CN116601291A - Modified angiotensin converting enzyme 2 (ACE 2) and uses thereof - Google Patents

Abstract

The present disclosure describes modified angiotensin converting enzyme 2 (ACE 2) polypeptides. The modified polypeptides include at least one amino acid substitution that enables the polypeptides to better bind coronavirus S surface glycoproteins that use ACE2 as a cell entry receptor by directly increasing affinity or improving ACE2 folding and expression. The use of the modified ACE2 polypeptides to inhibit CoV entry, replication and/or transmission, to prevent and treat CoV infection (e.g., covd-19) before and after CoV exposure is also described.

Description

Modified angiotensin converting enzyme 2 (ACE 2) and uses thereof

Cross-reference to related applications

The application claims the benefit of U.S. provisional application number 63/089,895, U.S. provisional application number 63/042,907, U.S. provisional application number 63/022,151, and U.S. provisional application number 62/989,976, U.S. provisional application number 16, month 3, and month 9, respectively, both of which are filed on days 10, 2020, 6, and 23, 2020, respectively, each of which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates to modified angiotensin converting enzyme 2 (ACE 2) proteins with improved folding and enhanced binding to SARS-CoV-2 and other coronaviruses using ACE2 as a cell entry receptor.

Government support statement

The present invention was completed with government support under grant No. 5R01AI129719-03 awarded by the national institutes of health. The government has certain rights in this invention.

Background

In month 12 2019, a new human and livestock co-human species closely related to bats coronavirus was transmitted to humans (Zhu et al, N Engl J Med.2020Feb 20;382 (8): 727-733; zhou et al, nature.2020Feb3;579 (7798): 270-273). The virus is called SARS-CoV-2 because it is similar to Severe Acute Respiratory Syndrome (SARS) coronavirus which resulted in a minor epidemic of burst size of the last 20 years (Peiris et al, lancet.2003Apr19;361 (9366): 1319-1325;Coronaviridae Study Group of the International Committee on Taxonomy of Viruses,Nat Microbiol.2020Mar 2;4 (5): 3), after which people rapidly spread worldwide, causing governments to take unusual containment measures (Patel et al MMWR Morb Mortal Wkly Rep.2020Feb 7;69 (5): 140-146). Stock market falls, travel is limited, public gathering is cancelled, and a large number of people are isolated. These events are different from any event experienced by several generations. Symptoms of coronavirus disease (COVID-19) in 2019 range from mild cough to dry cough, fever, pneumonia, and death, SARS-CoV-2 is devastating to the elderly and other vulnerable populations (Wang et al, J Med virol.2020Apr;92 (4): 441-447; huang et al, lancet.2020Feb 15;395 (10223): 497-506). There is currently no vaccine to prevent SARS-CoV-2 infection, nor is there an approved drug to specifically treat this viral infection. Thus, there is a need to develop effective therapies to treat SARS-CoV-2 infection.

Disclosure of Invention

Described herein are human ACE2 polypeptides that enhance binding to SARS-CoV-2S proteins by improving the folding and structural stability of ACE2, eliminating glycan modifications, or increasing affinity. The modified polypeptides are useful as diagnostic or therapeutic agents for detecting, preventing (pre-exposure or post-exposure prevention) or treating covd-19 or any disease caused by coronaviruses that utilize ACE2 as a cellular receptor.

Provided herein are modified ACE2 polypeptides, including ACE2 or fragments thereof, e.g., extracellular fragments. The polypeptide comprises at least one amino acid substitution as compared to wild-type ACE2 and has an enhanced ability to bind coronavirus S due to a direct or indirect way of affinity change (e.g. by stabilization of the S recognition structure). In a specific example, the ACE2 is human ACE2. In some embodiments, the at least one amino acid substitution is selected from any of the substitutions shown in table 1, table 2, and/or table 3. In some examples, the at least one amino acid substitution is a residue located at the ACE2 and S interface. In some examples, the at least one amino acid substitution is a residue located in an N90-glycosylation motif. In some examples, the at least one amino acid substitution is remote from the interface and enhances presentation of the S-recognition fold structure. In some examples, the modified ACE2 polypeptide is a dimer. In one example, the dimer ACE2 comprises the T27Y, L79T and N330Y amino acid substitutions.

Also provided herein are fusion proteins, including modified ACE2 polypeptides and heterologous polypeptides disclosed herein. In some embodiments, the heterologous polypeptide is an Fc protein or human serum albumin, e.g., a protein for recruiting effector functions and/or increasing serum stability. In some embodiments, the heterologous polypeptide is a protein that can be used as a diagnostic/detection reagent, such as a fluorescent protein (e.g., GFP) or an enzyme (e.g., horseradish peroxidase (HRP) or alkaline phosphatase).

Also provided is a method of inhibiting the entry of a coronavirus into a cell by contacting it with a modified ACE2 polypeptide or fusion protein as disclosed herein. Methods of inhibiting replication and/or transmission of a coronavirus in a subject are also provided. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of a modified ACE2 polypeptide or fusion protein disclosed herein. The modified ACE2 polypeptide may be administered prior to infection (e.g., in a subject at risk of infection) as a pre-exposure prophylactic treatment, shortly after infection as a post-exposure prophylactic treatment, or after the subject exhibits one or more signs or symptoms of infection.

Also provided are methods of treating a coronavirus infection (e.g., covd-19) in a subject by administering to the subject a therapeutically effective amount of a modified ACE2 polypeptide or fusion protein disclosed herein. The coronavirus may be any human or animal disease coronavirus including a newly emerging strain of coronavirus that utilizes ACE2 as a cell entry receptor. In some examples, the modified ACE2 polypeptide is administered intravenously, intratracheally, or by inhalation. The treatment may be a pre-exposure prophylactic treatment, a post-exposure prophylactic treatment or a method of treating covd-19.

Nucleic acid molecules and vectors encoding the modified ACE2 polypeptides or fusion proteins disclosed herein are also provided. Methods of inhibiting coronavirus replication and/or transmission (or treating CoV infection) in a subject by administering the nucleic acid molecules or vectors are also provided. In some examples, the nucleic acid molecule or vector is administered intravenously, intratracheally, or by inhalation.

Methods of detecting CoV in a biological sample are also provided. In some embodiments, the method comprises contacting the biological sample with a modified polypeptide or fusion protein disclosed herein; and detecting binding of the modified polypeptide or fusion protein to the biological sample.

Kits comprising the modified polypeptides or fusion proteins disclosed herein, bound to a solid support, are also provided.

The foregoing and other objects and features of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings.

Drawings

FIGS. 1A-1D: policy was chosen for ACE2 variants of RBD that highly bind SARS-CoV-2S. (FIG. 1A) the culture medium of the Expi293F cells secreting SARS-CoV-2S-RBD fused to sfGFP was collected and incubated with the ACE2 expressed myc tag by the Expi293F cells at different dilutions. Bound S-RBD-sfGFP was measured by flow cytometry. Dilutions of S-RBD-sfGFP containing medium used for FACS selection are indicated by arrows. (FIGS. 1B-1C) wild-type ACE2 plasmid diluted with excess vector DNA was transiently transfected into Expi293F cells. Under these conditions, the cells typically acquire no more than one encoding plasmid, and most cells are negative. Cells were incubated with medium containing S-RBD-sfGFP and co-stained with fluorescent anti-myc to detect surface ACE2 by flow cytometry. During the analysis, the first 67% was selected from ACE2 positive population (fig. 1B). The bound S-RBD was then measured relative to surface ACE2 expression (fig. 1C). (FIG. 1D) Expi293F cells were transfected with a library of ACE2 single site saturation mutations and analyzed as shown in FIG. 1B. During FACS, the first 15% cells with binding to S-RBD relative to ACE2 expression (nCoV-S-High sorting) were collected and the later 20% cells were collected (nCoV-S-Low sorting), respectively.

Fig. 2: mutation map of ACE2 for high binding signal of SARS-CoV-2S RBD. Log marking nCoV-S-High sorting from-3 (i.e., depleted/detrimental) to neutral to +3 (i.e., enriched) ₂ Enrichment ratio. ACE2 primary structure is located on the vertical axis and amino acid substitutions are located on the horizontal axis. * Is a stop codon.

Fig. 3A-3F: the data in independent replicates showed high consistency. (FIGS. 3A-3B) ACE2 mutant Log sorted by nCoV-S-High (FIG. 3A) and nCoV-S-Low (FIG. 3B) between two independent FACS experiments ₂ The enrichment ratio is highly consistent. 1/40 dilution of the medium containing S-RBD-sfGFP was used in repetition 1, and 1/20 dilution of the medium containing S-RBD-sfGFP was used in repetition 2. R is R ² Values were used for non-synonymous mutations. (FIG. 3C) average log of nCoV-S-High and nCoV-S-Low sorting ₂ The enrichment ratio tends to be inversely related. Nonsense mutations and small amounts of nonsense mutations are not expressed on the plasma membrane and are therefore depleted (i.e., fall below the diagonal) in both sorted populations. (FIGS. 3D-3F) a residue conservation score correlation plot repeating nCoV-S-High sorting (FIG. 3D) and a residue conservation score correlation plot repeating nCoV-S-Low sorting (FIG. 3E), and a residue conservation score correlation plot of two nCoV-S-High sorting average data versus two nCoV-S-Low sorting average data (FIG. 3F). According to the log of all amino acid substitutions at each residue position ₂ The mean of the enrichment ratios calculates a conservation score.

