Recombinant ACE2-FC fusion molecule, its preparation and use

Abstract

A method for preventing, reducing the risk of, or treating a viral infection in a subject, or for preventing or treating a symptom caused by a virus, the method comprising administering to the subject an effective amount of a fusion protein, the fusion protein comprising a variant angiotensin-converting enzyme 2 (ACE2) domain covalently fused to an Fc domain. The variant ACE2 domain comprises an N-terminal deletion and / or a C-terminal deletion compared to full-length wild-type ACE2 having SEQ ID NO: 1, and the variant ACE2 domain has ACE2 activity. The virus may be SARS-CoV, SARS-CoV-2, or MERS-CoV. The symptom may include severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), acute respiratory distress syndrome (ARDS), pulmonary arterial hypertension (PAH), or coronavirus disease 2019 (COVID-19).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application No. 63 / 086,593, filed October 1, 2020, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to the prevention or treatment of diseases, symptoms or conditions in which angiotensin-converting enzyme 2 (ACE2) is involved (e.g., coronavirus disease 2019 (COVID-19)) and related conditions. [Background technology]

[0003] Unless otherwise indicated herein, the material described in this section is not prior art to the claims of this application and is not admitted to be prior art by inclusion in this section.

[0004] COVID-19 is an infectious disease caused by severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2). Complications of COVID-19 can include long-term lung injury, pneumonia, acute respiratory distress syndrome (ARDS), peripheral and olfactory nerve damage, multiple organ failure, septic shock, and death. A study of the first 41 confirmed cases of COVID-19 published in The Lancet in January 2020 reported the earliest date of symptom onset as December 1, 2019. By March 11, 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic. As of September 26, 2020, more than 32.6 million cases and more than 990,000 deaths had been reported in 188 countries and territories, including more than 7.5 million cases and 205,000 deaths from the United States.

[0005] On December 2, 2020, the UK Medicines and Healthcare products Regulatory Agency (MHRA) granted temporary regulatory approval for the Pfizer-BioNTech mRNA vaccine, making it the first country to approve a vaccine and the first Western country to approve a COVID-19 vaccine for use. Since then, more vaccine types have been approved for public use by regulatory authorities in at least one country: two RNA vaccines (made by Pfizer-BioNTech and Moderna, respectively); three conventional inactivated vaccines (made by Sinopharm, Bharat Biotech, and Sinovac, respectively); three viral vector vaccines (made by Sputnik V, Oxford-AstraZeneca, and Janssen, respectively); and one peptide vaccine (EpiVacCorona).

[0006] As of August 16, 2021, CDC vaccine effectiveness studies provide evidence that available RNA COVID-19 vaccines are protective in real-world conditions as well as in clinical trial settings. The vaccines reduce the risk of COVID-19 infection, especially severe disease, in fully vaccinated individuals. A Washington state study showed that compared with fully vaccinated individuals, unvaccinated individuals were six times more likely to test positive for COVID-19, 37 times more likely to be hospitalized, and 67 times more likely to die. CDC data also shows that unvaccinated individuals are five times more likely to become infected, 10 times more likely to be hospitalized, and 11 times more likely to die.

[0007] Angiotensin-converting enzyme 2 (ACE2) is a zinc-containing metalloenzyme present primarily in the cell membranes of alveolar cells, small intestinal enterocytes, arterial and venous endothelial cells, arterial smooth muscle cells, and other cell systems in the lungs, arteries, heart, kidneys, intestines, and other tissues. ACE2 plays an important role in regulating the renin-angiotensin system and controlling blood pressure by opposing angiotensin-converting enzyme activity in the cardiovascular, renal, and respiratory systems. ACE2 plays a protective role in hypertension, cardiac function, and diabetic physiology. In acute respiratory distress syndrome (ARDS), ACE, Ang II, and AT1R promote disease development, while ACE2 and AT2R protect against ARDS. ACE2 has also been identified as a receptor for severe acute respiratory syndrome (SARS) coronavirus and plays an important role in the pathogenesis of SARS. At least three viruses in the coronavirus family (SARS-CoV, MERS CoV, and SARS-CoV-2) gain viral entry into human cells by binding to the ACE2 protein on the surface of human host cells with one of their viral proteins (also known as the spike).

[0008] SARS-CoV-2 is one of seven known coronaviruses infecting humans (the other two being SARS-CoV-1 and MERS-CoV, which caused SARS outbreaks in Asia in 2003 and the Middle East in 2012). The immune response to the SARS-CoV-2 virus involves a combination of cell-mediated immunity and antibody production. Although over 100 million infected individuals have recovered from COVID-19 (as of January 2021), it remains unclear whether natural immunity to the SARS-CoV-2 virus persists long-term within individuals. The continued accumulation of viral mutations could shift the virus's antigenic spectrum, raising concerns about reinfection with variants. As of January 2021, SARS-CoV-2 variants identified in Europe and South Africa appear to have spread rapidly. These variants may ultimately harbor mutations that enhance viral recognition and infection of host cells, resulting in increased infectivity and / or pathogenicity.

[0009] Another concern concerns the phenomenon of antibody-dependent enhancement (ADE). ADE occurs when inappropriate antibody binding promotes viral entry into host cells. In coronaviruses, antibodies targeting the viral spike (S) glycoprotein promote ADE (Wan et al., J. Virol. 2020). In the case of the SARS-CoV-1 virus, it has been discovered that antibodies that neutralize most variants can enhance the entry of mutant viruses into immune cells, thereby exacerbating the disease that vaccines are designed to prevent. Therefore, ADE threatens to hinder vaccine development because vaccines may trigger the production of inappropriate antibodies. In this context, non-vaccine prevention strategies are considered viable alternatives to avoid ADE, whether before or after exposure to the SARS-CoV-2 virus.

[0010] Early in the pandemic, the low number of infected people meant that there were few "mutating" variant viruses, providing little opportunity for escape mutations to emerge. Over time, SARS-CoV-2 began to evolve into many variants, each with increased transmissibility. Some SARS-CoV-2 variants are particularly important because they may increase infectivity, virulence, or reduce the effectiveness of vaccines against them (Planas et al., Nature, 2020; Kim et al., bioRxiv, 2021). SARS-CoV-2 variants are classified as "A" (ancestral type) and "B" (derived type). Type B mutated into further types, including B.1, the ancestor of the major global variant. The WHO has named the variants α (B.1.1.7, November 2020), β (B.1.351, January 2021), γ (P.1, January 2021), κ (B.1.617.1), δ (B.1.617.2, May 2021), λ (C.37), and others. Both the α and δ variants are significantly more transmissible than the original virus identified in early 2020.

[0011] Delta variants are approximately 40% more infectious than alpha variants and became the dominant strain in the spring of 2021. By late August 2021, delta variants accounted for 99% of U.S. cases, exposing unvaccinated individuals to a doubled risk of severe illness and hospitalization. Even RNA vaccine protection had declined from 91% to 66%. According to a CDC study, COVID-19 vaccines provided 55% protection against infection, 80% protection against symptomatic infection, and at least 90% protection against hospitalization. Recent studies have demonstrated vaccine efficacy declines of 53.1%, 42–76%, or 64.6%, likely due to waning immunity and reduced protection against the highly infectious delta strains (Nanduri, et al., MMWR. 2021; Puranik et al., medRxiv. 2021; Seppala et al., Eurosurveillance. 2021). The CDC reported that of the more than 75 million fully vaccinated people in the United States, 5,814 developed breakthrough infections (i.e., vaccinated individuals become infected with the same pathogen the vaccine is intended to protect against) and 74 died. Breakthrough infections and associated mortality rates can be very low, demonstrating vaccine efficacy. However, breakthrough infections are more likely to occur with novel virus strains, such as delta virus, as demonstrated in Israel. In Israel, more than half of the cases and hospitalizations in August 2021 occurred in fully vaccinated individuals (Wadman, M. Science 2021). Individuals with breakthrough infections, particularly frail elderly individuals, are at significantly higher risk for severe illness, delirium, hospitalization, and death (Antonelli, et al., Lancet, 2021).

[0012] The two primary medical interventions to mitigate the pathogenicity of SARS-CoV-2 include active and passive immunization (i.e., vaccination, monoclonal antibody therapy, and treatment with convalescent plasma from previously infected patients) (Taylor et al., Nat Rev Immunol., 2021; Yan et al., Pharmaceuticals., 2021). Each of these strategies relies on antibody binding and neutralization of viral antigens, particularly the receptor-binding domain of the spike protein, which mediates viral entry into host cells bearing the ACE2 receptor. Viral mutations that affect the structure of the spike protein can affect the ability of antibodies to bind and neutralize the spike, potentially reducing the effectiveness of most existing vaccines and therapeutics.

[0013] Therefore, there remains a great need for effective treatments or preventions for diseases or conditions involving angiotensin-converting enzyme 2 (ACE2) (e.g., SARS-CoV2, especially its more virulent variants). Summary of the Invention

[0014] The following summary is illustrative only and not intended to be limiting. In addition to the exemplary aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

[0015] The present application provides, inter alia, methods for preventing, reducing the risk of, or treating a viral infection, or for preventing or treating symptoms caused by a virus in a subject.

[0016] In one embodiment, the virus may be a coronavirus, hi one embodiment, the virus may be SARS-CoV, SARS-CoV-2, MERS-CoV, or a combination thereof.

[0017] In one embodiment, the symptom may be any symptom caused by a coronavirus. In one embodiment, the symptom may be Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), Acute Respiratory Distress Syndrome (ARDS), Coronavirus Disease 2019 (COVID-19), or a combination thereof. In one embodiment, the symptom (disease or condition) involves angiotensin-converting enzyme 2 (ACE2). In one embodiment, the symptom may be a viral infection, such as an infection with SARS-CoV-2, SARS-CoV, SARS spike protein, coronavirus, SARS virus, fragments thereof, or combinations thereof.

[0018] In one embodiment, the SARS-CoV-2 virus comprises substantially a strain δ. In one embodiment, the SARS-CoV-2 virus comprises a spike protein mutation. In one embodiment, the mutation is configured to improve the binding affinity of the virus to the ACE2 domain.

[0019] In one embodiment, the method comprises administering to the subject an effective amount of a fusion protein or fusion protein complex. In one embodiment, the fusion protein comprises a variant angiotensin-converting enzyme 2 (ACE2) domain covalently fused to an Fc domain. The variant ACE2 domain may comprise an N-terminal deletion and / or a C-terminal deletion compared to full-length wild-type ACE2 having SEQ ID NO: 1. The variant ACE2 domain has ACE2 activity.

