ACE2 modified protein and its application

Abstract

This invention relates to the field of genetic engineering technology. Specifically, it provides the use of a modified ACE2 protein in the preparation of drugs for treating and / or preventing diseases caused by SARS-like coronaviruses that use ACE2 as a receptor, or in the preparation of vaccines for preventing infection by SARS-like coronaviruses that use ACE2 as a receptor. The modified hACE2 fragment and hFc protein of this invention are antibody-like proteins, which can be widely used to treat diseases caused by the original and variant strains of SARS-CoV-2, and are expected to treat diseases caused by SARS-related coronaviruses or SARS-like coronaviruses that use ACE2 as a receptor.

Description

Technical fields:

[0001] This invention relates to the field of genetic engineering technology, and more specifically to the field of antibody-like proteins that have a neutralizing effect against the novel coronavirus. Background technology:

[0002] The COVID-19 pandemic poses a significant threat to human health and global public health security. Its pathogen, SARS-CoV-2, is the third coronavirus to cause infectious disease outbreaks in humans since the 21st century. Currently, although several vaccines and monoclonal antibody drugs targeting SARS-CoV-2 have received emergency use authorization, most studies indicate that their protective efficacy against currently widespread variants of the novel coronavirus (such as the UK strain N501Y.V1, the South African strain N501Y.V2, and the Brazilian strain N501Y.V3) is reduced. Furthermore, a large amount of research data indicates that animals such as bats carry multiple SARS-related coronaviruses or SARS-like coronaviruses, such as bat-derived RaTG13 and pangolin-derived GX / P2V / 2017 and GD / 1 / 2019. Current research results show that these three viruses can also utilize angiotensin-converting enzyme 2 (ACE2) as a receptor, suggesting that they have the potential to infect humans. Therefore, there is an urgent need to develop broad-spectrum drugs or vaccines against current SARS-like coronavirus variants and potential new SARS-like coronavirus outbreaks in the future. This is not only a major need for the country and the world under the current COVID-19 pandemic, but also an important measure related to global human health (One Health).

[0003] ACE2, a member of the renin-angiotensin system, maintains cardiovascular, renal, and respiratory function by regulating blood pressure and electrolyte balance. It also acts as a receptor for viruses such as SARS-CoV and SARS-CoV-2, mediating viral invasion. Studies have found that exogenous recombinant ACE2 protein (rACE2) can competitively bind to SARS-CoV and SARS-CoV-2 with endogenous ACE2, thereby inhibiting viral infection. Clinical trials have shown that rACE2 is safe in healthy individuals and patients with severe respiratory distress syndrome (ARDS). These findings suggest that rACE2 is a potential macromolecular drug for coronaviruses that use ACE2 as a receptor, making the design and development of highly effective rACE2 drugs potentially valuable for clinical applications. Currently, although several rACE2 inhibitors have been reported for inhibiting SARS-CoV and SARS-CoV-2 infection, with two in preclinical preparation and one in Phase II clinical trials, their effectiveness against currently prevalent SARS-CoV variants remains unknown. Summary of the Invention:

[0004] In view of this, the present invention provides the use of an ACE2 modified protein in the preparation of a medicament for the treatment and / or prevention of disease caused by SARS-like coronaviruses that use ACE2 as a receptor, or in the preparation of a vaccine for the prevention of infection with SARS-like coronaviruses that use ACE2 as a receptor.

[0005] In some specific embodiments of the present invention, the SARS-like coronavirus with ACE2 as the receptor is the original SARS-CoV-2 strain and / or the SARS-CoV-2 variant strain and / or the COVID-19 related coronavirus, etc.

[0006] In some specific embodiments of the present invention, the modified protein is the hACE2-hFc-M4-1 modified protein, the gene sequence of which is shown in SEQ ID No. 5 or the amino acid sequence of which is shown in SEQ ID No. 6.

[0007] In some specific embodiments of the present invention, the modified protein is the hACE2-hFc-M4-2 modified protein, the gene sequence of which is shown in SEQ ID No. 7 or the amino acid sequence of which is shown in SEQ ID No. 8.

[0008] In some specific embodiments of the present invention, the modified protein is the hACE2-hFc-M5 modified protein, the gene sequence of which is shown in SEQ ID No. 9 of the sequence list or the amino acid sequence of which is shown in SEQ ID No. 10 of the sequence list.

[0009] In some specific embodiments of the present invention, the original SARS-CoV-2 strain is SARS-CoV-2WT.

[0010] In some specific embodiments of the present invention, the SARS-CoV-2 variant strain is the British strain N501Y.V1, the South African strain N501Y.V2, or the Brazilian strain N501Y.V3.