Fig. 4A-4C: ACE2 residues highly bind the sequence preference of SARS-CoV-2S RBD. (FIG. 4A) the conservation score of nCoV-S-High sorting mapped to the S-RBD bound to the low temperature EM structure of ACE2 (surface) (PDB 6M 17). Left sideThe view is a top view of the substrate binding chamber, showing only a single protease domain for clarity. (fig. 4B) the average hydrophobic weighted enrichment ratio maps to RBD-bound ACE2 structure. (fig. 4C) a partially enlarged view of ACE 2. The accompanying heat map shows the log of substitution of ACE2-T27, ACE2-D30 and ACE2-K31 from +.ltoreq.3 (depletion) to +.gtoreq. +3 (enrichment) in nCoV-S-High sorting ₂ Enrichment ratio.

Fig. 5A-5C: the effect of single amino acid substitutions in ACE2 to enhance RBD binding was predicted to be small by deep mutation scanning. (FIG. 5A) full-length ACE2 expressing Expi293F cells were stained with RBD-sfGFP in medium and analyzed by flow cytometry. Data from wild-type ACE2 and single mutant (L79T) were compared. The RBD binding enhancement is most pronounced in cells expressing low levels of ACE2 (smaller gate). In this experiment ACE2 has an extracellular N-terminal myc tag upstream of residue S19 for detection of surface expression. (FIG. 5B) the RBD-sfGFP binding was measured for 30 amino acid substitutions in ACE 2. The data are the relative change in average fluorescence of GFP in the low expression gate, minus background fluorescence. n means = 2, error line indicates range. (fig. 5C) the upper panel shows the relative RBD-sfGFP binding measured for the total ACE2 positive population (larger gate in fig. 5A), while the lower panel plots the relative ACE2 expression measured by detecting extracellular myc tags. Binding of RBD-sfGFP to the total positive population correlated with total ACE2 expression, so the difference in binding between mutants was most pronounced only after control of expression levels, as shown in figure 5B.

Fig. 6A-6B: engineered sACE2 with enhanced binding to S. (FIG. 6A) the expression of sACE2-sfGFP mutants was assessed qualitatively by fluorescence of transfected cell cultures. (FIG. 6B) cells expressing full-length S were stained with a dilution of medium containing sACE2-sfGFP and analyzed for binding by flow cytometry.

Fig. 7A-7D: analytical Size Exclusion Chromatography (SEC) of purified sACE2 protein. (FIG. 7A) purified sACE2 protein (10. Mu.g) was separated on 4-20% SDS-polyacrylamide gel and stained with Coomassie. (FIG. 7B) analysis SEC of IgG1 fused wild-type sACE2 and sACE 2.v2. Molecular Weight (MW) of the standard is expressed above the peak in kD. The absorbance of the MW standard is scaled for clarity. (FIG. 7C) analysis of 8his marker proteins SEC. The main peak corresponds to the expected MW of the monomer. Dimer peaks were also observed, although their abundance varied between independent protein preparations (compare fig. 9D). (FIG. 7D) soluble ACE2-8h protein was incubated at 37℃for 40 hours and analyzed by SEC.

Fig. 8A-8E: a variant of sACE2 with high affinity for S. (FIG. 8A) full-length S-expressing Expi293F cells were incubated with purified wild-type sACE2 or sACE2.v2 fused to 8his (solid line) or IgG1-Fc (dashed line). After washing, the binding proteins were detected by flow cytometry. (FIG. 8B) 100nM wild-type sACE2-IgG1 (dotted line) competed with wild-type sACE2-8h or sACE2.v2-8 h. The competitor protein was added simultaneously to cells expressing full-length S and the binding protein was detected by flow cytometry. (fig. 8C) Biological Layer Interference (BLI) kinetics of wild-type sacce 2-8h association (t=0 to 120 seconds) and dissociation (t >120 seconds) with immobilized RBD-IgG 1. (FIG. 8D) kinetics of sACE2.v2-8h binding to immobilized RBD-IgG1 were measured by BLI. (FIG. 8E) recovered serum IgG from patients with COVID-19 competed with wild-type sACE2-8h or sACE2.v2-8h for binding to immobilized RBD in ELISA. Three different patient sera were tested (light to dark for P1 to P3).

Fig. 9A-9G: the high affinity sACE2 variants were optimized to increase yield. (FIG. 9A) dilutions containing sACE2-sfGFP medium were incubated with full-length S-expressing Expi293F cells. After washing, bound sACE2-sfGFP was analyzed by flow cytometry. (FIG. 9B) Coomassie stained SDS-polyacrylamide gel compares the yields of the sACE2-IgG1 variants purified from the expression medium with protein A resin. (FIG. 9C) Coomassie stained gel (10. Mu.g per lane) for purified sACE2-8h variants. (FIG. 9D) by analysis of SEC, sACE2.v2.4-8h was indistinguishable from wild-type sACE2-8 h. For clarity, the absorbance of the MW standard is scaled, MW expressed in kD above the elution peak. (FIG. 9E) analysis SEC was performed after 60 hours of storage at 37 ℃. Compared to sACE2.v2.4, the surface of the variant sACE2.v2.2 is more hydrophobic and has a higher tendency to partially aggregate, so that partial storage instability may be intrinsically linked to increased hydrophobicity. (fig. 9F) association (t=0 to 120 seconds) and dissociation (t >120 seconds) of wild-type sACE2-8h with immobilized RBD-IgG1 were measured by BLI. The data can be compared to the second independent preparation data for sACE2-8h shown in FIG. 8C. (FIG. 9G) BLI kinetics of sACE2.v2.4-8h association and dissociation with immobilized RBD-IgG 1.

Fig. 10A-10D: a dimeric sACE2 variant with improved binding to viral spike properties. (FIG. 10A) wild-type sACE2 ₂ -8h and sACE2 ₂ V2.4-8h analysis SEC after incubation at 37℃for 62 hours. (FIG. 10B) ELISA analysis of serum IgG (light to dark P1 to P3) binding to RBD from convalescence patients. Addition of dimer sACE2 ₂ (WT) -8h or sACE2 ₂ V2.4-8h to compete with antibodies recognizing the receptor binding site. The concentration is based on the monomer subunit. (FIG. 10C) measurement of RBD-8h with immobilized sACE2 by BLI ₂ Association (t=0 to 120 seconds) and dissociation of (WT) -IgG1 (t>120 seconds). (FIG. 10D) RBD-8h and immobilized sACE2 ₂ V 2.4-BLI kinetics of IgG1 binding.

Fig. 11: the engineered receptors enhance the neutralization of SARS-CoV-2 and SARS-CoV-1. In the micro-neutralization assay, monomeric (solid line) or dimeric (dashed line) sACE2 (WT) -8h or sACE2.v2.4-8h were pre-incubated with virus prior to addition of VeroE6 cells. The concentration is based on the monomer subunit. Data are mean ± SEM of n=4 (2 independent experiments, 2 technical replicates).

Fig. 12A-12C: binding of sACE2 glycosylation mutant to RBD of SARS-CoV-2. (FIG. 12A) the soluble ACE2 protease domain carrying the mutation T92Q was purified as an 8 his-tagged fusion. 6 μg was separated on coomassie stained 4-20% sds-polyacrylamide gel to assess purity. (fig. 12B) analysis of SEC shows elution as the main peak of monomer, the smaller fraction eluting at the expected MW of dimer. (FIG. 12C) RBD-IgG1 was immobilized to the sensor surface and the association and dissociation of sACE2-T92Q-8h were measured in a Biological Layer Interference (BLI) experiment. The reported affinity (80 nM) was tighter than the affinity of the equivalent wild-type protein (140-150 nM, FIG. 8C and FIG. 9F).

Fig. 13A-13C: flow cytometry measures the binding of sACE2 to myc-expressed marker S on plasma membranes. (FIG. 13A) Expi293F cells expressing full-length S (either tagged (FIG. 8A) or tagged with extracellular myc epitope tag) were gated by the forward scattering properties of the main cell population (gating region). (FIG. 13B) histograms show that flow cytometry analysis of representative raw data of myc-S-expressing cells incubated with 200nM wild-type sACE2-8h or sACE 2.v2. After washing, the binding protein was detected with a fluorescent anti-HIS-FITC secondary antibody. The fluorescence of myc-S expressing cells not treated with sACE2 was black. (FIG. 13C) binding of purified wild-type sACE2 or sACE2.v2 fused to 8his (solid line) or IgG1-Fc (dashed line) to myc-S expressing cells.

Fig. 14A-14D: dimer sACE2 ₂ Strongly binds to RBD. (FIG. 14A) purified dimer sACE2 ₂ SDS-PAGE of 8h proteins (10. Mu.g per lane, coomassie staining). (FIG. 14B) sACE2 ₂ Preparation of 8h protein SEC. The eluate of the NiNTA affinity chromatography was concentrated and injected into a gel filtration column. The absorbance of the MW standard was scaled and the kD was shown above the elution peak. (FIG. 14C) comparing full-length S-expressing Expi293F cells with wild-type and v2.4 sACE2 ₂ -8h of incubation, washing and staining with fluorescent anti-his. n means = 2, error line indicates range. Binding was similar to that observed for the sACE2 IgG1 fusion (FIGS. 8A and 13C), indicating dimeric sACE2 ₂ -strong interaction of 8h and trimer spikes on the cell surface. (FIG. 14D) dimer sACE2 ₂ (WT) -8h and sACE2 ₂ V2.4-8h BLI kinetics strongly binding to dimeric RBD-IgG1 immobilized on the sensor surface.