[0020] In one embodiment, the fusion protein comprises a variant angiotensin-converting enzyme 2 (ACE2) domain covalently linked to an Fc domain. In one embodiment, the variant ACE2 domain comprises an N-terminal deletion and / or a C-terminal deletion relative to full-length wild-type ACE2. In one embodiment, the full-length wild-type ACE2 domain comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 1. In one embodiment, the variant ACE2 domain has ACE2 activity.

[0021] In one embodiment, the variant ACE2 domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to an amino acid sequence segment of full-length wild-type ACE2. In one embodiment, the segment may begin at an amino acid residue selected from residues 1-17 of full-length wild-type ACE2. In one embodiment, the segment may end at an amino acid residue selected from residues 615-740 of full-length wild-type ACE2. For example, the variant ACE2 domain may comprise an amino acid sequence having at least 98% or 99% sequence identity to an amino acid sequence segment of full-length wild-type ACE2 from residues 1 to 615, residues 2 to 618, residues 2 to 740, residues 4 to 615, residues 17 to 615, residues 18 to 740, or other combinations of these starting and ending residues.

[0022] In one embodiment, the variant ACE2 domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:3.

[0023] In one embodiment, the variant ACE2 domain has a higher binding affinity for SARS-CoV or SARS spike protein than full-length wild-type ACE2. For example, the variant ACE2 domain may have a binding affinity (KD) for SARS-CoV or SARS spike protein of 0.1 nM to 100 nM.

[0024] In one embodiment, the variant ACE2 domain has a higher avidity for SARS-CoV or SARS spike protein than full-length wild-type ACE2. For example, the variant ACE2 domain may have an avidity for SARS-CoV or SARS spike protein of less than 10 nM (KD).

[0025] In one embodiment, the fusion protein has an avidity for the κ variant of less than 1.0E-12. In one embodiment, the fusion protein has a higher binding affinity for the δSARS-CoV-2 strain than the Wuhan-Hu-1 strain. In one embodiment, the binding affinity for the δSARS-CoV-2 strain is at least three times that of the Wuhan-Hu-1 strain.

[0026] In one embodiment, the Fc domain is derived from the Fc domain of an immunoglobulin. The immunoglobulin may be IgG1, IgG2, IgG3, IgG4, IgA1 (d-IgA1, S-IgA1), IgA2, IgD, IgE, or IgM. In one embodiment, the Fc domain may have an Fc hinge region. In one embodiment, the Fc hinge region may be engineered to be C220S. In one embodiment, the Fc domain may comprise a null mutation selected from K322A, L234A, and L235A compared to a wild-type Fc domain. In one embodiment, the wild-type Fc domain comprises an amino acid sequence having at least 98% or 99% sequence identity to SEQ ID NO:5.

[0027] In one embodiment, the Fc domain may lack effector functions. In one embodiment, the Fc domain may lack antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). In one embodiment, the Fc domain comprises an IgG1 Fc domain.

[0028] In one embodiment, the Fc domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:6.

[0029] In one embodiment, the fusion protein may comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7, 9, 11, 13, 15, 16, 17, 18, 19 or 21.

[0030] In one embodiment, the fusion protein can have a molecular weight of about 50 kDa to 250 kDa, hi one embodiment, the fusion protein can have a molecular weight of 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 120 kDa, 150 kDa, 180 kDa, 200 kDa, 250 kDa, or any number therebetween.

[0031] In one embodiment, the fusion protein complex is a homodimer of the fusion proteins disclosed herein. In one embodiment, the fusion protein complex comprises two variant ACE2 domains. In one embodiment, the fusion protein complex comprises at least two fusion proteins. In one embodiment, the two fusion proteins are paired via one or two disulfide bonds. In one embodiment, the disulfide bonds are in the hinge of the Fc domain.

[0032] In one embodiment, the fusion protein or fusion protein complex has binding affinity for SARS-CoV-2, SARS-CoV, SARS spike protein, or a fragment thereof, with an equilibrium dissociation constant (KD) of less than or equal to 0.1 nM, 0.5 nM, 1 nM, 2 nM, 3 nM, 5 nM, 10 nM, 20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 80 nM, or any number therebetween.

[0033] In one embodiment, the fusion protein or fusion protein complex has an avidity for SARS-CoV-2, SARS-CoV, SARS spike protein, or a fragment thereof, with an equilibrium dissociation constant (KD) of less than or equal to 1.0E-12, 0.001 nM, 0.01 nM, 0.05 nM, 1 nM, 2 nM, 3 nM, 5 nM, 10 nM, or any number therebetween.

[0034] In one embodiment, the fusion protein or fusion protein complex has a specific enzymatic activity of about 50 pmol / min / μg to about 5000 pmol / min / μg, hi one embodiment, the fusion protein has a specific enzymatic activity of about 568 pmol / min / μg.

[0035] The fusion protein is administered in an effective amount to treat and prevent the infections or diseases disclosed herein. In one embodiment, the dose of the fusion protein administered per treatment is about 1 mg / kg to about 200 mg / kg, about 5 mg / kg to about 100 mg / kg, about 3 mg / kg to about 70 mg / kg, or about 10 mg / kg to about 150 mg / kg of body weight.

[0036] In one embodiment, the daily dosage of the fusion protein is about 100, 120, 140, 150, 180, 200 mg / Kg body weight or less, hi one embodiment, the fusion protein is administered twice daily at a dose of about 25, 50, 70, 90, 100, 150, 200 mg / Kg body weight or less.

[0037] In one embodiment, the fusion protein is administered as a liquid formulation. In one embodiment, the fusion protein is administered as a suspension in a solution. In one embodiment, the solution may include a salt, a carbohydrate, a surfactant, or a combination thereof. In one embodiment, the salt may be sodium chloride, histidine hydrochloride, or a combination thereof. In one embodiment, the carbohydrate may be sucrose, glucose, or a combination thereof. In one embodiment, the surfactant may be polysorbate 80.

[0038] In one embodiment, the liquid formulation may comprise the fusion protein at a concentration of about 2 mg / ml to about 20 mg / ml, about 5 mg / ml to about 10 mg / ml, or about 5 mg / ml to about 20 mg / ml.

[0039] The methods disclosed herein can be used to treat or prevent viral infection, acute respiratory distress syndrome, pulmonary arterial hypertension, or acute lung injury in a subject. In one embodiment, administration of the fusion protein can prevent the subject from becoming infected with the SARS-CoV-2 virus. In one embodiment, administration of the fusion protein can reduce the subject's risk of SARS-CoV-2 virus infection. In one embodiment, administration of the fusion protein can prevent the subject with a SARS-CoV-2 virus infection from being hospitalized. In one embodiment, administration of the fusion protein can reduce the risk of hospitalization in a subject infected with the SARS-CoV-2 virus. In one embodiment, administration of the fusion protein can shorten the length of hospitalization in a subject infected with the SARS-CoV-2 virus. In one embodiment, administration of the fusion protein can prevent oxygenation and ventilation in a subject infected with the SARS-CoV-2 virus. In one embodiment, administration of the fusion protein can reduce the need for oxygenation and ventilation in a subject infected with the SARS-CoV-2 virus. In one embodiment, administration of the fusion protein can prevent death in a subject infected with the SARS-CoV-2 virus. In one embodiment, administration of the fusion protein can reduce the risk of death in a subject infected with the SARS-CoV-2 virus. In one embodiment, administration of the fusion protein can reduce the severity of COVID symptoms in a subject with a SARS-CoV-2 virus infection.

[0040] In one embodiment, the method may include administering the fusion protein or fusion protein complex intravenously, subcutaneously, intranasally (e.g., as a nasal spray), or pulmonary. In one embodiment, the fusion protein may be administered by daily infusion. In one embodiment, the fusion protein may be administered by daily intramuscular injection.

[0041] In one embodiment, the fusion protein may be co-administered with an antiviral agent, an immunomodulatory agent, or a combination thereof. In one embodiment, the antiviral agent may be favipiravir, ribavirin, galidesivir, remdesivir, or a combination thereof.

[0042] In one embodiment, the subject is a human. The method disclosed herein can be used on subjects with at least one risk factor selected from the group consisting of age 65 years or older, a moderately or severely weakened immune system, metabolic syndrome, allergy to COVID vaccines, and a low or absent immune response after COVID vaccination. In one embodiment, the subject suffers from cancer, chronic kidney disease, chronic lung disease, diabetes, or heart disease.

[0043] In a further aspect, the present invention provides a pharmaceutical composition for treating a disease or condition involving angiotensin-converting enzyme 2 (ACE2). In one embodiment, the pharmaceutical composition comprises a fusion protein or fusion complex disclosed herein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further comprises an antiviral agent. In one embodiment, the pharmaceutical composition comprises a protein complex disclosed herein and a pharmaceutically acceptable carrier.

[0044] In one embodiment, the present invention provides a liquid composition comprising the fusion protein disclosed herein, wherein the liquid composition contains about 100 mg to about 20,000 mg, about 200 mg to about 10,000 mg, about 100 mg to about 10,000 mg, or about 500 mg to about 10,000 mg of the fusion protein per dose.

[0045] In one embodiment, the liquid composition comprises the fusion protein at a concentration of about 0.1% to about 10% by weight, about 0.5% to about 5% by weight, about 0.5% to about 1% by weight, or about 0.5% to about 2% by weight. [Brief explanation of the drawings]

[0046] The foregoing and other features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings, in which: The present disclosure is described with additional specificity and detail through the use of the accompanying drawings, with the understanding that these drawings illustrate only some embodiments constructed in accordance with the present disclosure and therefore should not be considered limiting of its scope.

[0047] [Figure 1] Figure 1A is a diagram of recombinant fusion proteins (SI-69R2 and SI-69R4) of the ACE2 functional domain and modified Fc (null) fragment. Figure 1B shows the sequence of the SI-F019 fusion protein (post-translationally modified SI-69R2 lacking the N-terminal 17-amino acid signal peptide). Figure 1C is a size-exclusion chromatography image showing that the SI-F019 fusion protein complex is a homodimer. Figure 1D is a diagram of the SI-F019-spike protein complex.

[0048] [Figure 2] We show that SI-F019, but not SI-69R4, is resistant to TMPRSS2-dependent hydrolysis (2A), and that the enzymatic activity of SI-F019 can be quantified by an in vitro fluorescence assay (2B).

[0049] [Figure 3] In the study, we show that SI-F019 dose-dependent blockade of live SARS-CoV-2 infection of VeroE6 cells reached 100% at all three MOIs of virus.