[0011] In some specific embodiments of the present invention, the SARS-CoV-2 variant strain is the mink-circulating strain Y453F, the mink-circulating strain F486L, or the mink-circulating strain N501T.

[0012] The modified hACE2 fragment and the modified hFc protein of this invention are antibody-like proteins that can be used to treat diseases caused by the original and variant strains of the novel coronavirus. They can be widely applied to diseases caused by novel coronaviruses or SARS-like coronaviruses that use ACE2 as a receptor. Attached image description:

[0013] Figure 1.Structural diagram of the SARS-CoV-2 RBD-human ACE2 complex. Detailed implementation method:

[0014] Based on this, the inventors designed and screened a highly efficient recombinant human ACE2 protein that has the ability to neutralize the original SARS-CoV-2 strain and a variety of variant strains on a broad spectrum. At the same time, it eliminated the side effects caused by the enzymatic activity of ACE2 itself, indicating that it can be used as a potential broad-spectrum macromolecular drug against existing or future new variant strains of the novel coronavirus and new SARS-like coronaviruses that may emerge in the future.

[0015] First, based on the structure of the SARS-CoV-2 spike protein receptor-binding domain (RBD) and the human ACE2 (hACE2) complex ( Figure 1 The researchers discovered that the amino acids involved in RBD interactions on ACE2 are mostly hydrophobic or charged. Therefore, they selected a series of sites for mutation to enhance these hydrophobic or charged amino acids. Simultaneously, they fused these sites with the Fc segment (hFc) of human IgG1, ultimately forming a series of hACE2-hFc mutants. Then, by measuring the affinity of these hACE2-hFc mutants for the RBD of the original SARS-CoV-2 strain (SARS-CoV-2WT), they screened for hACE2-hFc mutants with enhanced affinity. These mutants were then combined to form combined mutants with even stronger affinity, including the four sites T27F, K31Y, L79W, and N330Y.

[0016] Furthermore, since ACE2 plays an important role in regulating blood pressure and electrolyte balance, in order to eliminate the potential side effects such as excessive blood pressure regulation caused by excessive exogenous ACE2 protein, according to literature reports, the key amino acid R273, which controls enzyme activity, is mutated (R273Q) to disrupt its enzyme activity without affecting its binding to SARS-CoV-2. These are referred to below as hACE2-hFc-M4-1 (including four mutation sites: T27F, K31Y, N330Y, and R273Q), hACE2-hFc-M4-2 (including four mutation sites: T27F, L79W, N330Y, and R273Q), and hACE2-hFc-M5 (including five mutation sites: T27F, K31Y, L79W, N330Y, and R273Q), and their coding sequences and amino acid sequences are SEQ ID No. 5-10, respectively.

[0017] After expressing and purifying hACE2-hFc-M4-1, hACE2-hFc-M4-2, and hACE2-hFc-M5 proteins, their affinity for the original SARS-CoV-2 virus strain and various variant strains (RBD) was determined. These variant strains included the UK strain N501Y.V1, the South African strain N501Y.V2, the Brazilian strain N501Y.V3, and three mink-circulating strains Y453F, F486L, and N501T. Compared to wild-type hACE2-hFc (hACE2-hFc... The researchers found that hACE2-hFc-M4-1 and hACE2-hFc-M4-2 both enhanced their ability to bind to the original and variant RBD strains of SARS-CoV-2 (Table 1). Furthermore, their ability to bind to SARS-CoV-2WT and the mink epidemic strain Y453F was similar, while their ability to bind to other variant strains differed. Therefore, the inventors combined hACE2-hFc-M4-1 and hACE2-hFc-M4-2 to form hACE2-hFc-M5.

[0018] The binding RBD of hACE2-hFc-M5 to the original and variant strains of SARS-CoV-2 is 3.7 to 29.5 times that of hACE2-hFc-WT, and slightly higher than that of hACE2-hFc-M4-1 and hACE2-hFc-M4-2. Its ability to bind variant strains (except for the mink epidemic strain Y486L) is higher than that of SARS-CoV-2WT (Table 1).

[0019] Compared to hACE2-hFc-WT, hACE2-hFc-M4-1 and hACE2-hFc-M4-2 showed significantly improved neutralization abilities against both the original SARS-CoV-2 strain and its variant pseudoviruses, increasing by 8.3–104.1 times and 6.5–44.6 times, respectively, particularly in neutralizing the South African strain 501Y.V2 and the Brazilian strain 501Y.V3 (Table 2). hACE2-hFc-M5's neutralization ability against the original SARS-CoV-2 strain and its variant pseudoviruses was 8.7–126 times that of hACE2-hFc-WT, and overall higher than or similar to hACE2-hFc-M4-1 and hACE2-hFc-M4-2, especially in neutralizing the South African strain 501Y.V2 and the Brazilian strain 501Y.V3 (Table 2), which is consistent with the affinity results.