Fig. 15A-15B: purified sACE2 ₂ IgG1 is a dimer. (FIG. 15A) purified sACE2 ₂ Coomassie stained gel image of IgG1 protein (10 μg per lane). (FIG. 15B) purified sACE2 ₂ Analysis SEC of IgG1, scaled absorbance of the MW standard (kD indicated above elution peak) was superimposed. Note that there is no high MW peak, which may correspond to the separation of the different subunits by sACE2 ₂ And IgG1 dimerization-mediated concatemers.

Fig. 16A-16D: unlabeled sACE2 expressed in non-human cells ₂ V2.4 is tightly bound to S. (FIG. 16A) for sACE2 purified in human Expi293F cells with 8h tag ₂ V2.4 SDS-PAGE comparison with unlabeled proteins produced by non-human ExpiCHO-S cell lines. 10 μg per lane. (FIG. 16B) sACE2 ₂ V2.4 analytical SEC before and after incubation at 37 ℃ for 146 hours. The absorbance of MW standard was scaled and marked as kD. (FIG. 16C) will express SIs combined with 100nM wild-type sACE2 ₂ IgG1 and increasing concentration of human cell-derived sACE2 ₂ (WT) -8h, human cell-derived sACE2 ₂ V2.4-8h or ExpiCHO-S derived sACE2 ₂ V2.4 co-cultivation. Measurement of bound his-tag protein (solid line) and sACE2 by flow cytometry ₂ (WT) -IgG1 (dashed line). (FIG. 16D) measurement of unlabeled sACE2 by BLI ₂ V2.4 strong binding to immobilized RBD-IgG 1.

Fig. 17: the catalytic activity of the engineered receptor is reduced. For human cell-derived sACE2 ₂ (WT) -8h (left side), human cell-derived sACE2 ₂ V2.4-8h (intermediate) and ExpiCHO-S derived sACE2 ₂ V2.4 (right side) measurement of cleavage of peptide substrate to release fluorescent product. Specific activity was determined at the initial time point where product release was linear and data for the highest concentration of wild-type protein (22 nM) was excluded. Time=0 minutes indicates the time at which fluorescence recording starts.

Fig. 18: SARS-associated coronaviruses have a high degree of sequence diversity at ACE2 binding sites. The RBD of SARS-CoV-2 (PDB 6M 17) is colored differently by the difference between the 7 SARS-associated CoV strains.

Fig. 19: the ACE2 binding site of SARS-associated beta coronavirus is a region of high sequence diversity. The 2 human betacoronavirus RBD sequences and the 5 bats betacoronavirus RBD sequences (SEQ ID NO: 3-9) using ACE2 as an entry receptor were aligned. Numbering is based on SARS-CoV-2 protein S. Asterisks indicate residues of SARS-CoV-2RBD, ACE2 in PDB 6M17 Within the range.

Fig. 20A-20C: FACS selection was performed on S variants with high or low binding signals for ACE 2. (FIG. 20A) flow cytometry analysis of Expi293F cells expressing SARS-CoV-2 full-length S with an N-terminal c-myc tag. Staining for myc epitope is shown on the x-axis, while binding to sACE2 ₂ Detection for 8h (2.5 nM) is shown on the y-axis. S plasmid is diluted 1500 times by weight with the vector DNA, so that the cell typically expresses no more than one coding variant; under these conditions, most cells are negative. (FIG. 20B) Single Site Saturation Mutagenesis (SSM) with RBDFlow cytometry of library transfected cells showed expression and sACE2 ₂ -8h of cells binding reduced S-variant. (fig. 20C) gating strategy of FACS. Gating of c-myc epitope-positive S-expressing cells and harvesting sACE2 relative to myc-S expression ₂ -8h of cells with the highest ("ACE 2-High") and lowest ("ACE 2-Low") 20% binding.

Fig. 21: RBD of SARS-CoV-2 full-length S and soluble ACE2 ₂ Mutation map of binding. Log of RBD in depth mutation scanning full-length S ₂ The enrichment ratio is drawn from less than or equal to-3 (depletion/harmful) to 0 (neutral) to more than or equal to +3 (enrichment). Wild type amino acids are shown in black. RBD sequences are on the vertical axis and amino acid substitutions are on the horizontal axis. * And (3) a stop codon.

Fig. 22A-22D: deep mutations indicate that the ACE2 binding site of SARS-CoV-2 is tolerant to many mutations. (FIG. 22A) map the surface expressed location score to the structure of SARS-CoV-2RBD (PDB 6M17, oriented as shown in FIG. 18). In FACS selection for surface S expression, several residues in the protein core are highly conserved (judged by mutation depletion of ACE2-High and ACE2-Low doors), while some surface residues are tolerant to mutations. (FIG. 22B) expression score (x-axis) for mutant selection for full-length S in human cells versus conservation score (log at residue position) for mutant selection in RBD isolated from yeast display ₂ Mean of enrichment ratio) (y-axis). Obvious outliers are indicated. (FIG. 22C) the conservation score of ACE2-High gated cell populations was mapped to RBD structure. (FIG. 22D) S-deep mutation (x-axis) and yeast surface RBD-deep mutation (. DELTA.K) in human cells _{D app} Is the average value of (2); y-axis) high ACE2 binding RBD conservation score correlation plot.

Fig. 23A-23C: substitution of alanine for disulfide-bonded cysteines in RBD reduces S-surface expression in human cells. (FIG. 23A) RBD is colored differently due to the deep mutation (conservative or mutation tolerant) expression score, forming a continuous hydrophobic core (PDB 6VSB strand B) with the remainder of the S1 subunit in the closed conformation. (FIG. 23B) based on surface immunostaining and flow cytometry analysis, the transfected Expi293F cells with myc-S cysteine mutants showed a decrease in both the percentage of myc positive cells (gating region) and the average fluorescence of the positive population. To embody both effects in a single number, the change in the mean fluorescence units (Δmfu) of the whole cell population compared to vector transfected control cells was calculated after first gating on living cells by scattering. (FIG. 23C) surface expression of myc-S cysteine mutants relative to wild-type myc-S. Data are mean ± range, n=2 independent replicates.

Fig. 24A-24G: a competition-based selection for identifying RBD mutations within SARS-CoV-2S that preferentially bind wild-type or engineered ACE2 receptors. (FIG. 24A) transfection of Expi293F cells with wild-type myc-S and competition with sACE2 ₂ (WT) -IgG1 (25 nM) and sACE2 ₂ V2.4-8h (20 nM) were incubated together. After immunostaining of each epitope tag, the binding proteins were detected by flow cytometry. (FIG. 24B) cells were transfected with RBD SSM library as described in FIG. 24A. Expression pair sACE2 ₂ V2.4 cell populations of specifically enhanced S variants are apparent (cells move to the upper left corner of the main population). (FIG. 24C) the gates used to FACS cells expressing the RBD SSM library. After removal of cells without binding protein, the cells directed against bound sACE2 were collected ₂ V2.4-8h (upper gate) and directed against binding to sACE2 ₂ (WT) -IgG1 (bottom gate) front 20% cells. (FIGS. 24D-24E) for expression pair sACE2 ₂ (WT) (FIG. 24D) or sACE2 ₂ V2.4 (FIG. 24E) specifically enhanced S variant cells, log of two independent FACS selections ₂ Consistency between enrichment ratios. Calculation of R for non-synonymous mutations ² Values. (FIGS. 24F-24G) conservation scores were based on log of all non-synonymous substitutions at a given residue position ₂ The mean value of the enrichment ratio is calculated. The correlation diagram shows that for sACE2 ₂ (WT) (FIG. 24D) or sACE2 ₂ V2.4 (fig. 24E) specific in-gate cells were independently selected twice, consistency of the conservation scores.

Fig. 25A-25C: mutations within RBD that are specific for wild-type ACE2 are rare. (FIG. 25A) SARS-CoV-2RBD was scored for specificity (in sACE2 ₂ (WT) and sACE2 ₂ Conservative score differences for the collected cells in v2.4 specific gates) and different colors. Some residues are hot spots of mutation, for sACE2 ₂ (WT) or pair sACE2 ₂ Specificity enhancement of v 2.4. ACE2 jointThe touch surface is shown as a strip, for sACE2 ₂ The mutation sites in v2.4 are marked and shown as spheres. (FIG. 25B) from sACE2 ₂ (WT) (x-axis) and sACE2 ₂ V2.4 (y-axis) mutant Log in S expressed by the cell population collected in the specific gate ₂ Enrichment ratio. The data are the mean of two independent sorting experiments. The test predicts the target pair sACE2 by targeted mutations in FIG. 26 ₂ (WT) S mutant with enhanced specificity. The test predicts the target pair sACE2 by targeted mutation in FIG. 27 ₂ V 2.4S mutant with enhanced specificity. (FIG. 25C) wild-type myc-S and both variants (N501W and N501Y) were expressed in an Expi293F cell and tested with sACE2 by flow cytometry ₂ (WT) -8h (dashed line) or sACE2 ₂ V2.4-8h (solid line). Wild-type sACE2 was observed ₂ The specific binding of (a) is slightly increased.