[0050] [Figure 4] This shows that addition of 10 fM or more of SI-F019 prevented a portion of Vero E6 cells from undergoing cytolysis 1 hour after infection with SARS-CoV-2 or SARS-CoV-1 virus at an MOI of 0.01.

[0051] [Figure 5]After preincubation with pseudovirus, SI-F019 inhibits viral infection in a dose-dependent manner, achieving complete inhibition at a relatively high concentration (IC50 = 32.56 nM).

[0052] [Figure 6] The results of the internalization / infection-mediated assay are shown. Specifically, when pretreated with the tested concentrations of SI-F019, there was no GFP signal incorporation, indicating the presence of pseudovirus (PsV), while low GFP signals were associated with SI-69C1 (anti-S1 antibody) and SI-69R3 (SARS-CoV-2 ACE-2 Fc WT), as well as medium, buffer, and ACE2-his (SI-69C1) (after 48 hours in THP1 (pH 7.2) (6A), THP1 (pH 6.0) (6B), and Daudi (6C)).

[0053] [Figure 7] We show that SI-F019 can compete with either native anti-SARS-CoV-2 antibodies or ACE2-Fc (wild-type) fusion protein to block Fc-mediated antibody-dependent enhancement (ADE), as measured by GFP signal, an indication of PsV infection.

[0054] [Figure 8] Flow cytometry analysis of HEK293-T cells expressing SARS-CoV-2 spike protein detected with anti-spike and anti-human Fc antibodies.

[0055] [Figure 9] Figure 1 shows the dose-dependent binding of SI-F019 to HEK293-T cells expressing the SARS-CoV-2 spike protein, as measured by geometric mean fluorescence intensity (gMFI).

[0056] [Figure 10]FACS analysis of an antibody-dependent cellular cytotoxicity (ADCC) assay shows that human anti-S1 antibody (SI-69C3) directs human NK cells to target HEK293-T cells expressing the SARS-CoV-2 spike protein, as measured by calcein-AM and propidium iodide staining.

[0057] [Figure 11] The dose-binding response of SI-F019 and control molecules against HEK293-T cells in an ADCC assay compared with a human anti-S1 antibody (SI-69C3) is shown (11A), demonstrating that SI-F019 did not mediate ADCC at therapeutic doses of 100 fM to 100 nM, whereas its wild-type Fc-containing variant (SI-69R3) mediated it in a dose-dependent manner, albeit at a lower level of activity (11B).

[0058] [Figure 12] We demonstrate that SI-F019, with its Fc null mutation, reduces serum-mediated complement-dependent cytotoxicity (CDC) in vitro, as measured by viability of HEK293-T cells expressing SARS-CoV-2 S protein.

[0059] [Figure 13] We measured the viability of HEK293-T cells expressing SARS-CoV-2 S protein after various treatments, and found that SI-F019 does not induce serum complement-dependent cytotoxicity (CDC) in vitro (13A), and that the Fc null mutation of SI-F019 does not affect cell proliferation after 96 hours of in vitro treatment (13B).

[0060] [Figure 14]SI-F019 does not induce the release of cytokines ((14A) IFN, (14B) TNF, (14C) GM-CSF, (14D) IL-2, (14E) IL-10, (14F) IL-6, (14G) IL-1, (14H) IL-12p70, and (14I) MCP-1) in PBMC cultures in either soluble or plate-bound formats.

[0061] [Figure 15] Biolayer interferometry shows the binding kinetics (affinity) of SI-F019 (15a) and neutralizing antibodies, including bamlanivimab (SI-69C4) (15b), casirivimab (SI-69C5) (15c), eteseevimab (SI-69C6) (15d), imdevimab (SI-69C7) (15e), silgavimab (SI-69C8) (15f), and tisagevimab (SI-69C9) (15g), to COVID-19 RBD variants.

[0062] [Figure 16] Biolayer interferometry shows the binding kinetics (avidity) of SI-F019 (16a) and neutralizing antibodies (including bamlanivimab (SI-69C4) (16b), casirivimab (SI-69C5) (16c), eteseevimab (SI-69C6) (16d), imdevimab (SI-69C7) (16e), silgavimab (SI-69C8) (16f), and tisagevimab (SI-69C9) (16g)) to RBD variants.

[0063] [Figure 17] We demonstrate the potency of SI-F019 in protecting ACE2-expressing 293T cells from viral inhibition using variants of S protein-packaged pseudovirus (NICPBP) in luciferase reporter assays (17a), a linear correlation between IC50 and binding affinity (17b), or avidity indicating competitive inhibition by SI-F019 (17c). DETAILED DESCRIPTION OF THE INVENTION

[0064] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically indicate like elements unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0065] The present invention relates, inter alia, to the generation and characterization of fusion proteins, such as recombinant human ACE2-Fc fusion proteins. In some embodiments, these fusion proteins can protect membranous ACE2 in human host cells from viral particles or viruses. In one embodiment, the viral particles or viruses can enter host cells after infection using viral spike proteins. In one embodiment, the viral particles include, but are not limited to, SARS-CoV-2, COVID-19, SARS-CoV-2 variants, and other coronaviruses. In one embodiment, the viruses can cause severe acute respiratory syndrome (SARS). In one embodiment, SARS can include coronavirus disease 2019 or COVID-19.

[0066] In one embodiment, the recombinant human ACE2-Fc fusion protein can be a fusion protein of the ACE2 zinc metallopeptidase domain (also known as the ACE2 extracellular domain, ACE2-ECD) and an IgG1 Fc fragment. In one embodiment, the fusion protein is SI-F019. SI-F019 is a fusion protein of the ACE2-ECD and an IgG1 Fc fragment containing the residues C220S, L234A, L235A, and K322A (EU numbering system) (Table 1 and Figure 1). An active ACE2-ECD retains the structural conformation required for host receptor-virus interaction. Each mutation in the IgG1 Fc fragment can dramatically attenuate a specific immune response. The C220S mutation can remove an unpaired cysteine ​​for heavy and light chain pairing, which provides technical advantages, particularly in preventing protein aggregation, improving protein stability, and facilitating manufacturing efficiency and scalability. The introduction of L234A and L235A can reduce Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The K322A mutation can reduce complement-dependent cytotoxicity (CDC) triggered by C1q binding. SI-F019 is designed to neutralize the SARS-CoV-2 virus and trigger fewer effector responses.

[0067] As used herein, the terms "a," "an," and "the" are defined to mean "one or more," and include the plural, unless the context is inappropriate.

[0068] The term "recombinant fusion protein" refers to a protein produced by genetic engineering of a fusion gene encoding two or more genes to encode separate proteins.

[0069] The term "ACE2-Fc" refers to a recombinant fusion protein of a human ACE2 protein fragment and an engineered fragment of the fragment crystallizable region (Fc region) of a human immunoglobulin, including, but not limited to, IgG1, IgG2, IgG3, IgG4, IgA1 (d-IgA1, S-IgA1), IgA2, IgD, IgE, and IgM.

[0070] The terms "spike," "spike s," "S protein," or variants refer to the proteins that allow the virus to attach (the "S1 subunit" or "S1 protein") and fuse (the "S2 subunit" or "S2 protein") with the host cell membrane. In the case of COVID-19, SARS-CoV-2 has sufficient affinity for the ACE2 receptors on human cells and uses them as a mechanism for cell entry. SARS-CoV-2 has a higher affinity for human ACE2 than the original SARS virus.

[0071] The terms "Fc domain," "Fc fragment," and "Fc region" refer to the same domain or fragment of the Fc region ("Fc domain" and "Fc fragment," respectively) in IgG, IgA, and IgD antibody isotypes, which are derived from the hinge and second and third constant domains (CH2-CH3) of the two heavy chains of the antibody.

[0072] The term "affinity" refers to a measure of the attractive force between two polypeptides (e.g., receptor / ligand, ACE2 / spike protein or variants thereof). The inherent attractive force between two polypeptides can be expressed as the binding affinity equilibrium constant (KD) of a particular interaction. The KD binding affinity constant can be measured, for example, by biolayer interferometry.

[0073] The term "avidity" refers to the cumulative strength of the affinities of multiple individual noncovalent interactions (e.g., a protein receptor and its ligand), commonly referred to as functional affinity. As such, avidity differs from affinity, which describes the strength of a single interaction.

[0074] The term "antigenic drift" refers to random genetic mutations in infectious viruses that result in new virus strains with slightly altered antigenicity, and antibodies that prevented infection with the previous virus strain may not be effective against this new virus strain.

[0075] The term "cytokine release syndrome" (CRS) refers to CRS in severe cases of COVID-19 associated with elevated levels of inflammatory mediators, including cytokines and chemokines (e.g., interleukin (IL)-2, IL-6, IL-7, IL-10, tumor necrosis factor (TNF), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP1; also known as CCL2), macrophage inflammatory protein 1 alpha (MIP1α; also known as CCL3), C-X-C chemokine ligand 10 (CXCL10), C-reactive protein, ferritin, and D-dimers, resulting from SARS-CoV-2 infection.

[0076] The term "neutralizing antibody" refers to an antibody that protects cells from pathogens or infectious particles by neutralizing all of their biological effects. Neutralization renders the particle non-infectious or non-pathogenic. Neutralizing antibodies are part of the adaptive immune system's humoral response to viruses, intracellular bacteria, and microbial toxins. By specifically binding to the surface structures (antigens) of infectious particles, neutralizing antibodies prevent the particles from interacting with host cells that they could infect and destroy. Neutralizing antibody immunity is also known as sterilizing immunity because the immune system eliminates infectious particles before infection can occur.

[0077] The term "vaccine" refers to a biological product that provides active acquired immunity against a specific infectious disease. Vaccines can be prophylactic (preventing or ameliorating the effects of future infection with a natural or "wild" pathogen) or therapeutic (combating disease that has already occurred).

[0078] The term "breakthrough infection" refers to a vaccinated person contracting the same disease that the vaccine is intended to prevent. The nature of the breakthrough infection depends on the virus itself. If a vaccinated person becomes infected, the symptoms will be milder and the infection will last for a shorter period than if they were infected naturally. Causes of breakthrough infection include age, mutations in the virus or neutralizing antibodies, and improper administration or storage of the vaccine.

[0079] "Sterilizing immunity" is immunity mediated by neutralizing antibodies that can bind to pathogens (e.g., all SARS-CoV-2 variants) and inhibit infectivity by blocking molecules required for cell entry (i.e., spikes encoded by the variants), thereby completely preventing infection. Due to breakthrough infection, neither COVID-19 vaccines nor neutralizing antibodies provide complete sterilizing immunity. According to these definitions, SI-F019 can be used as a therapeutic vaccine to achieve therapeutic sterilizing immunity against SARS-CoV-2 virus variants and other SARS viruses that utilize ACE2 to enter human cells.