[0020] When the novel coronavirus infects human cells, the modified hACE2 protein of this invention preferentially recognizes and binds to the virus. In addition, it is fused with the human IgG1 Fc fragment, thereby forming an hACE2-hFc antibody protein, which further reduces the half-life and prolongs the ability to neutralize the virus.

[0021] The three hFc fusion proteins of the modified hACE2 fragments of the present invention are particularly suitable for the treatment or prevention of mutant strains of the novel coronavirus, such as the British strain, the Brazilian strain, the South African strain, and the mink-circulating strain. It is speculated that they will also be effective against novel coronaviruses or SARS-like coronaviruses that use ACE2 as a receptor and new variant strains that may emerge in the future, and have potential for wide application value.

[0022] The modified hACE2 protein of the present invention can be obtained by methods conventionally used in the art. First, the wild-type hACE2 amino acid coding sequence and the hFc coding sequence are seamlessly ligated into the pCAGGS expression vector to form pCAGGS-hACE2-hFc. Then, site-directed mutagenesis of amino acid sites is performed on this vector to obtain expression plasmids of the modified hACE2 protein, namely pCAGGS-hACE2-hFc-M4-1, pCAGGS-hACE2-hFc-M4-2, and pCAGGS-hACE2-hFc-M5.

[0023] Example 1

[0024] Obtaining hACE2-hFc modified protein

[0025] First, the gene sequence encoding the wild-type hACE2 protein (as shown in SEQ ID No. 1 of the sequence listing) was ligated with the gene encoding the human IgG1 Fc fragment protein (as shown in SEQ ID No. 2 of the sequence listing). The resulting sequence was then artificially synthesized (synthesis services were provided by Genewiz Suzhou) to obtain the wild-type hACE2-hFc fusion protein gene (as shown in SEQ ID No. 2 of the sequence listing). The fusion protein gene was cloned into the pCAGGS eukaryotic expression vector (ID No. 3), resulting in the expression vector pCAGGS-hACE2-hFc for the wild-type hACE2-hFc fusion protein. Then, through site-directed mutagenesis, amino acid T at position 27 was mutated to amino acid F, amino acid K at position 31 to amino acid Y, amino acid L at position 79 to amino acid W, amino acid N at position 330 to amino acid Y, and amino acid R at position 273 to amino acid Q, yielding the pCAGGS-hACE2-hFc-M4-1 vector (containing mutation sites T27F, K31Y, N330Y, and R273Q, gene sequence as shown in SEQ ID No. 5) and the pCAGGS-hACE2-hFc-M4-2 vector (containing mutation sites T27F, L79W, N330Y, and R273Q, gene sequence as shown in SEQ ID No. 5). (as shown in ID No. 7), pCAGGS-hACE2-hFc-M5 vector (containing mutation sites T27F, K31Y, L79W, N330Y and R273Q, gene sequence as shown in SEQ ID No. 9).

[0026] Using the pCAGGS-hACE2-hFc vector and the pCAGGS-hACE2-hFc-M4-1, pCAGGS-hACE2-hFc-M4-2, and pCAGGS-hACE2-hFc-M5 vectors, the following proteins were expressed and purified: hACE2-hFc-WT fusion protein (amino acid sequence as shown in SEQ ID No. 4), hACE2-hFc-M4-1 modified protein (amino acid sequence as shown in SEQ ID No. 6), hACE2-hFc-M4-2 modified protein (amino acid sequence as shown in SEQ ID No. 8), and hACE2-hFc-M5 modified protein (amino acid sequence as shown in SEQ ID No. 10).

[0027] Methods for site-directed mutagenesis of amino acids:

[0028] Primers were designed to mutate specific amino acids in the wild-type hACE2 gene to target amino acids. The mutated target fragment was then amplified by PCR. The target fragment was then homologously recombinated with the linearized vector pCAGGS to form a circular vector containing the target fragment. Only after the sequence was correctly sequenced could the vector be used for subsequent expression of the target protein.

[0029] The mutation sites include: the 27th amino acid T codon (ACA) is mutated to the amino acid F codon (TTT), the 31st amino acid K codon (AAG) is mutated to the amino acid Y codon (TAC), the 79th amino acid L codon (CTT) is mutated to the amino acid W codon (TGG), the 330th amino acid N codon (AAT) is mutated to the amino acid Y codon (TAT), and the 273rd amino acid R codon (AGA) is mutated to the amino acid Q codon (CAG).