Fig. 26A-26B: screening for wild-type sACE2 by deep mutation prediction ₂ Specificity is higher than that of sACE2 ₂ V2.4 mutation of SARS-CoV-2S. (FIG. 26A) relative surface expression of myc-S mutants as determined in FIG. 23. Data are mean ± range, n=2 independent replicates. (FIG. 26B) sACE2 ₂ (WT) -IgG1 (x-axis) and sACE2 ₂ V2.4-8h (y axis) competitive binding on Expi293F cells expressing the indicated myc marker S protein. Cells expressing an S variant that has enhanced specificity for the wild-type receptor will move to the lower right; only minor movements were observed. Cells expressing the S variants (surface expression and/or ACE2 affinity reduction) have a lower percentage in the gating region. The results represent 2 replicates.

Fig. 27A-27B: screening for prediction of sACE2 by deep mutation ₂ V2.4 has a higher specificity than wild-type sACE2 ₂ Mutation of SARS-CoV-2S. (FIG. 27A) relative surface expression of myc-S mutants was measured by flow cytometry. Data are mean ± range, n=2. (FIG. 27B) flow cytometry analysis of cells expressing myc-S variants, which were compared with competing sACE2 ₂ (WT) -IgG1 (x-axis) and sACE2 ₂ V2.4-8h (y-axis) binding. Expression pair sACE2 ₂ V2.4 cells of specifically enhanced S will move to the upper left corner. The results represent 2 replicates.

Fig. 28A-28B: serum of sACE2 peptide after intravenous administrationHalf-life period. Unfused sACE2 ₂ V2.4 were injected into the tail veins of mice (3 male mice and 3 female mice per time point; 0.5 mg/kg). Serum was collected, analyzed by ACE2 ELISA (fig. 28A), and analyzed for proteolytic activity of fluorogenic substrate (fig. 28B). Data are mean ± s.e.

Fig. 29: pharmacokinetics of fusion of sACE2 with human IgG1 Fc following intravenous administration. 3 male mice were intravenously injected with 2.0mg/kg wild-type sACE2 at each time point ₂ IgG1 (open circles) or sACE2 ₂ V2.4-IgG1 (filled circles). Proteins in serum were quantified by human IgG1 ELISA. Data are mean ± s.e.

Fig. 30A-30D: sACE2 in serum after intravenous administration ₂ V 2.4-pharmacokinetics of IgG 1. sACE2 ₂ V2.4-IgG1 was intravenously administered to mice (3 males and 3 females per time point; 2.0 mg/kg). Serum was collected and analyzed by human IgG1 ELISA (fig. 30A), by ACE2 ELISA (fig. 30B), and by ACE2 catalytic activity (fig. 30C). Data are mean ± s.e. (FIG. 30D) serum samples from representative male mice were separated on a non-reducing SDS electrophoresis gel and probed with anti-human IgG 1. Standard 10ng purified sACE2 ₂ V2.4-IgG1. The expected molecular weight (excluding glycans) was 216kD.

Fig. 31A-31F: PK of ACE2 protein delivered directly to the lungs. (FIGS. 31A and 31B) administration of wild-type sACE2 intratracheal at a dose of 1.0mg/kg ₂ IgG1 (open circles) and sACE2 ₂ V2.4-IgG1 (filled circles). Lung tissue was collected, proteins extracted and analyzed by human IgG1 ELISA (fig. 31A) and ACE2ELISA (fig. 31B). Data are mean ± s.e., n=3 male mice per time point. (FIG. 31C) analysis of intratracheal administration of sACE2 by anti-human IgG1 immunoblotting under non-reducing conditions ₂ V2.4-IgG1 representative mouse lung extract. (FIGS. 31D and 31E) inhalation of aerosolized sACE2 by mice ₂ V2.4-IgG1. The lung tissue extracts were analyzed by ACE2ELISA (fig. 31D) and human IgG1 ELISA (fig. 31E). Data are mean ± s.e., n=3 male mice per time point. (FIG. 31F) receiving nebulized sACE2 by anti-human IgG1 immunoblotting analysis ₂ V2.4-IgG1 mouse lung tissueIs a representative extract of (a).

Fig. 32: neutralization by sACE2 ₂ -pseudovirus of IgG1 into human lung cells. Human A549 lung epithelial cells, human A549 lung epithelial cells and human lung endothelial cells over-expressing ACE2 receptor and VSV-SARS-CoV-2-luciferase-pseudotype virus and wild-type sACE2 ₂ IgG1 or engineered sACE2 ₂ v2.4-IgG1 were incubated together at the indicated concentrations. Each experiment contained no virus control (leftmost bar in each figure) and all other samples contained the virus dose at the indicated MOI. The extent of viral entry was quantified based on luciferase activity.

Fig. 33: sACE2 ₂ Efficacy of IgG1 in inhibiting pseudovirus entry into lung and liver in vivo infection models. K18-hACE2 mice express human ACE2 receptor in epithelial cells and were injected intravenously with sACE2 ₂ IgG1 (wild-type, middle bar; engineering v2.4, right bar) and intraperitoneal injection of VSV-SARS-CoV-2-luciferase pseudotype virus. Lungs and livers were collected at 24 hours and the extent of viral entry was quantified based on luciferase expression.

Sequence listing

The nucleic acid sequences and amino acid sequences listed in the accompanying sequence listing are indicated using standard letter abbreviations for nucleotide bases and amino acid three letter codes as defined by 37 c.f.r.1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included in any reference to the indicated strand. The sequence listing was submitted as an ASCII text file, created at 2021, month 3, 11, 43.7KB, which is incorporated herein by reference. In the attached sequence listing:

SEQ ID NO. 1 is the amino acid sequence of human ACE2 (also known as peptidyl-dipeptidase A; deposited under GenBank accession number NP-068576.1):

MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGA

NEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYML

EKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYS

FIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLA

LENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKS

ALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFN

FFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWL

IVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDDVQT

SF

SEQ ID NO. 2 is the amino acid sequence of the surface glycoprotein (protein S) of Severe acute respiratory syndrome coronavirus 2 (deposited under GenBank accession number YP_ 009724390.1):

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF

SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLI

VNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFL

MDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT

RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCAL

DPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAW

NRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAP

GQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIST

EIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPK

KSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDI

TPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQT

RAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSV

AYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNR

ALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK

VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITS

GWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTA

SALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRL

QSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPH

GVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIIT

TDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINAS

VVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIML

CCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO. 3-9 is the amino acid sequence of the human and bata beta coronavirus RBD sequence (see FIG. 19).

SEQ ID NO. 10 is sACE2 ₂ V2.4 amino acid sequence of sACE2 ₂ V2.4 consists of residues 19-732 of human ACE2 (including protease domain and dimerization domain) with three amino acid substitutions (T27Y, L T and N330Y) relative to human ACE 2.

STIEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKE

QSTTAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVC

NPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNE

MARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVR

AKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQA

WDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFR

ILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAA

TPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQ

WMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEA

LCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPL

LNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEM

YLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTE

VEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLG

SEQ ID NO. 11 is from sACE2 ₂ sACE2 comprising v2.4 fused to human IgG1 Fc ₂ V 2.4-amino acid sequence of IgG 1.

STIEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKE

QSTTAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVC

NPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNE

MARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVR

AKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQA

WDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFR

ILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAA

TPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQ

WMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEA

LCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPL

LNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEM

YLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTE

VEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGSDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK

NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ

QGNVFSCSVMHEALHNHYTQKSLSLSPGK

Detailed Description

I abbreviation

ACE2 angiotensin converting enzyme 2

BLI biological layer interference technique

CoV coronavirus

COVID-19 coronavirus disease 2019

IT intratracheal

IV vein

MOI multiplicity of infection

RBD receptor binding domains

sACE2 soluble angiotensin converting enzyme 2

SARS severe acute respiratory syndrome

SARS-CoV-2 SARS coronavirus 2

SEC size exclusion chromatography

sfGFP superfolder green fluorescent protein

II terminology and methods

Unless otherwise indicated, technical terms are used according to conventional usage. The definition of commonly used terms in molecular biology is found in Benjamin lewis, genes X, published by Jones & Bartlett Publishers,2009; and Meyers et al (eds.), the Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in16volumes,2008; and other similar references.

As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly dictates otherwise. For example, the term "an antigen" includes single or multiple antigens and may be considered equivalent to the phrase "at least one antigen". As used herein, the term "comprising" means "including. It is also to be understood that any and all base sizes or amino acid sizes, as well as all molecular weights or molecular weight values given for a nucleic acid or polypeptide are approximate, and are intended to be descriptive, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particularly suitable methods and materials are described below. In case of conflict, the present specification, including definitions of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate examination of various embodiments, the following term explanations are provided:

Aerosol: a suspension of fine solid particles or droplets in a gas (e.g., air).

And (3) application: the agent, e.g., a modified human ACE2 polypeptide, is provided or administered to the subject by any effective route. Exemplary routes of administration include, but are not limited to, injection routes (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous), transdermal routes, intranasal routes, intratracheal routes, and inhalation routes.