[0080] Example

[0081] Example 1: Cloning, expression and purification of recombinant ACE2-Fc fusion protein

[0082] Human membranous ACE-2 is an important receptor mediating SARS-CoV virus entry into human host cells. The human ACE2 protein has at least three functional domains: a signal peptide (residues 1-17), a zinc metallopeptidase domain (residues 18-615), and a TMPRSS2 protease cleavage site (residues 697-716). SEQ ID NO: 1 is the full-length human ACE2 protein sequence from Genbank number: NP_001358344.1. Among these, the SARS-CoV viral spike protein interacts with the zinc metallopeptidase domain (SEQ ID NO: 3 is the protein sequence of truncated ACE2 from residues 1 to 615). Meanwhile, the Fc region of human antibodies (SEQ ID NO: 5) can interact with Fc receptors (FcRs) on many immune cells and several proteins of the complement system. Each Fc fragment of the IgG1 Fc region contains a cysteine ​​at C220 (EU numbering system), which can essentially form a disulfide bond with either the kappa or lambda light chain. To reduce the risk of protein destabilization and / or inactivation due to the inclusion of a free cysteine, C220 can be substituted with serine (C220S) or other amino acids. To reduce Fc binding to FcγR and C1q, other point mutations, such as K322A, L234A, and L235A, can be introduced into the wild-type IgG1 Fc fragment. The IgG1 Fc fragment with these four mutations is collectively referred to as IgG1 Fc null (SEQ ID NO: 6).

[0083] Recombinant human ACE2-Fc fusion proteins (Table 1) were engineered to produce soluble fusion proteins. Among them, SI-69R2 (SEQ ID NO: 7) is a recombinant fusion protein of a truncated ACE2 fragment lacking the TMPRSS2 protease cleavage site with an IgG1 Fc null fragment. Other recombinant fusion proteins were constructed to provide Fc fragments of Ig isotypes (e.g., SI-69R2-G4 (IgG4 Fc, SEQ ID NO: 9), SI-69R2-A1 (IgA1 Fc, SEQ ID NO: 11), SI-69R2-A2 (IgA2 Fc, SEQ ID NO: 13), or a wild-type IgG1 Fc fragment (IgG1 Fc, SEQ ID NO: 19). Additionally, a recombinant fusion protein of a truncated ACE2 containing all three domains with a wild-type IgG1 Fc fragment was constructed (SI-69R4, 1-740, SEQ ID NO: 21). In all recombinant ACE2-Fc fusion proteins, the signal peptide (ACE2 residues 1-17) can be replaced with other signal peptides of different lengths without affecting the function of other domains of the human ACE protein or the ACE2-Fc fusion protein.

[0084] Recombinant fusion genes encoding the fusion proteins listed in Table 1 were cloned into pCGS3.0 (e.g., SI-69R2) or pTT5 expression vectors (e.g., SI-69R4 and SI-69R10) and expressed in ExpiCHO cells. All fusion proteins were purified according to standard protein expression protocols, sterilized through a 0.22 μm filter, and stored in a frozen storage solution at 4°C. During the expression and purification process, each recombinant fusion protein may undergo post-translational modifications, including N-glycosylation and cleavage of the N-terminal signal peptide (17 amino acids). In the case of SI-69R2, the purified fusion protein was newly designated SI-F019.

[0085] As shown in Figures 1A and 1B, SI-F019 contains a truncated ACE2 fragment (residues 18-615) containing the zinc metallopeptidase domain (residues 19-611) of human ACE2, but not the TMPRSS2 protease cleavage site. SI-F019 also contains an IgG1 Fc null fragment that lacks Fcγ receptor binding. Thus, the soluble form of SI-F019 is not expected to bind to target cells in peripheral blood.

[0086] The SI-F019 fusion protein may undergo post-translational modifications such as N-glycosylation and homodimerization through two disulfide bonds in the Fc region. As shown in Figure 1C, the actual molecular weight of the SI-F019 dimer was assessed using analytical size-exclusion chromatography (SEC) combined with multi-angle light scattering (MALS), absorbance (UV), and / or refractive index (RI) concentration detector techniques. This method combines chromatographic separation by molecular size with determination of absolute molar mass by light scattering (LS) without the limitation of molecular weight standard calibration. The average total molecular weight of SI-F019 was 209.6 kDa (main peak), of which the molecular weights of the SI-F019 dimer and its modifier (i.e., glycan) were 189.3 kDa and 20.3 kDa, respectively. Theoretical calculation of its amino acid structure suggests that the molecular weight of the SI-F019 monomer is 95.1 kDa. The purified SI-F019 fusion protein complex is a homodimer. SI-F019 protein complex refers to the protein-protein interaction between SI-F019 as a monomer or dimer and other proteins, such as spike proteins and effector proteins. The organization of the SI-F019-spike protein complex (Figure 1D) underlies a mechanism. According to this mechanism, SI-F019 is a candidate inhibitor that prevents the SARS-CoV-2 virus from docking to membranous ACE2 and entering human host cells.

[0087] Example 2: Binding of SI-F019 to spikes, Fc receptors, and C1q

[0088] SI-F019 was designed to block SARS-CoV viral entry into humans by preventing spike protein binding to the membranous ACE2 protein on human host cells. The spike is the most distinctive feature of coronaviruses and is a knob-like structure responsible for their corona- or halo-like surface. Spike proteins are typically composed of glycoproteins, with each spike consisting of a trimer of S protein, which is composed of S1 and S2 subunits. The homotrimeric spike protein mediates receptor binding and membrane fusion between the virus and host cells. The S1 subunit forms the head of the spike and contains the receptor-binding domain (RBD). The S2 subunit forms the stem that anchors the spike to the viral envelope and enables fusion upon protease activation. In a functionally active state, the S1 and S2 subunit complex is cleaved into individual subunits during virus binding and fusion with host cells under the action of proteases (e.g., host cell cathepsin family and transmembrane protease serine 2 (TMPRSS2)). The spike plays a key role in viral entry during the infection process by coronaviruses. In the case of COVID-19, the SARS-CoV-2 virus docks with the membrane-bound ACE2 receptor on the host cell surface, and the interaction between the spike and the functional domain of ACE2 triggers fusion between the viral envelope and the host cell membrane, resulting in the release of the viral nucleocapsid into the host cell cytoplasm.

[0089] The binding affinity and avidity of SI-F019, an ACE-Fc fusion protein, to viral spike proteins were evaluated. In biolayer interferometry, spike protein samples included the SARS-CoV-2 spike trimer, SARS-CoV-2 S1 protein, SARS-CoV-2 S1 protein RBD domain, and SARS-CoV-1 RBD domain (Table 2). These reagents were purchased from ACROBiosystems. The binding affinity assay measured the binding of SI-F019 immobilized on an anti-human IgG Fc capture biosensor chip (AHC) to the spike protein or its subunits in solution. The avidity assay measured the binding of biotinylated spike protein immobilized on a streptavidin biosensor chip (SA) to SI-F019 in solution. Data analysis utilized a 1:1 fitting model to calculate both binding affinity and avidity. The results indicate that the binding affinity and avidity of SI-F019 for these spike proteins, fragments, or domains are within the nanomolar (nM) scale, respectively (Table 2). This unique and informative data may be a useful reference for measuring SI-F019 protein complexes with viral spike protein variants, which may indicate potential antigenic drift among SARS-CoV-2 variants. Indeed, this type of viral mutation has been identified in certain strains of SARS-CoV-2, such as D614G in the spike protein, which alters the viral affinity for membranous ACE2 and viral entry into host cells (Zhang et al., 2020).

[0090] In parallel with binding to the spike, we evaluated the binding of SI-F019 to human FcγR, C1q, and FcRn by Bio-Layer Interferometry. As shown in Table 3, no binding was detected to FcγRs, including FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa, nor to C1q. However, SI-F019 bound to FcRn with a binding affinity of 37.6 nM (KD), comparable to that of the human IgG1 Fc region.

[0091] Example 3: SI-F019 is resistant to TMPRSS2 protease activity.

[0092] Human ACE2 undergoes membrane protease hydrolysis by TMPRSS2, resulting in monomeric extracellular ACE2 being exported from cells and easily detectable in serum. In the recombinant ACE2-Fc fusion protein, a truncated ACE2 domain was fused to an Fc fragment but still retained binding affinity for the viral spike protein.

[0093] SI-F019 was engineered to lack the TMPRSS2 cleavage site in its truncated ACE2 domain. As shown in Figure 1, SI-F019 contains residues 18-615, while SI-69R4 encodes all three ACE2 domains (residues 1-740, SEQ ID NO: 21), including the TMPRSS2 cleavage site. SI-69R4 was used as a control to demonstrate that SI-F019 is not subject to TMPRSS2-specific proteolysis. To assay for TMPRSS2-specific hydrolysis, the TMPRSS2 (106-492) catalytic domain was cloned, expressed, and purified according to Genbank: NP_001358344.1. As shown in Figure 2A, in the absence of TMPRSS2, both SI-F019 and SI-69R4 migrated stably at their respective molecular weights (as monomers under reducing and denaturing conditions). When TMPRSS2 was added, SI-F019 exhibited resistance to TMPRSS2, whereas SI-69R4 underwent proteolysis and exhibited sensitivity to TMPRSS2, as expected. Thus, SI-F019 is stable and resistant to TMPRSS2-mediated protease activity.

[0094] Example 4: SI-F019 exerts the enzymatic activity of ACE2.

[0095] SI-F019 is a fusion protein of a truncated ACE2 (residues 18-615) and an IgG1 FC null fragment. The truncated ACE2 encodes a zinc metallopeptidase, and its enzymatic activity can be assessed using established assays. The ACE2 enzymatic activity of SI-F019 was measured using a peptide substrate of ACE2 tagged with MCA (7-methoxycoumarin-4-acetic acid) fluorescently [MCA-YVADAPK(Dnp)-OH fluorescent peptide substrate]. MCA molecules were prepared as a standard curve calibration for free fluorophore quantification, and the substrate was diluted to 0.97 mg / ml in DMSO. SI-F019 was diluted to 100, 200, and 300 ng / ml and used to cleave the fluorescent peptide to release free MCA in vitro. The assay was incubated at room temperature for 20 minutes, and data were collected for the fluorescent signal at 2-minute intervals.