[0030] Methods for expression purification:

[0031] The recombinant target plasmid was transfected into HEK293F cells at a cell density of approximately 2 × 10⁶ cells. 6 / ml. Cell supernatant was collected 5-7 days after transfection, centrifuged and filtered. The supernatant containing the modified hACE2-hFc-WT, hACE2-hFc-M4-1, hACE2-hFc-M4-2, and hACE2-hFc-M5 proteins was passed through a Protein A affinity chromatography column at 4°C using a peristaltic pump to ensure complete binding of the modified proteins to the column. Impurities were eluted with binding buffer (20mM Na3HPO4, pH 8.0), and the modified proteins were eluted with elution buffer (0.1M Glycine, pH 3.0). 1M Tris-HCl (pH 9.0) buffer was pre-added to the protein collection tube to prevent protein inactivation in an overly acidic environment. Finally, the protein was transferred to PBS buffer via a molecular sieve.

[0032] Example 2

[0033] Affinity of hACE2 modified protein to RBD of SARS-CoV-2 mutant strain

[0034] The inventors used surface plasmon resonance (SPR) technology to detect the affinity of the RBD of the original SARS-CoV-2 strain (SARS-CoV-2WT) and its variants (UK strain 501Y.V1, South African strain 501Y.V2, Brazilian strain 501Y.V3, mink epidemic strain 453F, mink epidemic strain 486L, and mink epidemic strain N501T) for the modified proteins hACE2-hFc-WT, hACE2-hFc-M4-1, hACE2-hFc-M4-2, and hACE2-hFc-M5, respectively.

[0035] Instruments and materials:

[0036] RBD proteins: The RBD proteins of the original SARS-CoV-2 strain, the UK strain 501Y.V1, the South African strain 501Y.V2, the Brazilian strain 501Y.V3, the mink epidemic strain Y453F, the mink epidemic strain F486L, and the mink epidemic strain N501T were all expressed and purified in the applicant's laboratory.

[0037] Equipment: BIAcore 8K, CM5 chip (GE Healthcare)

[0038] The specific experimental steps are as follows:

[0039] a. Chip pretreatment: The secondary antibody recognizing human IgG1 Fc was immobilized on the surface of the CM5 chip using amino-coupled coupling. The CM5 chip has 8 channels, each containing two flow cells (Fc 1 and 2). Fc 2 is injected with the mobile phase protein sample, and Fc 1 serves as a control.

[0040] b. Sample injection: First, dilute the sample with running buffer (20mM HEPES, 150mM NaCl, 0.005% (vol / vol) Tween 20, pH 7.4). This experimental sample contains RBDs of different mutant strains.

[0041] c. Chip regeneration: Regenerate the chip with regeneration buffer (10mM Glycine, pH 1.5).

[0042] d. Data Analysis: The data was analyzed using BIAcore 8K evaluation software to obtain the binding constant Ka, dissociation constant Kd, and equilibrium dissociation constant K. D .

[0043] Table 1. Comparison of Affinity between ACE2 Modified Proteins and Wild-Type Proteins

[0044]

[0045]

[0046] The results showed that, compared with wild-type hACE2-hFc (hACE2-hFc-WT), the three modified hACE2 proteins (hACE2-hFc-M4-1, hACE2-hFc-M4-2, and hACE2-hFc-M5) all had enhanced ability to bind to the original strain and the variant RBD of the SARS-CoV-2 virus, especially hACE2-hFc-M5, which showed the most significant improvement.

[0047] Example 3

[0048] Neutralizing effect of hACE2-hFc modified protein

[0049] The neutralizing effects of modified hACE2-hFc-WT, hACE2-hFc-M4-1, hACE2-hFc-M4-2, and hACE2-hFc-M5 proteins on the original SARS-CoV-2 strain (SARS-CoV-2WT) and variant strains (including the UK strain N501Y.V1, the South African strain N501Y.V2, the Brazilian strain N501Y.V3, and mink-circulated strains Y453F, F486L, and N501T) pseudoviruses in Vero E6 cells were determined.

[0050] The following groups were set up: Vero E6 cells (cell negative control group), Vero E6 cells + pseudovirus + culture medium (protein-free negative control group), Vero E6 cells + pseudovirus + hACE2-hFc wild-type protein (hACE2-hFc-WT group), Vero E6 cells + pseudovirus + hACE2-hFc-M4-1 protein (hACE2-hFc-M4-1 group), Vero E6 cells + pseudovirus + hACE2-hFc-M4-2 protein (hACE2-hFc-M4-2 group), and Vero E6 cells + pseudovirus + hACE2-hFc-M5 protein (hACE2-hFc-5 group).

[0051] Experimental instruments and materials:

[0052] Vero E6 cells (preserved in the applicant's laboratory), hACE2-hFc-WT protein gene (synthesized by Suzhou Genewiz, its coding sequence is SEQ ID No. 3), hACE2-hFc-M4-1, hACE2-hFc-M4-2, and hACE2-hFc-M5 protein sequences were all obtained in Example 1, and the original SARS-CoV-2 strain and its variant pseudoviruses were packaged and obtained in the applicant's laboratory.