Biological sample: a sample obtained from a subject (e.g., a human subject or an animal subject). Biological samples include, for example, fluid samples, cell samples, and/or tissue samples. In some embodiments herein, the biological sample is a fluid sample. Fluid samples include, but are not limited to, serum, blood, plasma, urine, stool, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage (BAL), nasal swab, or other bodily fluids. Biological samples may also refer to cellular tissue or tissue samples, such as biopsy samples or tissue sections.

Contact: an arrangement in which physical association is directly performed; including both solid and liquid forms.

Coronavirus: a large family of plus-sense single-stranded RNA viruses can infect humans and non-human animals. Coronaviruses are named as coronal spikes on their surface. The viral envelope consists of a lipid bilayer containing the viral membrane (M) protein, the envelope (E) protein and the spike (S) protein. Most coronaviruses cause mild to moderate upper respiratory diseases such as the common cold. However, three coronaviruses have emerged that can lead to more serious human diseases and death: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2 and middle east respiratory syndrome coronavirus (MERS-CoV). Other human-infected coronaviruses include human coronavirus HKU1 (HKU 1-CoV), human coronavirus OC43 (OC 43-CoV), human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL 63-CoV). In some embodiments of the present disclosure, "coronavirus" includes any human coronavirus or human-animal co-coronavirus that utilizes ACE2 as a cellular receptor, including known and emerging strains of coronavirus. Human and animal co-affected coronaviruses include, but are not limited to, bats coronaviruses and rodent coronaviruses.

Fusion protein: a protein comprising at least a portion of two different (heterologous) proteins. In some embodiments, the fusion protein consists of a modified ACE2 polypeptide and an Fc protein (e.g., the Fc of human IgG 1).

Heterologous: derived from a separate genetic source or species.

Separating: an "isolated" biological component (e.g., a nucleic acid or protein) has been substantially separated or purified from other biological components (e.g., other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles) in the environment in which the component naturally occurs (e.g., a cell). "isolated" nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids.

Atomizer: a device for converting a therapeutic agent (e.g., a polypeptide) in liquid form into a mist or fine spray (aerosol) that is inhaled into the respiratory system (e.g., the lungs). Atomizers are also known as "nebulizers".

Pharmaceutically acceptable carrier (carrier): the pharmaceutically acceptable vehicle used is a conventional vehicle. Remington, the Science and Practice of Pharmacy, the University of the Sciences in Philadelphia, editor, lippincott, williams, &Wilkins,Philadelphia,PA,21 ^st Edition (2005), describes compositions and formulations suitable for drug delivery of the polypeptides and other compositions disclosed herein. Generally, the nature of the delivery will depend on the particular mode of administration employed. For example, parenteral formulations typically comprise injectable fluids including pharmaceutically and physiologically acceptable fluids as vehicles, e.g., water, physiological saline, balanced salt solutionsLiquid, glucose aqueous solution, glycerin, etc. For solid compositions (e.g., powders, pills, tablets, or capsules), conventional non-toxic solid vehicles may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to bio-neutral inclusion bodies, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Polypeptides, peptides and proteins: refers to amino acid polymers of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also includes modified amino acid polymers; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other treatment, such as conjugation to a labeling component. As used herein, the term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics.

Preventing, treating or ameliorating a disease: "preventing" a disease refers to inhibiting the overall progression of the disease. "treatment" refers to a therapeutic intervention that ameliorates signs or symptoms of a disease or pathological condition after it has begun to develop. "ameliorating" refers to reducing the number or severity of signs or symptoms of a disease. "prophylactic" treatment is treatment of a subject that exhibits no sign of disease or only early signs, with the aim of reducing the risk of developing a condition. The prophylactic treatment may be performed either before or after exposure.

Prevention of: medical treatment is used to prevent (or reduce the risk of developing) a disease or infection, such as CoV infection or covd-19. In the case of viral infection, pre-exposure prevention refers to treatment performed prior to exposure of the subject to the virus, while post-exposure prevention refers to treatment performed immediately or shortly after exposure to the virus, but prior to the appearance of signs or symptoms of infection.

And (3) purifying: the term "purified" does not require absolute purity; rather, it is a relative term. Thus, for example, a purified polypeptide preparation is one in which the polypeptide is more enriched than in its natural environment (e.g., in a cell). In one embodiment, the preparation is purified such that the polypeptide is at least 50% of the total peptide or protein content of the preparation. By substantially purified is meant purification from other proteins or cellular components. The purity of the substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98%. Thus, in one specific non-limiting example, the substantially purified protein is 90% free of other proteins or cellular components.

Sequence identity: similarity between amino acid or nucleic acid sequences is expressed as similarity between sequences, also known as sequence identity. Sequence identity is typically measured in terms of percent identity (or similarity or homology); the higher the percentage, the more similar the two sequences. Homologs or variants of a polypeptide or nucleic acid molecule will have a higher degree of sequence identity when aligned using standard methods.

Sequence alignment methods for comparison are well known in the art. Various procedures and alignment algorithms are described in the following documents: smith and Waterman, adv.appl.Math.2:482,1981; needleman and Wunsch, J.mol.biol.48:443,1970; pearson and Lipman Proc.Natl.Acad.Sci.U.S.A.85:2444,1988; higgins and Sharp, gene 73:237,1988; higgins and Sharp, CABIOS 5:151,1989; corpet et al Nucleic Acids Research 16:10881,1988; and Pearson and Lipman, proc.Natl.Acad.Sci.U.S. A.85:2444,1988.Altschul et al, nature Genet.6:119,1994, taking into account sequence alignment and homology calculations in detail.

NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J.mol. Biol.215:403,1990) is available from a variety of sources, including the national center for Biotechnology information (NCBI, bethesda, MD) and the Internet, for use in conjunction with sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how this procedure can be used to determine sequence identity is available at the NCBI website on the internet.

Homologs and variants of polypeptides, such as modified human ACE2 polypeptides, are generally characterized as possessing at least about 75%, such as at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, using NCBI Blast 2.0 to count in full length alignments with antibody amino acid sequences, with empty blastp set as default parameters. For amino acid sequences greater than about 30 amino acids, the Blast 2 sequence function was used, using a default BLOSUM62 matrix (gap present cost of 11, gap cost per residue of 1) set as a default parameter. When aligning short peptides (less than about 30 amino acids), the alignment should be performed using Blast 2 sequence functions, using PAM30 matrix, set to default parameters (create gap penalty 9, extend gap penalty 1). Proteins that are more similar to a reference sequence will exhibit an increase in percent identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, when assessed by this method. When comparing sequence identity of less than the entire sequence, homologues and variants generally have at least 80% sequence identity within a short window of 10-20 amino acids, and may have at least 85% or at least 90% or 95% sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on the NCBI website on the internet. Those skilled in the art will appreciate that these ranges of sequence identity are provided for guidance only; it is entirely possible to obtain highly significant homologs outside the ranges provided.

The subject: living multicellular vertebrate organisms, i.e., classes including human and animal subjects, including humans and non-human mammals.

Therapeutically effective amount of: an amount of a particular substance (e.g., a modified human ACE2 polypeptide) sufficient to achieve a desired effect in a subject being treated. For example, this may be an amount necessary to inhibit CoV replication or reduce CoV titer in the subject. In one embodiment, a therapeutically effective amount is an amount required to inhibit CoV replication by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% (as compared to untreated conditions). In another embodiment, a therapeutically effective amount is an amount required to reduce CoV titer in a subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% (as compared to untreated conditions). A therapeutically effective amount may also be an amount necessary to reduce or eliminate one or more symptoms of CoV infection, for example, an amount necessary to reduce or eliminate fever, cough, or shortness of breath. Similarly, in some embodiments, a prophylactically effective amount is an amount necessary to reduce the risk of infection with CoV or a disease (e.g., covd-19) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% (as compared to untreated conditions).

Vector (vector): introducing a nucleic acid molecule into a host cell, thereby producing a transformed host cell. A vector may include a nucleic acid sequence, such as an origin of replication, that allows it to replicate in a host cell. The vector may also include one or more selectable marker genes and other genetic elements known in the art. In some embodiments, the vector is a viral vector, such as a lentiviral vector.