[0096] The cleaved MCA was quantified in moles using an MCA standard curve. Enzyme activity was determined based on the slope of the linear curve (MCA quantity versus time) shown in Figure 2B. SI-F019 showed good linearity (R2 > 0.99) at three concentrations, indicating that stable cleavage of the peptide was concentration-dependent. To calculate the enzymatic activity, the slope was divided by the mass of SI-F019 (μg). The final specific enzymatic activity was 568 pmol / min / μg. The fact that SI-F019 retains the enzymatic activity of membranous ACE2 indicates that this independent domain of ACE2 also retains the structural conformation required for host receptor-virus interaction.

[0097] Example 5: SI-F019 inhibits live SARS-CoV-2 infection of VeroE6 cells.

[0098] SI-F019 was tested for its ability to inhibit live SARS-CoV-2 infection and lysis of VeroE6 (ATCC: CRL-1586) cells in vitro. Test concentrations ranging from 1.5 nM to 1200 nM of SI-F019 were preincubated with three concentrations of live SARS-CoV-2 virus (USA-WA1 / 2020 strain, representing a 100x range of multiplicity of infection (MOI)) for 1 hour and then added to 90% confluent VeroE6 cell monolayers. After 1 hour, the virus-containing medium was removed and replaced with medium containing SI-F019 at the matching test concentration. Tests were performed in triplicate. Cell viability was measured by neutral red dye uptake after 72 hours, and the percent inhibition of lytic virus infection was determined relative to wells containing virus at each MOI (without SI-F019). The median inhibitory concentration (IC50) for each virus concentration (1 MOI = 40,000 virus particles) was calculated using GraphPad Prism software and is shown in each graph. Preincubation of SI-F019 with live SARS-CoV-2 resulted in a dose-dependent blockade of infection against the test virus of 100% at all three MOIs. As shown in Figure 3, SI-F019 neutralized as many as 40,000 virus particles at a 1.0 MOI with an IC50 of 97.62 nM. At 0.1 and 0.01 MOIs, SI-F019 was able to block infection with IC50s of 79.95 nM and 36.5 nM, respectively.

[0099] Example 6: SI-F019 reduces viral replication and reinfection.

[0100] SI-F019 was tested for its ability to inhibit replication and reinfection (i.e., further infection of VeroE6 cells previously infected with SARS-CoV-2 or SARS-CoV-1 virus at a low MOI). Ninety-percent confluent monolayers (approximately 20,000 cells) of VeroE6 cells were exposed to SARS-CoV-2 (strain USA-WA1 / 2020) or SARS-CoV-1 (strain Urbani 2003000592) at an MOI of 0.01 (calculated as 400 viral infectious particles) for 1 hour. After washing away free viral particles, SI-F019 was added to the cells in triplicates at concentrations ranging from 10 fM to 100 nM, and the cell cultures were maintained for 72 hours. Cell viability was determined by neutral red dye uptake, and % inhibition of viral cytotoxicity was calculated. The absorbance values ​​for each plate were normalized by the maximum absorbance in the no virus or drug (NVND) condition, which represents 100% cell viability, and the mean absorbance value for no virus / drug (VND) to establish maximum cell death was calculated using the following formula:

[0101] %Cell Viability=[(Well OD 540 -VND OD 540 ) / (NVND OD 540 -VND OD 540 )]*100

[0102] As shown in Figure 4, the addition of SI-F019 at a concentration of 10 fM protected Vero E6 cells from secondary infection. Cultures infected with SARS-CoV-2 or SARS-CoV-1 virus at an MOI of 0.01 for 1 hour resulted in at least a 20% reduction in cell lysis. However, when the concentration of SI-F019 was increased tenfold up to 100 nM, no significant improvement in protection was observed in this assay. This finding demonstrates that the addition of SI-F019 to cells infected with low-titer viruses can reduce viral spread and the degree of cytotoxicity, even at low concentrations.

[0103] Example 7: SI-F019 inhibits pseudovirus infection of HEK293T-ACE2 cells.

[0104] A HEK293T (ATCC: CRL-3216)-3D4 clonal cell line was generated by lentiviral transduction of human ACE2 protein. The functionality of the expressed human ACE2 was confirmed by enzyme substrate conversion assay and specific antibody binding by FACS. SARS-CoV-2 S protein-packaged pseudovirus containing a luciferase reporter gene was obtained from the National Institute for Drug and Biological Products Control. Tests were performed according to the manufacturer's instructions. The S pseudovirus stock solution was diluted 20 MRD in culture medium to obtain a virus load of 300 TCID50 / well. SI-F019 at concentrations ranging from 0.07 nM to 1500 nM was preincubated with the diluted virus solution for 1 hour. HEK293T-3D4 cells were dispersed in a 96-well plate. After 1 hour, the mixture was added to the cell plate. After 24 hours of incubation, infected cells were measured by testing luciferase activity. The 50% inhibitory concentration (IC50) of the defined viral load was calculated using GraphPad Prism software. As shown in Figure 5, after preincubation with the pseudovirus, SI-F019 inhibited viral infection in a dose-dependent manner, achieving complete inhibition at higher concentrations (IC50 = 32.56 nM).

[0105] Example 8: SI-F019 reduces the incidence of ADE.

[0106] Antibody-dependent enhancement (ADE) is a phenomenon in which virus binding to inappropriate antibodies enhances virus entry into host cells. In the case of COVID-19, secondary SARS-CoV-2 infection in patients with anti-SARS-CoV-2 antibodies due to primary infection or in vaccinated individuals can lead to enhanced virus uptake by monocytes and B cells. Antiviral antibodies that come into contact with the virus can bind to Fc receptors expressed on specific immune cells or on some complement proteins. The latter binding is dependent on the Fc region of the antibody. Typically, viruses are degraded by a process called phagocytosis, in which viral particles are engulfed by host cells through the plasma membrane. However, if the virus is not neutralized by antibodies, antibody binding can cause viral escape due to low binding affinity or targeting a non-neutralizing epitope. This results in antibody-mediated enhancement of infection.

[0107] Antibodies generated by either natural immunization or vaccination possess wild-type Fc regions. SI-F019 can compete with anti-spike antibodies to bind to the SARS-CoV2 virus, whereas the IgG1 Fc null fragment cannot bind to either Fc receptors or C1q (see Table 3). To demonstrate its comparative advantage in mitigating the effects of ADE, we evaluated the role of SI-F019 in internalization, replication, and reinfection.

[0108] In an assay to measure Fc-mediated internalization, the SARS-CoV-2 S protein was packaged into a GFP-expressing pseudovirus (PsV) and two cell lines expressing Fc receptors and complement receptor 2 (CR2)—THP1 (monocytes) and Daudi (B cells)—were used to test the FcRg and CR2-mediated ADE mechanism. SI-69R3 was used as a control for SI-F019 and contains wild-type Fc, whereas SI-F019 contains an IgG1 Fc null modification (see Table 1). After 48 hours of exposure to PsV, green fluorescent signals from cells were quantified as an indicator of PsV infection. In conditions treated with PsV and SI-69C1, anti-S1 antibody, or SI-69R3, low levels of green fluorescence were measured in THP1 (pH 7.2) (6A), THP1 (pH 6.0) (6B), and Daudi (6C) cells after 48 hours. These results suggest that some PsV transfer may occur via Fc receptors. In contrast, conditions containing the indicated concentrations of SI-F019 did not result in PsV uptake by THP1 or Daudi cells, and the green fluorescent signal was comparable to that measured in negative control conditions, such as assay media, formulation buffer, and SI-69C1 (Figure 6). As can be seen from the results of treating cells with doses ranging from 1 pM to 100 nM, the effect of Fc-mediated ADE was dose-dependent. This indicates that PsV uptake may occur via either the FcγR or CR2 mechanism.

[0109] Example 9: SI-F019 reduces PsV viral load.

[0110] SI-F019 may not mediate the internalization of GFP-expressing pseudoviruses (PsVs) packaged with the S protein due to the lack of a functional Fc fragment. To confirm whether SI-F019 could inhibit pseudovirus uptake, we used SI-F019 as a co-treatment with SI-69R3 or a natural anti-SARS-CoV-2 antibody in a competitive mode. Before infecting the same set of target cells, PsVs were incubated with SI-F019 at doses ranging from 1 pM to 100 nM, along with 10 pM of anti-SARS-CoV-2 (S1) antibody or 10 pM of SI-69R3, for 1 hour. The GFP signal derived from PsV was detected as the viral load of infection. SI-F019 was able to inhibit the viral load of PsV in target cells from 10 fM (Figure 7).

[0111] Both antibodies, such as anti-SARS-CoV-2 (S1) antibodies, and the truncated ACE2-wild-type Fc fragment fusion protein SI-69R3, were able to mediate internalization of SARS-CoV-2 spike-pseudotyped lentiviruses, whereas SI-F019, lacking a functional Fc fragment, was unable to mediate internalization of SARS-CoV-2 spike-pseudotyped lentiviruses. Here, SI-F019 was effective in reducing PsV viral loads even at concentrations as low as 10 fM in the presence of either 10 pM anti-SARS-CoV-2 (S1) antibodies or 10 pM SI-69R3. These results demonstrate that SI-F019 can reduce the incidence of ADE induced by FcRg- and CR2-dependent mechanisms in THP1 monocytes and Daudi B cells, respectively.

[0112] Example 10: HEK293-T cells expressing SARS-CoV-2 spike protein

[0113] We established HEK293-T cells (ATCC: CRL-3216) stably expressing the SARS-CoV-2 spike protein by transducing lentivirus packaged with a cDNA encoding the SARS-CoV-2 spike protein (Accession No. YP_009724390.1) and selecting for puromycin resistance via IRES expression driven by the same expression construct (LPP-CoV219-Lv105-050, GeneCopoeia). Expression of the SARS-CoV-2 spike protein was confirmed by binding of the human IgG clone AM001414 specific for the SARS-CoV-2 spike protein, "Anti-Spike" (SKU938701, Biolegend), and the human IgG isotype-matched clone QA16A12 (SKU403502, Biolegend) was used as an "isotype" control. Bound protein was quantified by secondary incubation with polyclonal anti-human Fc AF647 Fab (SKU109-607-008, Jackson Immuno Research), and the FACS evaluation is shown in Figure 8.

[0114] HEK293-T cells expressing either SARS-CoV-2 spike protein and parental HEK293 cells were stained with the indicated materials in the presence of the internalization inhibitor sodium azide for 30 minutes at 37°C. After removing free SI-F019, SI-F019 was detected and quantified using anti-human Fc AF647 Fab (SKU109-607-008, Jackson ImmunoResearch) and flow cytometry analysis. Binding of SI-F019 to the target cell line was quantified by the geometric mean signal intensity, as shown in Figure 9. HEK293-T cells expressing either SARS-CoV-2 spike protein can be used as a model for COVID-19-infected cells.

