thereof having at least 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 fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

5. The fusion protein according to any one of claims 1 to 4, wherein the human IgG Fc protein or its functional variant comprises one or more Fc region sequences or functional variants and fragments selected from human IgG1 Fc, IgG2 Fc, IgG3 Fc, and IgG4 Fc.

6. The fusion protein according to any one of claims 1 to 5, wherein the human IgG Fc protein or its functional variant is selected from human IgG1 Fc region sequences or their functional variants and fragments.

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

9. A fusion protein according to any one of claims 1 to 8, which optionally includes 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 a fragment thereof and the human IgG Fc protein or a functional variant thereof are either directly bound or bound via the peptide linker.

11. The fusion protein according to claim 10, wherein the peptide linker is selected from an arginine-serine peptide linker (-RS-), a valine-serine linker (-VS-), and a glycine-serine linker (-GS-).

12. A fusion protein according to any one of claims 1 to 11, wherein the fusion protein optionally includes a mutant sequence.

13. A fusion protein according to claim 1, comprising an amino acid sequence shown in any of SEQ ID NO: 1-18, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith, or a combination thereof.

14. A fusion protein according to claim 1, comprising an amino acid sequence shown in any of SEQ ID NO: 1-4 and 15-18, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith, for example, an amino acid sequence shown in any of SEQ ID NO: 1-4 or 15-18, or an amino acid sequence or fragment thereof having at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

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

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

17. The pharmaceutical composition according to claim 16, comprising the fusion protein according to any one of claims 1 to 15 in an amount of about 0.1 mg / ml to about 100 mg / ml, for example, about 0.50 mg / ml to about 20.00 mg / ml, 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 one or more combinations of buffering agents and osmotic pressure regulators.

19. The pharmaceutical composition according to claim 18, wherein the buffering agent is selected from sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dihydrate, or a combination thereof.

20. The pharmaceutical composition according to claim 18 or 19, wherein the osmotic pressure adjusting agent comprises one or more combinations selected from the group consisting of sodium chloride, potassium chloride, glycerin, glucose, sorbitol, sucrose, xylitol, or mannitol.

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

22. The pharmaceutical composition according to any one of claims 18 to 21, wherein the pharmaceutically acceptable carrier optionally comprises one or more combinations selected from the group consisting of proteins, peptides or their hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), and amino acids (e.g., monosodium glutamate), for example, a stabilizer containing sucrose.

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

24. The pharmaceutical composition according to any one of claims 18 to 23, wherein the composition is in a dosage form applicable to intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intra-articular, intraperitoneal, transnasal, sublingual, tonsillar, oral / pharyngeal, or other extraintestinal or mucosal administration routes, preferably a dosage form suitable for transnasal administration such as a transnasal spray.

25. Application 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, for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for preventing the transmission of coronavirus SARS-CoV-2 and its variants to a previously infected subject.

26. The coronavirus SARS-CoV-2 and its variants are not limited to SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, mu, omicron, and JN. 1, but also include unknown coronavirus SARS-CoV-2 variants, as described in claim 25, for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants to a previously infected subject, or for the pharmaceutical composition described in any one of claims 1 to 15, or as described in any one of claims 16 to 24.

27. The fusion protein or pharmaceutical composition is administered via intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intra-articular, intraperitoneal, transnasal, sublingual, tonsillar, oral cavity / pharynx, or other extraintestinal and mucosal administration routes, preferably transnasally, for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for preventing transmission of coronavirus SARS-CoV-2 and its variants in previously infected subjects, according to any one of claims 1 to 15 or the pharmaceutical composition according to any one of claims 16 to 24.

28. The fusion protein or pharmaceutical composition is administered intranasally in a single dose or in multiple doses separated by hours, days, or weeks, for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for preventing transmission of coronavirus SARS-CoV-2 and its variants in previously infected subjects, as described in any one of claims 25 to 27, as described in any one of claims 1 to 15, or as described in any one of claims 16 to 24.

29. The interval is 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours, for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants in a previously infected subject, 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.

30. The fusion protein or pharmaceutical composition is administered once or more times a day, for example, once / day or twice / day, for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants in a previously infected subject, as described in any one of claims 25 to 29, as described in any one of claims 1 to 15, or as described in any one of claims 16 to 24.

31. Application 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, in the manufacture of a pharmaceutical for the prevention and treatment of infection by coronavirus SARS-CoV-2 and its variants, and / or for the prevention of transmission of coronavirus SARS-CoV-2 and its variants in a previously infected subject.

32. The use described in claim 31, wherein the coronavirus SARS-CoV-2 and its variants are not limited to SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, mu, omicron, and JN. 1, but also include variants of unknown coronavirus SARS-CoV-2.

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

34. The method according to claim 33, wherein the coronavirus SARS-CoV-2 and its variants are not limited to SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, mu, omicron, and JN. 1, but also include variants of unknown coronavirus SARS-CoV-2.

35. The method according to claim 33 or 34, wherein the fusion protein or pharmaceutical composition is administered via intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intra-articular, intraperitoneal, transnasal, sublingual, amygdala, oral cavity / pharynx, or other extraintestinal and mucosal administration routes, preferably transnasal administration.

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 as a single dose or as multiple doses separated by hours, days, or weeks.

37. The method according to claim 36, wherein the interval 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 a day, for example, once a day or twice a day.

39. 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 A kit for the prevention and treatment of infection with coronavirus SARS-CoV-2 and its variants, and / or for preventing transmission of coronavirus SARS-CoV-2 and its variants in a previously infected subject, comprising, optionally, an accompanying leaflet or label indicating prevention and / or treatment.

40. The kit according to claim 39, wherein the coronavirus SARS-CoV-2 and its variants are not limited to SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, mu, omicron, and JN. 1, but also include variants of unknown coronavirus SARS-CoV-2.

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

42. A carrier comprising the nucleic acid described in claim 41.

43. The carrier according to claim 33, wherein the carrier is phFC(IM).

44. A host cell comprising the nucleic acid according to claim 41, or the carrier according to claim 42.

45. The host cell according to claim 44, wherein the host cell is a CHO cell (e.g., GH-CHO).

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