Modified ACE2 polypeptides and methods of use

The spike (S) glycoprotein of SARS-CoV-2 binds to angiotensin converting enzyme 2 (ACE 2) on host cells. S is a trimeric class I viral fusion protein, proteolytically processed into S1 and S2 subunits, which remain non-covalently associated in the pre-fusion state (Walls et al, cell.2020Mar6; 181 (2): 281-292.e6;Hoffmann et al, cell.2020Mar4; 181 (2) 271-280.e8;Tortorici and Veesler,Adv Virus Res.Elsevier;2019;105:93-116). Upon binding of the Receptor Binding Domain (RBD) in S1 to ACE2 (Wong et al, J Biol Chem;200 Jan 30;279 (5): 3197-3201), conformational rearrangement occurs, leading to S1 shedding, S2 cleavage by the host protease and exposure of the fusion peptide adjacent to the S2' proteolytic site (Tortorici and Veesler, adv Virus Res. Elsevier;2019;105:93-116; madu et al, J Virol;2009Aug;83 (15): 7411-7421; walls et al, proc Natl Acad Sci USA;2017Oct 17;114 (42): 11157-11162;Millet and Whittaker,Proc Natl Acad Sci USA;2014Oct 21;111 (42): 15214-15219). The favorable folding of the S into the post-fusion conformation is related to host cell/viral membrane fusion and cytoplasmic release of viral RNA. Atomic contact with RBD is limited to the protease domain of ACE2 only (Yan et al, science.2020Mar4: eabb2762; li et al, science.2005Sep16;309 (5742): 1864-1868), and removal of soluble ACE2 (sACE 2) from the neck and transmembrane domains is sufficient to bind S and neutralize infection (Li et al, nature.2003Nov 27;426 (6965): 450-454;Hofmann et al, biochem Biophys Res Commun.2004Jul 9;319 (4): 1216-1221; lei et al, bioRxiv.2020Jan 1: 2020.02.01.929976;Moore et al, J Virol;2004Oct;78 (19): 10628-10635). In principle, the virus has limited potential to escape the sACE 2-mediated neutralization without simultaneously reducing affinity for the native ACE2 receptor, thereby attenuating virulence. Furthermore, fusion of sACE2 to the Fc region of human immunoglobulins can result in increased affinity while recruiting immune effector functions and enhancing serum stability, which is a particularly desirable advantage if used for prophylaxis (Moore et al, J Virol;200 Oct;78 (19): 10628-10635; liu et al, kidney int.2018Jul;94 (1): 114-125), and recombinant sACE2 has been demonstrated to be safe for healthy human subjects (Haschke et al, clin pharmacokinet.2013 ep;52 (9): 783-792) and for patients with lung disease (Khan et al, crit Care.2017Sep7;21 (1): 234).

Rapid spread of SARS coronavirus 2 (SARS-CoV-2) and continual upgrades have resulted in sudden public health events, and no approved therapeutic methods or vaccines are currently available. The viral spike protein S binds to membrane anchored ACE2 on pulmonary host cells, triggering a molecular event, ultimately releasing the viral genome in the cell. The extracellular protease domain of ACE2 inhibits the entry of SARS and SARS-2 coronavirus into cells by acting as a soluble decoy to the receptor binding site on S, the best option for therapeutic and prophylactic development. The efficacy and manufacturability of ACE2 can be improved by mutations that increase the affinity and expression of folding functional proteins. The present disclosure solves this difficulty using deep mutations and in vitro selection, thereby determining enhanced binding of variants of ACE2 to the S receptor binding domain on the cell surface. Mutations exist throughout the protein-protein interface and they can enhance folding and presentation of the buried sites of the interacting epitope. In some embodiments herein, the N90-glycan on ACE2 is removed because it impedes association with S. Mutation maps blueprints were drawn for engineering high affinity ACE2 receptors to address this unprecedented challenge. The disclosed ACE2 polypeptides are advantageous because the risk of developing resistance to these receptor decoys by SARS-CoV-2 or any other ACE2 binding coronavirus is very small.

Described herein are ACE2 polypeptides (e.g., human ACE2 polypeptides) that improve their binding properties to CoV S protein. In particular, provided herein are modified ACE2 polypeptides, including human ACE2 or fragments thereof, e.g., extracellular fragments thereof. The polypeptide comprises at least one amino acid substitution relative to wild type human ACE2 (SEQ ID NO: 1).

In some embodiments, the at least one (e.g., at least one, at least two, at least three, at least four, at least five, or more) amino acid substitution is selected from any of the substitutions shown in table 1.

In some embodiments, the at least one (e.g., at least one, at least two, at least three, at least four, at least five, or more) amino acid substitution is selected from any of the substitutions shown in table 2.

In some embodiments, the at least one (e.g., at least one, at least two, at least three, at least four, at least five, or more) amino acid substitution is selected from any of the substitutions shown in table 3.

In some embodiments, the at least one amino acid substitution is at residue 19, 23, 24, 25, 26, 27, 29, 30, 31, 33, 34, 35, 39, 40, 41, 42, 65, 69, 72, 75, 76, 79, 82, 89, 90, 91, 92, 324, 325, 330, 351, 386, 389, 393, and/or 518 of human ACE2 of SEQ ID No. 1.

In some embodiments, the modified polypeptide contains only a single amino acid substitution relative to wild-type human ACE2 (SEQ ID NO: 1), such as one of the amino acid substitutions listed in Table 1. In other examples, the modified polypeptide comprises two, three, four, five or more amino acid substitutions, e.g., two, three, four, five or more amino acid substitutions listed in table 1. In specific examples, the modified polypeptide comprises only a single substitution at residues 19, 23, 24, 25, 26, 27, 29, 30, 31, 33, 34, 35, 39, 40, 41, 42, 65, 69, 72, 75, 76, 79, 82, 89, 90, 91, 92, 324, 325, 330, 351, 386, 389, 393, or 518 of human ACE2 of SEQ ID No. 1. In other specific examples, the modified polypeptide comprises two, three, four, five or more amino acid substitutions at residues selected from the group consisting of: residues 19, 23, 24, 25, 26, 27, 29, 30, 31, 33, 34, 35, 39, 40, 41, 42, 65, 69, 72, 75, 76, 79, 82, 89, 90, 91, 92, 324, 325, 330, 351, 386, 389, 393 or 518 of human ACE2 of SEQ ID No. 1. In other examples, the modified polypeptides include the combinations of substitutions listed in table 4.

In some embodiments, the modified polypeptide is a full length human ACE2 polypeptide. In some examples, the amino acid sequence of the polypeptide is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to SEQ ID No. 1 and comprises at least one amino acid substitution disclosed herein.

In other embodiments, the modified polypeptide consists of an extracellular fragment of human ACE 2. For example, the modified polypeptide may consist of the complete extracellular protease domain of human ACE2, e.g., amino acid residues 19-615 of SEQ ID NO:1, or the modified polypeptide may consist of a portion of the extracellular domain, e.g., about 50 amino acids, about 75 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 350 amino acids, about 400 amino acids, about 450 amino acids, about 500 amino acids, about 550 amino acids, or about 590 amino acids of the extracellular domain. In some examples, the amino acid sequence of the extracellular fragment is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to residues 19 to 615 of SEQ ID NO. 1, and includes at least one amino acid substitution disclosed herein.

In some embodiments, the modified polypeptide consists of a fragment of human ACE 2. In some examples, the modified polypeptide is a polypeptide of SEQ ID NO of 1 about 50 amino acids, about 75 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 350 amino acids, about 400 amino acids, about 450 amino acids, about 500 amino acids, about 550 amino acids, about 590 amino acids, about 596 amino acids, about 600 amino acids, about 650 amino acids, about 700 amino acids, about 714 amino acids, about 722 amino acids, about 732 amino acids, about 740 amino acids, about 750 amino acids, or about 800 amino acids, and includes at least one amino acid substitution disclosed herein. In specific non-limiting examples, the amino acid sequence of the polypeptide is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a fragment of human ACE2 (e.g., residues 1-732, 19-732, or 19-740 of SEQ ID NO: 1), and comprises at least one amino acid substitution disclosed herein. In a specific example, the modified polypeptide consists of amino acid residues 1-732, 19-732 or 19-740 of SEQ ID NO. 1 and includes at least one amino acid substitution disclosed herein.

In some examples, the modified polypeptide comprises: T27Y, L T and N330Y amino acid substitutions; amino acid substitutions H34A, T92Q, Q P and a 386L; T27Y, L79T, N Y and a386L amino acid substitutions; L79T, N amino acid substitutions of 330Y and A386L; T27Y, N Y and a386L amino acid substitutions; T27Y, L T and a386L amino acid substitutions; amino acid substitutions a25V, T27Y, T Q, Q325P and a 386L; amino acid substitutions H34A, L79T, N Y and a 386L; amino acid substitutions a25V, T92Q and a 386L; or the amino acid substitutions T27Y, Q42L, L T, T92Q, Q325P, N330Y and A386L, wherein said amino acid substitutions are referred to SEQ ID NO. 1.

Dimers of the modified polypeptides disclosed herein are also provided. In some embodiments, the dimeric polypeptide comprises residues 1-732 or 19-732 of SEQ ID NO. 1, and at least one amino acid substitution disclosed herein, e.g., 1, 2, 3, 4 or 5 amino acid substitutions. In some examples, the dimer is a dimer of a sACE2v.2.4 variant having the amino acid sequence of SEQ ID NO. 10.

Fusion proteins comprising a modified ACE2 polypeptide as disclosed herein and a heterologous polypeptide are also provided. In some embodiments, the heterologous polypeptide is an Fc protein, such as a human Fc protein, e.g., the Fc of human IgG 1. In a specific non-limiting example, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO. 11. In other embodiments, the heterologous polypeptide is a protein that can be used as a diagnostic/detection reagent, such as a fluorescent protein (e.g., GFP) or an enzyme (e.g., alkaline phosphatase, HRP, or luciferase). In some embodiments, the heterologous polypeptide is an antibody or antigen binding protein for strong binding to a second CoV antigen. In some embodiments, the heterologous polypeptide is an antibody or antigen binding protein for anchoring to a cell or cell periphery (e.g., for recruiting immune cells). In some embodiments, the heterologous polypeptide is a cytokine, ligand, or receptor for eliciting a biological response. In some embodiments, the heterologous polypeptide is a protein that increases serum half-life (e.g., antibody Fc or serum albumin).