[0115] Example 11: Effect of SI-F019 on antibody-dependent cellular cytotoxicity (ADCC)

[0116] Antibody-dependent cellular cytotoxicity (ADCC) is one of the key immune responses against viral infections, such as SARS-CoV-2 infection in the case of COVID-19. After initial viral infection, antiviral antibodies directly bind to viral particles, neutralizing and agglutinating them. Binding of virus-antibody complexes to Fc receptors on phagocytes can trigger phagocytosis and destroy the virus, while binding to Fc receptors on immune effector cells, such as monocytes, neutrophils, eosinophils, and NK cells, can trigger the release of cytotoxic factors (e.g., antiviral interferons) and create a microenvironment hostile to viral replication.

[0117] To distinguish the effects of SI-F019 from anti-spike antibodies, HEK293-T cells expressing the SARS-CoV-2 spike protein were loaded with Calcein-AM and co-cultured with purified human NK cells at an effector-to-target ratio of 5:1. The treatments tested included SI-F019 and the S1-specific human IgG clone SI-69C3, a human antibody clone CC12.3 isolated from a hospitalized COVID-19 patient (10.1126 / science.abc7520). After 12 hours of co-culture, cells were stained with propidium iodide to assess viability. Calcein-AM expression and the reduction in viable target cell frequency (population 3) based on propidium iodide staining were assessed as measures of cytolysis, as shown in Figure 10.

[0118] NK cell-mediated ADCC could be directed against HEK293-T cells expressing SARS-CoV-2 proteins upon exposure to the S1-specific human IgG clone SI-69C3 (clone CC12.3). SI-F019 did not mediate ADCC compared to SI-69C3 within the therapeutic range of 100 nM to 100 fM. Under these assay conditions, the SI-F019 drug variant containing the wt Fc (SI-69R3) was able to mediate ADCC in a dose-dependent manner, although the level of activity was lower than that of the S1-specific human IgG clone CC12.3, as shown in Figures 11 and 12. These data demonstrate that, unlike the SARS-CoV-2 S1-specific human IgG antibody, SI-F019 does not mediate NK cell-mediated ADCC.

[0119] Example 12: Effect of SI-F019 on complement-dependent cytotoxicity (CDC)

[0120] The role of the complement cascade in mediating antibody-based cell and tissue injury in COVID-19 patients is evident in both the innate immune response and neutralizing antibody-based therapy (Perico et al., 2021). Immune complexes formed between the virus and specific IgG mediate complement-induced blood coagulation, thromboembolism, and systemic microangiopathy. These widespread complications in COVID-19 patients are potentially life-threatening and depend on complement proteins binding to IgG. Viral immune complexes that crosslink red blood cells via C1q and platelets bearing FcγRIIA are mediators of thromboembolism in COVID-19 patients (Nazy et al., 2020). Immune complex fixation to the endothelial vessel wall and complement-mediated coagulation are major concerns in COVID-19 patients, where endothelial cell activation is part of the thromboembolic cascade.

[0121] Unlike natural IgG antibodies, SI-F019 cannot bind to C1q, as shown in Table 3. This feature eliminates the risk of inducing cell death in infected epithelial and endothelial cells that can transiently express the SARS-CoV-2 spike protein on their surface. This protective effect of SI-F019 has been demonstrated in comparison with anti-spike human IgG antibodies.

[0122] To demonstrate the protective effect of SI-F019, HEK293-T cells expressing the SARS-CoV-2 spike protein were cultured in serum-free medium (Optimem) and treated for 30 minutes before the addition of human serum complement at a serum-to-medium ratio of 1:10. The treatments tested included SI-F019 and S1-specific human IgG clone AM001414 (BioLegend). After 3 hours of incubation at 37°C, propidium iodide staining was performed, and positively stained cells were counted in each well. As shown in Figures 12 and 13, red blood cells counted using Incucyte Zoom software after 3 hours were assessed as a measure of CDC. Total cell confluence after 96 hours was assessed as a measure of CDC effect, as shown in Figure 13.

[0123] Protection of tissue cells from complement damage is further confirmed by the ability of these cells to further proliferate after human serum complement challenge. Human serum complement-mediated CDC was assessable in HEK293T cells expressing SARS-CoV-2 S protein when exposed to an S1-specific human IgG clone (clone AM4141) at a volume-to-volume ratio of 1:10 in serum-free medium. Consequently, both human soluble monomeric ACE2 and SI-F019 did not mediate CDC, whereas SI-69R3 only increased CDC activity in a dose-dependent manner compared to human IgG antibodies. Based on full confluence after 96 hours of treatment, CDC cytolysis was reflected in a decrease in cell proliferation.

[0124] Example 13: Cytokine release induced by soluble or plate-bound SI-F019 in PBMC cultures

[0125] SARS-CoV-2 has an affinity for ACE2-expressing epithelia in the respiratory tract and small intestine. Clinical laboratory findings of elevated levels of IL-2, IL-6, IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ-inducible protein 10 (IP-10), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), and tumor necrosis factor α (TNF-α), indicative of cytokine release syndrome (CRS), suggest an underlying immunopathology. CRS is a major side effect that may limit the usefulness of biologic treatments and was tested by in vitro cytokine release assays.

[0126] SI-F019 is a fusion protein composed of human ACE2 and a mutant human IgG1 Fc that cannot bind to Fcγ receptors. Therefore, SI-F019 is expected not to bind to target cells in peripheral blood or induce cytokine release. White blood cells (WBCs), including neutrophils, isolated from five healthy donors were placed in culture wells containing plate-bound or soluble SI-F019 at concentrations of 2000 nM and 200 nM.

[0127] The TGN1412 antibody was used as a positive control at the same concentration and in the same format, due to its well-documented ability to induce cytokine release in the plate-bound format of this assay. Comparison with SI-69R3 assessed the potential contribution of the IgG1 Fc null fragment to reduced cytokine release. SI-69R3 possesses a wild-type Fc fragment that can bind to Fcγ receptors expressed by several cell types in peripheral blood. WBC cultures containing only SI-F019 formulation buffer at a similar dilution were used as a negative control. Culture supernatants were collected at 24 and 48 hours, and the presence of nine cytokines was detected using the Meso Scale Discovery (MSD) platform.

[0128] The cytokine panel included the T cell-associated cytokines IFNγ, TNFα, GM-CSF, IL-2, and IL-10, as shown in Figures 14A-14E. Levels of the proinflammatory non-T cell-associated cytokines IL-1β, IL-12p70, and IL-6, and the monocyte chemotactic protein MCP-1, as shown in Figures 14F-14I, were also tested. Each well was measured in duplicate for each donor, and the results were averaged. Box plots showing 95% confidence intervals and outliers were generated using JMP14 software.

[0129] As can be seen from the results, SI-F019, at concentrations of 200 nM and 2000 nM, in either plate-bound or soluble formats, did not induce exposure of WBCs to any of the tested cytokines. Cytokine levels in SI-F019-treated samples were similar to those in the buffer control in all conditions. The positive control TGN1412, in plate-bound but not soluble format, strongly induced most cytokines, consistent with previously published results. When plate-bound ACE2-Fc wild-type was used to stimulate leukocytes, intermediate production of IFNγ, GM-CSF, and TNFα was detected, indicating the improved safety of the Fc null fragment of SI-F019.

[0130] The pathogenic role of humoral responses to the SARS-CoV-2 virus has recently been suggested by patients receiving interventional IgG therapy (Weinreich et al., 2021; Chen et al., 2021). Hyperinflammatory responses in small vessels underlie adverse events such as thrombocytosis, pruritus, fever, and hypertension. This study demonstrates that SI-F019 can provide virus-neutralizing effects comparable to IgG therapy while protecting tissues and organs from multiple pathways of dysfunction. Therefore, SI-F019 can be used to treat, prevent, or alleviate viral infections, particularly to prevent and manage the progression of COVID-19 and reduce clinical complications, as well as acute respiratory distress syndrome, pulmonary arterial hypertension, or acute lung injury.

[0131] Example 14: Binding kinetics (affinity) of SI-F019 to SARS-CoV-2 RBD variants As the pandemic continues, mutation and selection drive the evolution of the SARS-CoV-2 virus to acquire higher binding affinity for ACE2 and thus increase viral transmission, resulting in mutant strains, including the newly emerged, highly infectious delta variant. In fact, both the alpha and delta variants are more infectious than the original SARS-CoV-2 virus. The spread of SARS-CoV-2 variants remains an unresolved challenge in developing treatments and prevention strategies. While SI-F019 is a potential neutralizing agent, the FDA has approved the use of b) bamlanivimab (Eli Lilly's LY-CoV555; SI-69C4, SEQ ID NOs: 29 and 30), c) casirivimab (Regeneron's REGN10933; SI-69C5, SEQ ID NOs: 31 and 32), d) eteseevimab (Eli Lilly's CB-6; SI-69C6, SEQ ID NOs: 33 and 34); e) imdevimab (Regeneron's REGN10987; SI-69C7, SEQ ID NOs: 35 and 36); f) silgavimab (AstraZeneca's AZD1061; SI-69C8, SEQ ID NOs: 37 and 38), and g) tisagevimab (AstraZeneca's Several neutralizing antibodies have been approved, including AZD8895; SI-69C9, SEQ ID NOs: 39 and 40).

[0132] To determine the comparative advantage of the recombinant ACE2-Fc fusion protein SI-F019 over these neutralizing antibodies, we quantified the strength of binding interactions to SARS-CoV-2 RBD variants mimicking COVID-19 variants using Octet Red 384 biolayer interferometry. These variant proteins were purchased from Sino Biological. First, 10 μg / ml of SI-F019 protein in assay buffer (PBS containing 1% BSA and 0.05% Tween 20) was loaded onto the AHC sensor for 180 seconds. After a 180-second baseline step, the loaded protein was allowed to associate with a 1:2 serial dilution of the RBD variant protein in assay buffer (highest concentration 50 nM) for 180 seconds, followed by a 300-second dissociation step in assay buffer. Regeneration was performed with 10 mM glycine (pH 1.5). The data were globally fitted to a 1:1 binding model using the full association and the first 60 seconds of the dissociation phase, and the kinetic parameter K was calculated. D , k on and k dis The binding affinities of the same variant and wild-type RBD proteins shown in Tables 2 and 4 are comparable, and readings may vary depending on the vendor (ACROBiosystem and Sino Biological).