[0053] Protein stock solution preparation: The modified hACE2-hFc-WT, hACE2-hFc-M4-1, hACE2-hFc-M4-2, and hACE2-hFc-M5 proteins were filtered through a 0.22 μm sterile filter in a clean bench, and the concentration was determined by the BCA method. The solutions were prepared to a concentration of 60 μg / ml using DMEM medium containing 2% FBS.

[0054] Preparation of gradient dilution buffers for modified proteins: Each hACE2 modified protein stock solution was serially diluted 2-fold using DMEM medium containing 2% FBS, resulting in a total of 11 dilutions. Each dilution was applied in 8 replicates, with 50 μl per well.

[0055] Pseudovirus dilution: The original SARS-CoV-2 strain and the variant strain pseudovirus solution were quantified on Vero E6 cells, and the dilution at which 1000 FFU appeared was used as the amount of virus used in the neutralization experiment.

[0056] The specific steps are as follows:

[0057] a. Seed Vero E6 cells in 96-well cell culture plates one day in advance, so that the cell confluence density reaches 80-90% the next day.

[0058] b. Take the gradient dilutions of each of the above-mentioned hACE2 modified proteins, add an equal volume (50 μl) of the above-mentioned pseudovirus dilution to each well, mix well, and incubate at 37°C for 1 h.

[0059] c. Carefully discard the supernatant from the 96-well cell culture plate, add the above protein-virus mixture (100 μl / well), and continue culturing in an incubator for 15 h.

[0060] b. Use a microscope to count the number of infected cells, calculate the inhibition rate at each concentration, and then use GraphPad to calculate the IC50. 50 .

[0061] Table 2. Comparison of neutralizing activity between ACE2 modified protein and wild-type protein.

[0062]

[0063] The results showed that, compared with hACE2-hFc-WT, the three modified hACE2 proteins (hACE2-hFc-M4-1, hACE2-hFc-M4-2, and hACE2-hFc-M5) significantly improved their ability to neutralize the original SARS-CoV-2 strain and variant pseudoviruses, especially against the original strain, the South African strain 501Y.V2, the Brazilian strain 501Y.V3, and the mink-circulating strain F486L.

[0064] Overall, the neutralizing activity of hACE2-hFc-M5 was higher than or similar to that of hACE2-hFc-M4-1 and hACE2-hFc-M4-2, but its ability to neutralize the South African strain 501Y.V2 and the Brazilian strain 501Y.V3 was further enhanced, which is consistent with the affinity results.

[0065] Example 4

[0066] Protective effect of hACE2-hFc modified protein in mouse models

[0067] Modified hACE2 proteins, including hACE2-hFc-WT, hACE2-hFc-M4-1, hACE2-hFc-M4-2, and hACE2-hFc-M5, were injected into mouse models. The efficacy of ACE2 modified proteins in treating SARS-CoV-2 infection was evaluated by measuring viral load in the lungs and HE staining of lung tissue.

[0068] The following groups were set up: virus + PBS group (negative control group), virus + hACE2-hFc-WT protein (hACE2-hFc-WT group), virus + hACE2-hFc-M4-1 protein (hACE2-hFc-M4-1 group), virus + hACE2-hFc-M4-2 protein (hACE2-hFc-M4-2 group), and virus + hACE2-hFc-M5 protein (hACE2-hFc-M5 group).

[0069] Experimental materials:

[0070] The live SARS-CoV-2 virus (P3 laboratory, Institute of Microbiology, Chinese Academy of Sciences), BALB / c mice (purchased from Vital River Laboratory Animal Company), and the modified proteins hACE2-hFc-WT, hACE2-hFc-M4-1, hACE2-hFc-M4-2, and hACE2-hFc-M5 were obtained in Example 1.

[0071] Equipment conditions: P3 laboratory, Institute of Microbiology, Chinese Academy of Sciences

[0072] The experimental steps are as follows:

[0073] a. Injecting COVID-19: 5×10 5 TCID 50 The novel coronavirus was injected into mice via nasal drops, with 5 mice in each group.

[0074] b. Injection of ACE2 modified protein: On the second day after challenge, the ACE2 modified protein was injected into mice via intraperitoneal injection at a dose of 10 mg / kg.

[0075] c. Tissue testing: On the fifth day after challenge, lung tissue was taken from mice. Three mice in each group were used to extract viral RNA and measure viral load, while the other two were used to make tissue sections and stain with hematoxylin and eosin (HE).

[0076] d. Data analysis: Based on the viral load in lung tissue and HE staining results, the protective effect of the modified ACE2 protein against SARS-CoV-2 infection in mice was determined.