Also provided are compositions comprising a modified ACE2 polypeptide or fusion protein thereof and a pharmaceutically acceptable carrier. In some embodiments, the modified ACE2 polypeptide or fusion protein is formulated for intratracheal or inhalation administration. The intratracheal or inhalation article may be a liquid (e.g., a solution or suspension) and includes a mist, spray, aerosol, or the like. In specific examples, the composition is formulated for administration using a nebulizer. In other embodiments, the modified ACE2 polypeptide or fusion protein is formulated for intravenous administration.

Also provided are in vitro methods of inhibiting replication of CoV by contacting CoV with a modified ACE2 polypeptide or fusion protein disclosed herein. In some examples, coV-infected cells (e.g., cultured cell lines or primary cells) are contacted with the modified ACE2 polypeptide to test the effect of the modified polypeptide on CoV replication.

Methods of inhibiting replication and/or transmission of CoV in a subject are also provided. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of a modified ACE2 polypeptide, fusion protein or composition disclosed herein. Also provided is a method of treating a CoV infection (e.g., covd-19 or SARS) in a subject comprising administering to the subject a therapeutically effective amount of a modified ACE2 polypeptide, fusion protein or composition disclosed herein. In some examples, the subject is an elderly person or a patient suffering from an underlying medical condition (e.g., heart disease, lung disease, obesity, or diabetes). In some examples, the subject has a covd-19. In some examples, the subject is a healthcare worker. In some examples, the modified ACE polypeptide is administered intravenously. In other examples, the modified ACE polypeptide is administered Intratracheally (IT) or by inhalation (e.g., by using a nebulizer). In a specific non-limiting example, the modified ACE2 polypeptide, fusion protein or composition is administered by at least two routes, such as IV and IT, or IV and inhalation. Other routes of administration to the lungs or respiratory tract include bronchial, intranasal, or other inhalation routes, such as direct instillation in the nasal airways or endotracheal tubes of intubated patients. In a specific non-limiting example, the amino acid sequence of the modified ACE2 polypeptide comprises or consists of SEQ ID No. 10 or the amino acid sequence of the fusion protein comprises or consists of SEQ ID No. 11.

Also provided is a method of prophylactically treating (e.g., preventing) a CoV infection in a subject, comprising administering to the subject a prophylactically effective amount of a modified ACE2 polypeptide, fusion protein or composition disclosed herein. Prophylactic treatment includes pre-exposure prophylaxis and post-exposure prophylaxis. In some examples, the subject is an elderly person or a patient suffering from a potential medical condition. In some examples, the underlying condition is heart disease, lung disease, obesity, or diabetes. In some examples, the subject has been exposed to a patient with covd-19. In some examples, the subject is a healthcare worker. In some examples, the modified ACE polypeptide is administered intravenously. In other examples, the modified ACE polypeptide is administered intratracheally or by inhalation (e.g., by using a nebulizer). Other routes of administration to the lungs or respiratory tract include bronchial, intranasal, or other inhalation routes, such as direct instillation in the nasal airways or endotracheal tubes of intubated patients. In a specific non-limiting example, the amino acid sequence of the modified ACE2 polypeptide comprises or consists of SEQ ID No. 10 or the amino acid sequence of the fusion protein comprises or consists of SEQ ID No. 11.

In some examples of prophylactic treatment, the treatment includes pre-exposure prophylaxis. For example, a subject (e.g., a health care worker or a primary worker) exposed to a high risk environment may be administered a modified ACE polypeptide, fusion protein, or a combination thereof to reduce the risk of developing infection with SARS-CoV-2 and/or covd-19. In particular non-limiting examples, the pre-exposure prophylactic treatment comprises intratracheal or by inhalation (e.g., by using a nebulizer) administration of the polypeptide, fusion protein, or composition.

In some examples of prophylactic treatment, the treatment includes post-exposure prophylaxis. In this type of method, the modified ACE polypeptide, fusion protein or composition thereof is administered to the subject immediately or shortly after exposure to SARS-CoV-2, e.g., within 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours or 24 hours. In particular non-limiting examples, the post-exposure prophylactic treatment comprises intratracheal or by inhalation (e.g., by using a nebulizer) administration of the polypeptide, fusion protein, or composition.

Nucleic acid molecules and vectors encoding the modified ACE2 polypeptides or fusion proteins disclosed herein are also provided. In some examples, the nucleic acid molecules and vectors have different codon usage or may be codon optimized for expression in a particular cell type (e.g., mammalian cell). In some examples, the nucleic acid molecules and vectors bear natural human polymorphisms.

Also provided are compositions comprising a nucleic acid molecule or vector disclosed herein and a pharmaceutically acceptable carrier.

Also provided are methods of inhibiting CoV replication and/or transmission in a subject by administering a therapeutically effective amount (or a prophylactically effective amount of a pre-or post-exposure prophylactic method) of a nucleic acid molecule, vector or composition disclosed herein. Also provided are methods of treating CoV infection in a subject comprising administering to the subject a therapeutically effective amount of a nucleic acid molecule, vector or composition disclosed herein. In some examples, the nucleic acid or vector is administered intravenously. In other examples, the nucleic acid or vector is administered intratracheally or by inhalation (e.g., by using a nebulizer). In certain non-limiting embodiments, the nucleic acid or vector is administered using at least two routes, such as IV and IT, or IV and inhalation. Other routes of administration to the lungs or respiratory tract include bronchial, intranasal, or other inhalation routes, such as direct instillation in the nasal airways or endotracheal tubes of intubated patients. In some examples, the subject is an elderly person or a patient suffering from an underlying medical condition (e.g., heart disease, lung disease, obesity, or diabetes). In some examples, the subject has a covd-19. In some examples, the subject is a healthcare worker.

In some embodiments, one or more doses of a modified ACE2 polypeptide, fusion protein, nucleic acid or composition disclosed herein are administered to the subject. For example, one or more, two or more, three or more, four or more, or five or more doses may be administered to the subject, e.g., twice daily, once every other day, twice weekly, once weekly, or once monthly. One of ordinary skill in the art can select an appropriate number of doses and administration times based on factors such as the subject being treated, the condition of the subject, and the underlying condition.

Also provided herein are methods of detecting CoV in a biological sample. In some embodiments, the method comprises contacting the biological sample with a modified polypeptide or fusion protein disclosed herein; and detecting binding of the modified polypeptide or fusion protein to the biological sample. In some examples, the biological sample is a blood, saliva, sputum, nasal swab, or bronchoalveolar lavage sample.

In some embodiments of the methods disclosed herein, the coronavirus is any human or animal coronavirus that utilizes ACE2 as an entry receptor, including emerging strains of coronavirus. In some examples, the coronavirus is a human coronavirus. In specific examples, the human coronavirus is SARS-CoV, SARS-CoV-2, MERS-CoV, human coronavirus HKU1 (HKU 1-CoV), human coronavirus OC43 (OC 43-CoV), human coronavirus 229E (229E-CoV) or human coronavirus NL63 (NL 63-CoV). In other examples, the coronavirus is a human-animal co-patient coronavirus, such as a human-animal co-patient coronavirus that may cross-infect humans. In specific examples, the coronavirus is a bat coronavirus or a rodent coronavirus. In a specific non-limiting example, the hepialus coronavirus is LYRa11, rs4231, rs7327, rs4084, or RsSHC014.

Kits are also provided, comprising the modified polypeptides or fusion proteins disclosed herein bound to a solid support.

The following examples are provided to illustrate certain specific features and/or embodiments. These examples should not be construed as limiting the disclosure to the particular features or embodiments described.

Examples

Example 1

Since human ACE2 has not evolved to recognize SARS-CoV-2S, it is hypothesized that some mutations may be found that enhance affinity for therapeutic and diagnostic applications. By introducing degenerate codons, the coding sequence of full length ACE2 with an N-terminal c-myc epitope tag was diversified to create a library containing all possible single amino acid substitutions at 117 sites, spanning the entire interface with S and arranged within the substrate binding cavity. S binding is independent of ACE2 catalytic activity (Moore et al, J Virol;200 Oct;78 (19): 10628-10635) and occurs on the outer surface of ACE2 (Yan et al, science.2020Mar4: eabb2762; li et al, science.2005Sep16;309 (5742): 1864-1868), whereas angiotensin substrates bind in deep gaps containing active sites (Towler et al, J Biol Chem;200 Apr 23;279 (17): 17996-18007). Thus, substitution within the substrate binding cleft of ACE2 was expected as a control with minimal impact on S interactions, but might help engineer substrate affinity to improve in vivo safety. However, the advantage of treating COVID-19 with catalytically inactivated sACE2 is questioned (Kruse, F1000Res;2020;9 (72): 72).