[0133] Figure 15(a) shows the sensorgrams of SI-F019 and neutralizing antibodies when bound to the RBD variants, and Table 4(a) shows the extracted binding kinetics (affinity) parameters. As a result, compared to SI-F019 (15a), bamlanivimab (SI-69C4) failed to bind to the kappa, gamma, beta, and lambda variants (15b), casirivimab (SI-69C5) showed significantly reduced binding affinity for the gamma and beta variants (15c), and eteseevimab (SI-69C6) also showed significantly reduced binding affinity for the gamma and beta variants (15d). Other monoclonal antibodies, such as imdevimab (SI-69C7) (4e), silgavimab (SI-69C8) (4f), and tisagevimab (SI-69C9) (4g), may not exhibit significant changes in their binding affinity, but sensorgrams revealed reduced binding responses for some variants (Figures 15e, 15f, and 15g). In contrast to neutralizing antibodies, SI-F019 exhibited higher binding affinity for new variants, including the highly transmissible δ variant, than for the ancestral wild-type RBD (Figure 15a, Table 4a). This observation supports the notion that the ACE2 portion of SI-F019 retains the wild-type full-length ACE2 conformation (the target for selection of high-affinity variants) required for viral entry into human cells. Therefore, SI-F019 has a comparative advantage over FDA-approved neutralizing antibodies and can be used as a binder and blocker.

[0134] Example 15: Binding kinetics (avidity) of SI-F019 and neutralizing antibodies to variants The binding affinity assay described above measures the binding of SARS-CoV-2 RBD protein variants in solution to SI-F019 immobilized on an anti-human IgG Fc capture biosensor chip (AHC) surface, while the avidity assay measures the binding of SI-F019 in solution to biotinylated SARS-CoV-2 RBD protein variants immobilized on a streptavidin biosensor chip surface.

[0135] The strength of the binding interaction between SI-F019 and the SARS-CoV-2 S protein variant RBD domain was quantified by biolayer analysis using Octet Red 384. The reagent was purchased from Sino Biological and chemically biotinylated via NHS ester activation (biotin / protein stoichiometry: 2:1). First, 2 μg / ml of biotinylated RBD or its variant protein in assay buffer (PBS containing 1% BSA and 0.05% Tween 20) was loaded onto the SA sensor for 180 seconds. After a 180-second baseline step, the loaded protein was allowed to associate with a 1:2 serial dilution (maximum concentration: 50 nM) of SI-F019 protein (GMP lot) in assay buffer for 300 seconds, followed by a 600-second dissociation step in assay buffer. Regeneration was performed with 10 mM glycine (pH 1.5). The data were globally fitted to a 1:1 binding model using the complete association and dissociation phases, and the kinetic parameter K D , k on and k dis The binding affinities of the same mutant and wild-type RBD proteins shown in Tables 2 and 5 are comparable, and the readings may vary depending on the vendor (ACROBiosystem and Sino Biological).

[0136] Figure 16(a) shows the sensorgrams of SARS-CoV-2 RBD protein variants bound to SI-F019, and Table 5(a) shows the extracted binding kinetics (avidity) parameters. Notably, SI-F019 binds with higher avidity to the variant forms of RBD compared to wild-type RBD, and the increased avidity is primarily driven by a slower dissociation rate. The results show that, compared to wild-type RBD, the binding affinity of SI-F019 to the RBD mutants is increased, the dissociation rate is decreased, and the efficacy of some neutralizing antibodies is reduced or diminished.

[0137] Example 16: Comparative superiority of SI-F019 over neutralizing antibodies To demonstrate the comparative superiority of SI-F019 over neutralizing antibodies, binding responses or values of responses were used. The response was measured as the nm shift in the interference pattern as shown in Figures 15 and 16, and is proportional to the number of molecules bound to the surface of the biosensor. The response is the maximum binding signal achieved when associating with the maximum concentration of analyte, and is a complex function that depends on kinetic binding parameters, protein size, and assay conditions, and should be equivalent in a particular assay as long as the sizes and formats of the interacting proteins are similar. Differences in responses can indicate differences in the effective strength of protein-protein interactions, with a high response indicating a strong interaction and a low response indicating a weak interaction.

[0138] Table 6 shows the values of responses (maximum concentration of analyte) extracted from the binding affinity and avidity of SI-F019 and neutralizing antibodies in Figures 15 and 16. To evaluate changes in the binding kinetics of each neutralizing agent and variant, the following definitions were applied. 1) When the response < 10% of WT, there is no binding; 2) When the response < 30% of WT, there is minimal binding; 3) When the response < 75% of WT, there is low binding; 4) In the case of complex kinetics, including dissociation of the rise and non-specific binding. When these criteria are applied to each response, each neutralizing antibody shows low binding or no binding at all to at least one variant. In particular, in the avidity assay, bamlanivimab (SI-69C4) showed a decreased binding response to δ, κ, γ, β, λ, etesevimab (SI-69C6) showed a decreased binding response to γ and β, and casirivimab (SI-69C5), imdevimab (SI-69C7), cilgavimab (SI-69C8) and tisagenevimab (SI-69C9) all showed a decreased binding response to β. In contrast, SI-F019 retains binding affinity and avidity for all variants.

[0139] Example 17: SI-F019 functions as a blocker that can inhibit the viral infectivity of novel coronavirus infection (COVID-19) RBD variants. To test the ability of SI-F019 to prevent viral infection, viral infectivity was assessed by luciferase reporter assay. SARS-CoV-2 S protein-packaged pseudovirus (NICPBP) (wild-type or mutant strain, Sino Biological) containing a luciferase reporter gene was co-incubated with ACE2-overexpressing 293T cells (clone 3D4) and 1:3 serial dilutions of SI-F019 (starting from 30 μg / ml). ACE2 expression on transfected cells was confirmed by enzyme and FACS assays. The pseudovirus can enter ACE2-positive cells via the S protein, which binds to ACE2, triggering luciferase expression. Therefore, luminescence is used as a readout of viral infectivity.

[0140] Specifically, a 10x stock solution of S protein pseudovirus was prepared in culture medium to achieve a final viral load of 227–394 TCID50 / well. SI-F019 was serially diluted 3-fold in culture medium to a maximum concentration of 150 μg / ml (final 30 μg / ml). 3D4 cells were harvested using trypsin-free dissociation buffer. Pseudovirus (20 μl) and SI-F019 (30 μl) were mixed in wells of a 96-well plate and incubated at room temperature for 1 hour. Next, 100 μl of harvested 3D4 cells were added to each well (20,000 / well) and incubated at 37°C, 5% CO2 for 18 hours. After incubation, the supernatant was removed, and 50 μl of luciferase substrate solution was added, mixed, and incubated at room temperature for 1 minute. Luminescence was read using a 3x plate reader. The luminescence signal in RLU (relative light units) represents the infectivity of the S protein pseudovirus.

[0141] The percent inhibition of infectivity can be determined by calculating the decrease in luminescence compared to conditions without SI-F019. This data was then fitted to a sigmoid function in GraphPad Prism 6.0 to extract the IC50 values ​​for SI-F019 inhibiting pseudovirus infectivity when the pseudovirus contained different variants of the S protein. The viral inhibition data are shown in Figure 17a, and the IC50 values ​​are listed in Table 7. As a result, SI-F019 was able to inhibit the infectivity of all variants with a 2- to 15-fold increase in potency compared to its potency against the wild-type strain. The linear correlation between the IC50 and KD values ​​of affinity (or avidity) (Figures 17b and 17c) indicates competitive inhibition by SI-F019. In vitro experimental data show that ACE2 proteins, such as the SI-F019 fusion protein for membrane-bound ACE2 proteins, can not only strongly bind to but also inhibit SARS-CoV-2 viral variants with increased affinity of the RBD-ACE2 interaction that evolves with increased infectivity and transmissibility.

[0142] Example 18: SI-F019 (NCT04851444) in a Phase I Clinical Trial in Healthy Participants in China Currently, three anti-SARS-CoV-2 monoclonal antibody products have received Emergency Use Authorization (EUA) from the Food and Drug Administration (FDA) for the treatment of mild to moderate COVID-19 in non-hospitalized patients. These patients have laboratory-confirmed SARS-CoV-2 infection and are at high risk of progressing to severe disease and hospitalization. The first, bamlanivimab + eteseevimab, are neutralizing monoclonal antibodies that bind to distinct but overlapping epitopes in the SARS-CoV-2 spike protein RBD. The second, casirivimab + imdevimab, are recombinant human monoclonal antibodies that bind to non-overlapping epitopes in the SARS-CoV-2 spike protein RBD. The third, sotrovimab, is a monoclonal antibody first identified in a SARS-CoV survivor in 2003. It targets an epitope in the spike protein RBD that is conserved between SARS-CoV and SARS-CoV-2.

[0143] Unlike monoclonal antibodies that bind to a single epitope, SI-F019 has the technical advantage of competing with the ACE2 protein on human cells for binding to all docking sites on the SARS-CoV-2 spike protein RBD, some of which may overlap. To reduce the burden and technical difficulties of combining two or more monoclonal antibodies as a treatment plan, SI-F019, currently undergoing clinical trials, offers the advantage of acting as a single therapeutic agent effective for the treatment of mild to moderate COVID-19 in individuals at high risk of progressing to severe COVID-19 and for post-exposure prophylaxis (PEP) of SARS-CoV-2 infection. SI-F019 can compete with other coronaviruses (e.g., SARS-CoV-1) that target the membrane-bound ACE2 protein on human cells (Figure 4). In addition to COVID-19, SI-F019 can also be used to treat severe acute respiratory syndrome (SARS) caused by SARS-CoV-1 virus, Middle East respiratory syndrome (MERS) caused by MERS-CoV virus, acute respiratory distress syndrome (ARDS), and other lung injuries. In such cases, SI-F019 can also be used in combination with other therapeutic agents as needed.

[0144] The objective of this Phase 1 study is to evaluate the safety, tolerability, and pharmacokinetic properties of a single intravenous administration of SI-F019. The study is double-blind, placebo-controlled, and randomized, with dose escalation from 3 mg / kg to 70 mg / kg (Tables 8a and 8b). The fusion protein will be administered as a suspension in histidine / histidine hydrochloride, sodium chloride, sucrose, and polysorbate 80. A total of 36 participants received a single dose on Day 1 and were followed through Day 29. Treatment-emergent adverse events (TEAEs), treatment-related adverse events (TRAEs), severity, and laboratory abnormalities were captured and graded using NCI-CTCAE v5.0. As of September 16, 2021, the electronic data capture (EDC) database has not yet been locked. Based on blinded review of the data, of the 36 participants, 21 experienced 44 adverse events (AEs), including 31 TRAEs in 16 of them. All AEs were grade 1, and no significant association with SI-F019 dose was found (Tables 9a and 9b). The overall good tolerability and safety of SI-F019 support further exploration as a preventative and therapeutic agent for COVID-19 and other related diseases.