[0077] The fusion protein of the modified hACE2 fragment and hFc of this invention is an antibody-like protein that can be widely used to treat diseases caused by the original and variant strains of the novel coronavirus, and can also be used to treat diseases caused by most COVID-19-related coronaviruses or SARS-like coronaviruses that use ACE2 as a receptor.

[0078] The fusion protein of the modified hACE2 fragment and hFc of this invention is particularly suitable for the treatment or prevention of variant strains of the novel coronavirus, such as the UK strain, Brazilian strain, South African strain, and mink-borne strain. It can also be widely used in clinical and epidemiological investigations and the preparation of corresponding drugs or vaccines. Sequence Listing <110> Institute of Microbiology, Chinese Academy of Sciences <120> ACE2 Modified Protein and Its Application <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2220 <212> DNA <213> hACE2 <400> 1 atgtcaggct ctttctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60 attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120 tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180 aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240 caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300 cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360 aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420 tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480 aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540 gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600 gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660 cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720 catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780 tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840 ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900 gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960 cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020 aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080 tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140 tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200 catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260 attggtcttc tgtcacccga ttttcaaga gacaatga cagaata cttcctgctc aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat cagatgattc ttttgggga ggaggatgtg cgagtggcta atttgaacc aagaatctcc 2040 tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100 gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gatgacac 2160 agcctagagt ttctgggat acagccaaca cttggacctc ctaccagcc ccctgttcc 2220 <210> 2 <211> 681 <212> DNA <213> IgG1 Fc <400> 2 gataaaactc accatgcc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60 ttcctcttcc cccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgg ccacgaagac cctgaggtca agttcactg gtacgtggac 180 ggcgtggagg tgcataatgc cagacaaag ccgcgggagg agcagtaca cagcacgtac 240 cgtgtgtca gcgtcctcac cgtcctgcac caggactggc tgaatggca ggagtacaag 300 tgcaaggtct ccaaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ctccgggtaa a 681 <210> 3 <211> 2904 <212> DNA <213> hACE2‑hFc <400> 3 atgtcaggct ctttctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60 attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120 tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180 aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240 caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300 cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360 aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420 tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480 aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540 gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600 gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660 cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720 catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780 tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840 ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900 gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960 cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020 aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080 tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140 tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200 catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260 attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320 aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380 tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440 aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500 gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560 taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620 aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680 ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740 gtaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800 aattctttg tgggatggag taccgactgg agtccatatg cagaccaag catchaagtg 1860 aggataagcc taaaatcagc tctggagat aaagcatatg atggaacga caatgaatg 1920 tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980 cagatgattc ttttgggga ggaggatgtg cgagtggcta atttgaacc aagaatctcc 2040 tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100 gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gatgacac 2160 agcctagagt ttctgggat acagccaaca cttggacctc ctaccagcc ccctgttcc 2220 gataaaactc accatgcc accgtgccca gcacctgaac tcctgggggg accgtcagtc 2280 ttcctcttcc cccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 2340 tgcgtggtgg tggacgtg ccacgaagac cctgaggtca agttcaactg gtacgtggac 2400 ggcgtggagg tgcataatgc cagacaaag ccgcgggagg agcagtaca cagcacgtac 2460 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 2520 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 2580 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 2640 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 2700 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 2760 gacggctcct tcttccta cagcaagctc accgtggaca agagcaggtg gcagcagggg 2820 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 2880 ctctccctgt ctccgggtaa atga 2904 <210> 4 <211> 967 <212> PRT <213> hACE2‐hFc‐WT <400> 4 Put Ser Gly Ser Phe Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620 Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met 625 630 635 640 Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655 Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670 Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685 Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700 Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn 705 710 715 720 Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735 Pro Pro Val Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 740 745 750 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 755 760 765 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 770 775 780 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 785 790 795 800 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 805 810 815 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 820 825 830 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 835 840 845 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 850 855 860 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 865 870 875 880 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 885 890 895 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 900 905 910 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 915 920 925 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 930 935 940 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 945 950 955 960 Leu Ser Leu Ser Pro Gly Lys 965 <210> 5 <211> 2904 <212> DNA <213> hACE2‑hFc‑M4‑1 <400> 5 atgtcaggct ctttctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60 attgaggaac aggccaagtt ttttttggac tactttaacc acgaagccga agacctgttc 120 tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180 aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240 caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300 cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360 aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420 tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480 aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540 gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600 gattatgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660 cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720 catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780 tgcctccctg ctcatttgct tggtgatatg tggggtcagt tttggacaaa tctgtactct 840 ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900 gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960 cctaatatga ctcaaggatt ctgggaatat tccatgctaa cggacccagg aaatgttcag 1020 aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080 tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140 tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200 catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260 attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320 aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380 tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440 aagcgagaga tagttggggt ggtgaacct gtgcccatg atgaacata ctgtgacccc 1500 gcatctctgt tccatgtttc taatgattac tcattcattc gatttacac aaggacctt 1560 taccaatttcc agttcaaga agcacttgt caagcagcta aacatgaagg cccctgcac 1620 aaatgtgaca tctcaactc tacagaagct ggacagaac tgttcaat gctgaggctt 1680 ggaaaatcag aaccctggac cctagcattg gaaatgttg taggagcaa gaacatgaat 1740 gtaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800 aattctttg tgggatggag taccgactgg agtccatatg cagaccaag catchaagtg 1860 aggataagcc taaaatcagc tctggagat aaagcatatg atggaacga caatgaatg 1920 tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980 cagatgattc ttttgggga ggaggatgtg cgagtggcta atttgaacc aagaatctcc 2040 tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100 gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gatgacac 2160 agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 2280 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 2340 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 2400 ggcgtggagg tgcataatgc caacaaag ccgcgggagg agcagtacaa cagcacgtac 2460 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggatcaag 2520 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 2580 gggcagcccc gagaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 2640 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 2700 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 2760 gacggctcct tcttctctca cagcaagctc accgtggaca agagcaggtg gcagcagggg 2820 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 2880 ctctccctgt ctccgggtaa atga 2904 <210> 6 <211> 967 <212> PRT <213> hACE2‐hFc‐M4‐1 <400> 6 Met Ser Gly Ser Phe Trp Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Gln Ser Thr Ile Glu Glu Gln Ala Lys Phe Phe Leu Asp Tyr Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Type Asn With Glu Glu Asn Val Gln Asn Met Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 75 80 Gln Met Tyr Pro With Gln Glu And Gln Asn With Thr Val Lys With Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Gln Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Tyr Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620 Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met 625 630 635 640 Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655 Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670 Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685 Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700 Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn 705 710 715 720 Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735 Pro Pro Val Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 740 745 750 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 755 760 765 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 770 775 780 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 785 790 795 800 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 805 810 815 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 820 825 830 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 835 840 845 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 850 855 860 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 865 870 875 880 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 885 890 895 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 900 905 910 Thr Thr Pro Pro Val Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 915,920,925 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 930,935,940 How Does Glu Ala Leu Discover Tyr Thr Gln Lys Ser? 945 950 955 960 Leu Ser Leu Ser Pro Gly Lys 965 <210> 7 <211> 2904 <212> DNA <213> hACE2‐hFc‐M4‐2 <400> 7 atgtcaggct ctttctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60 attgaggaac aggccaagtt ttttggac aagtttacc acgaagccga agacctgttc 120 tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gatgtccaa 180 aacatgaata atgctgggga caatggtct gcctttta aggaacagtc cacatgggcc 240 caaatgtatc cactacaaga aattcagaat ctcacagtca agctcagct gcaggctctt 300 cagcaaatg ggtcttcagt gctctcagaa gatagagca aacggttgaa cacaattcta 360 aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420 tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480 aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540 gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600 gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660 cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720 catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780 tgcctccctg ctcatttgct tggtgatatg tggggtcagt tttggacaaa tctgtactct 840 ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900 gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960 cctaatatga ctcaaggatt ctgggaatat tccatgctaa cggacccagg aaatgttcag 1020 aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080 tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140 tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200 catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260 attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320 aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380 tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440 aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500 gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560 taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620 aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680 ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740 gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800 aattctttg tgggatggag taccgactgg agtccatatg cagaccaag catchaagtg 1860 aggataagcc taaaatcagc tctggagat aaagcatatg atggaacga caatgaatg 1920 tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980 cagatgattc ttttgggga ggaggatgtg cgagtggcta atttgaacc aagaatctcc 2040 tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100 gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gatgacac 2160 agcctagagt ttctgggat acagccaaca cttggacctc ctaccagcc ccctgttcc 2220 gataaaactc accatgcc accgtgccca gcacctgaac tcctgggggg accgtcagtc 2280 ttcctcttcc cccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 2340 tgcgtggtgg tggacgtg ccacgaagac cctgaggtca agttcaactg gtacgtggac 2400 ggcgtggagg tgcataatgc cagacaaag ccgcgggagg agcagtaca cagcacgtac 2460 cgtgtgtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 2520 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 2580 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 2640 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 2700 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 2760 gacggctcct tcttccta cagcaagctc accgtggaca agagcaggtg gcagcagggg 2820 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 2880 ctctccctgt ctccgggtaa atga 2904 <210> 8 <211> 967 <212> PRT <213> hACE2‐hFc‐M4‐2 <400> 8 Put Ser Gly Ser Phe Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Phe Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Trp Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Gln Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Tyr Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620 Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met 625 630 635 640 Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655 Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670 Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685 Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700 Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn 705 710 715 720 Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735 Pro Pro Val Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 740 745 750 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 755 760 765 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 770 775 780 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 785 790 795 800 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 805 810 815 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 820 825 830 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 835 840 845 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 850 855 860 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 865 870 875 880 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 885 890 895 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 900 905 910 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 915 920 925 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 930 935 940 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 945 950 955 960 Leu Ser Leu Ser Pro Gly Lys 965 <210> 9 <211> 2904 <212> DNA <213> hACE2‑hFc‑M5 <400> 9 atgtcaggct ctttctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60 attgaggaac aggccaagtt ttttttggac tactttaacc acgaagccga agacctgttc 120 tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180 aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacatgggcc 240 caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300 cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360 aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420 tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480 aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540 gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600 gattatgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660 cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720 catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780 tgcctccctg ctcatttgct tggtgatatg tggggtcagt tttggacaaa tctgtactct 840 ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900 gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960 cctaatatga ctcaaggatt ctgggaatat tccatgctaa cggacccagg aaatgttcag 1020 aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080 tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140 tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200 catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260 attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320 aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380 tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440 aagcgagaga tagttggggt ggtgaacct gtgcccatg atgaacata ctgtgacccc 1500 gcatctctgt tccatgtttc taatgattac tcattcattc gatttacac aaggacctt 1560 taccaatttcc agttcaaga agcacttgt caagcagcta aacatgaagg cccctgcac 1620 aaatgtgaca tctcaactc tacagaagct ggacagaac tgttcaat gctgaggctt 1680 ggaaaatcag aaccctggac cctagcattg gaaatgttg taggagcaa gaacatgaat 1740 gtaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800 aattctttg tgggatggag taccgactgg agtccatatg cagaccaag catchaagtg 1860 aggataagcc taaaatcagc tctggagat aaagcatatg atggaacga caatgaatg 1920 tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980 cagatgattc ttttgggga ggaggatgtg cgagtggcta atttgaacc aagaatctcc 2040 tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100 gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gatgacac 2160 agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 2280 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 2340 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 2400 ggcgtggagg tgcataatgc caacaaag ccgcgggagg agcagtacaa cagcacgtac 2460 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggatcaag 2520 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 2580 gggcagcccc gagaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 2640 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 2700 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 2760 gacggctcct tcttctctca cagcaagctc accgtggaca agagcaggtg gcagcagggg 2820 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 2880 ctctccctgt ctccgggtaa atga 2904 <210> 10 <211> 967 <212> PRT <213> hACE2‑hFc‑M5 <400> 10 Met Ser Gly Ser Phe Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Phe Phe Leu Asp Tyr Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Trp Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Gln Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Tyr Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620 Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met 625 630 635 640 Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655 Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670 Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685 Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700 Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn 705 710 715 720 Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735 Pro Pro Val Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 740 745 750 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 755 760 765 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 770 775 780 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 785 790 795 800 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 805 810 815 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 820 825 830 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 835 840 845 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 850 855 860 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 865 870 875 880 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 885 890 895 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 900 905 910 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 915 920 925 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 930 935 940 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 945 950 955 960 Leu Ser Leu Ser Pro Gly Lys 965