[0145] table

[0146] Table 1: Cloning, expression, and purification of recombinant ACE2-Fc fusion proteins [Table 1]

[0147] Table 2: Affinity and avidity of SI-F019 to viral proteins [Table 2]

[0148] Table 3: Effect of Fc null mutations on binding to Fc receptors [Table 3]

[0149] Table 4: Binding kinetics (affinity) of SI-F019 (4a) and neutralizing antibodies (including bamlanivimab (SI-69C4) (4b), casirivimab (SI-69C5) (4c), eteseevimab (SI-69C6) (4d), imdevimab (SI-69C7) (4e), silgavimab (SI-69C8) (4f), and tisagevimab (SI-69C9) (4g)) to various variants of the S protein RBD. SI-F019 bound with higher affinity to the mutant forms of the RBD compared to the wild-type RBD, primarily due to a slower dissociation rate, whereas at least three neutralizing antibodies lost binding to at least one of the mutant forms (ND). [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0150] Table 5: Quantification of the binding kinetics (avidity) of SI-F019 (5a) and neutralizing antibodies (bamlanivimab (SI-69C4) (5b), casirivimab (SI-69C5) (5c), eteseevimab (SI-69C6) (5d), imdevimab (SI-69C7) (5e), ​​silgavimab (SI-69C8) (5f), and tisagevimab (SI-69C9) (5g)) to various variants of the S protein RBD by biolayer interferometry. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0151] Table 6a: Comparative analysis of maximum binding responses (affinity) of SI-F019 and neutralizing antibodies against SARS-CoV-2 viral RBD Four of these antibodies ranged from low to no response to at least one variant, whereas SI-F019 showed increased responses to all variants. [Table 6-1]

[0152] Table 6b: Comparative analysis of the binding response (avidity) of SI-F019 or neutralizing antibodies against SARS-CoV-2 viral RBD The antibodies ranged from low to no response, whereas SI-F019 showed no significant change. [Table 6-2]

[0153] Table 7: IC50 values ​​for inhibition of viral infectivity in luciferase reporter assays using S protein-packaged pseudovirus (NICPBP) to infect ACE2-expressing 293T cells. Based on the lower IC50 values, SI-F019 inhibition of pseudoviruses containing mutant forms of the S protein is more potent than that of pseudoviruses containing the wild-type S protein. [Table 7]

[0154] Table 8a: Demographic summary (age, weight) of the Phase I clinical trial of SI-F019 (NCT04851444) [Table 8-1]

[0155] Table 8b: Dose Escalation and Allocation Design for the Phase I Clinical Trial of SI-F019 (NCT04851444) [Table 8-2]

[0156] Table 9a: Summary of all adverse events (AEs) by system organ class / preferred term (SOC / PT) for the Phase 1 clinical trial of SI-F019 (NCT04851444) [Table 9-1] *N: Number of patients who experienced an AE.

[0157] Table 9b: Summary of all treatment-related adverse events (TRAEs) by system organ class / preferred term (SOC / PT) for the Phase 1 clinical trial of SI-F019 (NCT04851444) [Table 9-2] *N: Number of patients who experienced TRAE

[0158] Sequence Listing [Table 10]

[0159] >Sequence ID 1: huACE2 full length protein sequence (Genbank_number:NP_001358344.1, TMPRSS2 protease cutting site ) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIP RTEVEKAIRMSRSRINDAFR LNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDDVQTSF >Sequence ID 2: huACE2 full length DNA sequence (Genbank_number: NM_021804.3) >Sequence ID 3: huACE2 functional domain (residue:1-615) protein sequence MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD >Sequence ID 4: huACE2 functional domain (residue:1-615) DNA sequence >Sequence ID 5: Fc wild type IgG1 Fc (EU numbering 216-447) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 6: Fc null version (EU numbering 216-447, with mutations: C220S , L234A , L235A , and K322A ) EPKS S DKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC A VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 7: SI-69R2_huACE2 functional domain (residue:1-615)- IgG1 Fc (null) protein sequence (EU numbering 216-447, with mutations: C220S, L234A, L235A, and K322A) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 8:SI-69R2: huACE2 functional domain (residue:1-615)- IgG1 Fc (null) DNA sequence >Sequence ID 9: huACE2 functional domain (residue:1-615)- IgG4 Fc protein sequence MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK >Sequence ID 10: huACE2 functional domain (residue:1-615)- IgG4 Fc DNA sequence CCA TGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA >Sequence ID 11: huACE2 functional domain (residue:1-615)- IgA1 Fc Protein sequence MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY >Sequence ID 12: huACE2 functional domain (residue:1-615)- IgA1 Fc DNA sequence >Sequence ID 13: huACE2 functional domain (residue:1-615)- IgA2 Fc Protein sequence MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADSQDVTVPCRVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTYAVTSILRVAAEDWKKGETFSCMVGHEALPLAFTQKTIDRMAGKPTHINVSVVMAEADGTCY >Sequence ID 14: huACE2 functional domain (residue:1-615)- IgA2 Fc DNA sequence >Sequence ID 15: SI-F019_huACE2 functional domain (residue:18-615)- IgG1 Fc (null) protein sequence (with mutations at C220S, L234A, L235A, and K322A, EU numbering) QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 16: huACE2 functional domain (residue:18-615)- IgG4 Fc protein sequence QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK >Sequence ID 17: huACE2 functional domain (residue:18-615)- IgA1 Fc Protein sequence QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY >Sequence ID 18: huACE2 functional domain (residue:18-615)- IgA2 Fc Protein sequence QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADSQDVTVPCRVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTYAVTSILRVAAEDWKKGETFSCMVGHEALPLAFTQKTIDRMAGKPTHINVSVVMAEADGTCY >Sequence ID 19: SI-69R3_human ACE2-ECD-1-615-Fc-w2(EU numbering 216-447)-protein sequence MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 20: SI-69R3_human ACE2-ECD-1-615-Fc-w2-DNA sequence >Sequence ID 21: SI-69R4-human ACE2-ECD-1-740( TMPRSS2 protease cutting site )-Fc-w2(EU numbering 216-447)-protein sequence MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIP RTEVEKAIRMSRSRINDAFR LNDNSLEFLGIQPTLGPPNQPPVSEPKS SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 22: SI-69R4_human ACE2-ECD-1-740-Fc-w2-DNA sequence >Sequence ID 23: SI-69R1_huACE2 functional domain (residue:1-615)- 6XHis protein sequence MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADHHHHHH >Sequence ID 24: SI-69R1_huACE2 functional domain (residue:1-615)- 6XHis DNA sequence >Sequence ID 25: SI-69R10_Human TMPRSS2 protein, His-tagged (106-492)- protein sequence MYRMQLLSCIALSLALVTNSWKFMGSKCSNSGIECDSSGTCINPSNWCDGVSHCPGGEDENRCVRLYGPNFILQVYSSQRKSWHPVCQDDWNENYGRAACRDMGYKNNFYSSQGIVDDSGSTSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVNLNSSRQSRIVGGESALPGAWPWQVSLHVQNVHVCGGSIITPEWIVTAAHCVEKPLNNPWHWTAFAGILRQSFMFYGAGYQVEKVISHPNYDSKTKNNDIALMKLQKPLTFNDLVKPVCLPNPGMMLQPEQLCWISGWGATEEKGKTSEVLNAAKVLLIETQRCNSRYVYDNLITPAMICAGFLQGNVDSCQGDSGGPLVTSKNNIWWLIGDTSWGSGCAKAYRPGVYGNVMVFTDWIYRQMRADGHHHHHH >Sequence ID 26: SI-69R10_Human TMPRSS2 protein, His-tagged (106-492)- DNA sequence >Sequence ID 27: IgJ chain MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD >Sequence ID 28: Secretory Component KSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITLISSEGYVSSKYAGRANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLLNDTKVYTVDLGRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRLDIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCRQSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSVVITGLRKEDAGRYLCGAHSDGQLQEGSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLLVDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAAGSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPR >Sequence ID 29: Bamlanivimab_Heavy_Chain QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYEARHYYYYYAMDVWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 30: Bamlanivimab_Light_Chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLSWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTITSLQPEDFATYYCQQSYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 31: Casirivimab_Heavy_Chain QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYITYSGSTIYYADSVKGRFTISRDNAKSSLYLQMNSLRAEDTAVYYCARDRGTTMVPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 32: Casirivimab_Light_Chain DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAPKLLIYAASNLETGVPSRFSGSGSGTDFTFTISGLQPEDIATYYCQQYDNLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 33: Etesevimab_Heavy_Chain EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGGSTFYADSVKGRFTISRDNSMNTLFLQMNSLRAEDTAVYYCARVLPMYGDYLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 34: Etesevimab_Light_Chain DIVMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPEYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 35: Imdevimab_Heavy_Chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYAMYWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRTEDTAVYYCASGSDYGDYLLVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > Sequence ID 36: Imdevimab_Light_Chain QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQSEDEADYYCNSLTSISTWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS >Sequence ID 37: Cilgavimab_Heavy_Chain EVQLVESGGGLVKPGGSLRLSCAASGFTFRDVWMSWVRQAPGKGLEWVGRIKSKIDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTAGSYYYDTVGPGLPEGKFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 38: Cilgavimab_Light_Chain DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLMYWASTRESGVPDRFSGSGSGAEFTLTISSLQAEDVAIYYCQQYYSTLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 39: Tixagevimab_Heavy_Chain QMQLVQSGPEVKKPGTSVKVSCKASGFTFMSSAVQWVRQARGQRLEWIGWIVIGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAAPYCSSISCNDGFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 40: Tixagevimab_Light_Chain EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSRGWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A liquid composition comprising a fusion protein, the fusion protein comprises a variant angiotensin-converting enzyme 2 (ACE2) domain covalently fused to an Fc domain; The variant ACE2 domain comprises an N-terminal deletion and / or a C-terminal deletion compared to the full-length wild-type ACE2 having SEQ ID NO:1, the variant ACE2 domain has ACE2 activity, The fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 15, 16, 17 and 18.

2. 3. The liquid composition of claim 2, wherein the content of the fusion protein per dose is from about 100 mg to about 10,000 mg.

3. 3. The liquid composition of claim 2, wherein the concentration of the fusion protein is from about 0.5% to about 1% by weight.

Images