Claims

1. The use of the modified protein in the preparation of medicaments for the treatment and / or prevention of disease caused by SARS-like coronaviruses using ACE2 receptors, or in the preparation of vaccines for the prevention of infection with SARS-like coronaviruses using ACE2 receptors, characterized in that, The modified protein is hACE2-hFc-M4-1 modified protein, hACE2-hFc-M4-2 modified protein, or hACE2-hFc-M5 modified protein; The amino acid sequence of the modified hACE2-hFc-M4-1 protein is shown in the sequence list SEQ ID No. 6; The amino acid sequence of the modified hACE2-hFc-M4-2 protein is shown in the sequence list SEQ ID No. 8; The amino acid sequence of the modified hACE2-hFc-M5 protein is shown in the sequence list SEQ ID No.

10.

2. The application according to claim 1, characterized in that, The SARS-like coronaviruses that use ACE2 as a receptor are the original SARS-CoV-2 strain and / or SARS-CoV-2 variant strains.

3. The application according to claim 2, characterized in that, The original SARS-CoV-2 strain mentioned above is SARS-CoV-2WT.

4. The application according to claim 2, characterized in that, The SARS-CoV-2 variant strains mentioned are the UK strain N501Y.V1, the South African strain N501Y.V2, or the Brazilian strain N501Y.V3.

5. The application according to claim 2, characterized in that, The SARS-CoV-2 variant strains mentioned are mink-circulating strain Y453F, mink-circulating strain F486L, or mink-circulating strain N501T.

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