In the case of usually no more than one coding variant per cell, the ACE2 library is transiently expressed in human Expi293F cells, thus closely linking genotype and phenotype (Heredia et al, J Immunol;2018Apr20;200 (11): ji1800343-3839; park et al, J Biol Chem;2019;294 (13): 4759-4774). Cells were then incubated with a sub-saturated dilution of medium containing SARS-CoV-2 RBD (amino acids 333-529 of SEQ ID NO: 2) fused at the C-terminus to super-folded GFP ((sfGFP) (P delacq et al., nat Biotechnol.2006Jan;24 (1): 79-88)), the levels of bound S-RBD-sfGFP correlated with the surface expression levels of myc-labeled ACE2 measured by two-color flow cytometry, many variants in the ACE2 library failed to bind S-RBD compared to cells expressing wild-type ACE2 (FIG. 1C), whereas the higher bound ACE2 variants appeared to be less abundant (FIG. 1D.) cells expressing higher or lower bound ACE2 variants were collected by Fluorescence Activated Cell Sorting (FACS), referred to as "nCoV-S-High" and "nCoV-S-Low" sorting populations, respectively, which were consistently reduced in the range of fluorescent affinity between RBCoV-S-sGFP and sGFP-sFW as compared to cells expressing wild-type ACE2 (FIG. 1C), and the additional affinity between the two-RBF 2 variants was consistently reduced by fluorescent activated cell sorting (FIG. 1D) and the range of "nCoV-S-Low" FACS "sorting" was consistently reduced to "10 B.7", which was consistently reduced to the range of the affinity between the two-CoV-sFv-sFW 2 variants as compared to 6.

Transcripts in the selection population were depth sequenced and the frequency of variants compared to the natural plasmid library to calculate the enrichment or depletion of all 2,340 coding mutations in the library (figure 2). This method of tracking in vitro selection or evolution by deep sequencing is known as deep mutation (Fowler and Fields, nat methods.2014Aug;11 (8): 801-807). The enrichment ratio (fig. 3A and 3B) and the residue conservation score (fig. 3D and 3E) were very consistent between the two independent sorting experiments, giving confidence in the data. In most cases, the enrichment ratio (FIG. 3C) and conservation score (FIG. 3F) in the nCoV-S-High sorting are inversely related to the nCoV-S-Low sorting, except for nonsense mutations that are properly depleted from both gates. This suggests that most (but not all) of the non-synonymous mutations in ACE2 do not eliminate surface expression. The library is biased towards solvent exposed residues and there is little substitution of buried hydrophobic residues that may have a greater impact on plasma membrane transport (Park et al, J Biol Chem;2019;294 (13): 4759-4774).

Mapping the experimental conservation score of nCoV-S-High sorting to the structure of S-RBD binding ACE2 (Yan et al, science.2020mar4;: eabb 2762) suggests that residues buried in the interface tend to be conserved, while residues in the interface periphery or substrate binding cleft are mutation tolerant (fig. 4A). The ACE2 region around the C-terminus of the ACE2 α1 helix and β3- β4 chain is less tolerant to polar residues, whereas the amino acids at the N-terminus of α1 and the C-terminus of α2 are more prone to hydrophobicity (fig. 4B), possibly partially preserving the hydrophobic stacking effect between α1- α2. These discrete patches contact the bulbous RBD folds and the long protruding loops of the RBD, respectively.

The two ACE2 residues (N90 and T92) that together form the consensus N-glycosylation motif are significant hot spots for enrichment mutations (fig. 2 and 4A). Virtually all substitutions of N90 and T92, except T92S which retains N-glycans, are highly favorable for S-RBD binding, and thus N90-glycans are predicted to partially block the S/ACE2 interaction.

Mining data identified a number of ACE2 mutations that enriched S-RBD binding. For example, 122 mutations at 35 positions in the library, log in nCoV-S-High ₂ Enrichment ratio>1.5. Table 1 lists these mutations. Table 2 lists log ₂ Enrichment ratio>2.0. Table 3 lists log ₂ Enrichment ratio>2.5.

At least ten ACE2 mutations at the structural feature interface enhance S-RBD binding and may contribute to engineering highly specific and tight binders of SARS-CoV-2S, particularly for point-of-care diagnostics. The molecular basis of how some of these mutations enhance S-RBD binding can be reasonably explained from the S-RBD binding low temperature-EM structure (fig. 4C): the hydrophobic substitution of ACE2-T27 enhances hydrophobic packing with S-RBD aromatic residues, ACE2-D30E extends the acidic side chain to S-RBD-K417, and the aromatic substitution of ACE2-K31 contributes to the formation of interfacial clusters of aromatic residues. However, engineered ACE2 receptors with mutations at the interface may present binding epitopes that are completely different from native ACE2, such that virus escape mutants may occur, or they may be strain specific and lack breadth. In contrast, mutations in the second shell (shell) and further shells are noted, which do not directly contact the S-RBD, but rather have a putative structural role. For example, proline substitutions are enriched at five library positions (S19, L91, T92, T324 and Q325), which may be the first turn of the helix to be entropy stable. Proline is also enriched at H34, which may strengthen the central bulge in α1. Multiple mutations are also enriched at buried sites, which will alter local packing (e.g., a25V, L29F, W69V, F Y and L351F). Thus, selection of ACE2 variants with high binding signals reported not only affinity, but also presentation of the SARS-CoV-2S recognition fold structure on the membrane. The presence of enriched structural mutations in the sequence profile is particularly notable given that ACE2 libraries are biased towards solvent exposure positions.

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Example 2

The highly enriched 30 individual substitutions in the nCoV-S-High selection were verified by the targeted mutations (FIG. 5). The binding of RBD-sfGFP to the full-length ACE2 mutant was enhanced but the improvement was not great compared to wild-type ACE2, and was most pronounced on cells expressing low ACE2 levels (fig. 5A). The differences in ACE2 expression between mutants were also correlated with the overall level of binding RBD-sfGFP (fig. 5C), indicating that caution must be exercised in interpreting the deep mutation scan data, as mutations may affect activity and expression. To rapidly evaluate mutations whose format is more relevant to therapy development, the soluble ACE2 protease domain was fused to sfGFP. The expression level of sACE2-sfGFP was assessed qualitatively by fluorescence of the transfected cultures (FIG. 6A), and binding of sACE2-sfGFP to full-length S expressed at the plasma membrane was measured by flow cytometry (FIG. 6B). The elimination of the single substitution of N90 glycans (T92Q) resulted in a slight increase in binding signal (fig. 6B), as demonstrated by analysis of the purified protein (fig. 12). Focusing on the most enriched substitutions in the selection for S binding, these substitutions were also spatially separated to minimize negative epistasis (Heredia et al, J Virol93 (11): e00219-19, 2019), the combination of mutations in sACE2 greatly enhanced S binding (table 4 and fig. 6B). Although this assay only provides relative differences, combining mutants has enhanced binding by at least one order of magnitude. Combinations of mutations that have not been studied may have a greater impact.

Table 4 Combined mutant of sACE2

Variants	Mutation
		sACE2.v1	H34A、T92Q、Q325P、A386L
sACE2.v2	T27Y、L79T、N330Y、A386L
		sACE2.v2.1	L79T、N330Y、A386L
sACE2.v2.2	T27Y、N330Y、A386L
		sACE2.v2.3	T27Y、L79T、A386L
sACE2.v2.4	T27Y、L79T、N330Y
		sACE2.v3	A25V、T27Y、T92Q、Q325P、A386L
sACE2.v4	H34A、L79T、N330Y、A386L
		sACE2.v5	A25V、T92Q、A386L
sACE2.v6	T27Y、Q42L、L79T、T92Q、Q325P、N330Y、A386L

Individual variants of sace2.v2 were selected for purification and further characterization (fig. 7). This variant was chosen because it fused well to sfGFP expression and maintained N90-glycans, thus presenting a surface that more closely matched native sACE2 to minimize immunogenicity. When purified as 8 his-tagged protein (20% lower) or IgG1-Fc fusion protein (60% lower) yields of sace2.v2 were lower than wild-type protein yields and small fractions of sace2.v2 were found to aggregate after incubation for 40 hours at 37 ℃ by analytical Size Exclusion Chromatography (SEC) (fig. 7D). Otherwise, SEC was unable to distinguish sce 2.v2 from wild type (fig. 7C).

In flow cytometry experiments with purified 8 his-labeled sACE2, only sACE2.v2-8h was found to bind strongly to full-length S on the cell surface, indicating that wild-type sACE2 has a high dissociation rate, resulting in dissociation during sample washing (fig. 8A and 13). The difference between wild type and variant was less pronounced in the case of IgG1-Fc fusion (fig. 8A and 13), indicating that affinity masks the enhancement of mutant binding, again indicating the difference in dissociation rates between wild type and variant sACE2. Soluble ACE2.v2-8h outperformed wild-type sACE2-IgG1 in binding to S-expressing cells, whereas wild-type sACE2-8h did not outperform sACE2-IgG1 even at 10-fold higher concentrations (FIG. 8B). Furthermore, only engineered sACE2.v2-8h competed effectively with anti-RBD IgG in serum from three recovered COVID-19 patients when tested by ELISA (FIG. 8E). This is consistent with the results of the study, i.e., although sACE2 is very effective in inhibiting SARS-CoV-2 replication in Cell lines and organelles, extremely high concentrations are required (Moneil et al, cell DOI:10.1016/j. Cell.2020.04.004:1-28,2020). Using Biological Layer Interference (BLI), the affinity of sACE2.v2 for immobilized RBD was found to be 65-fold higher than that of wild-type protein, almost entirely due to the slower dissociation rate (Table 5 and FIGS. 8C and 8D).

Table 5. Summary of bli kinetic data

Soluble analytes	Immobilized ligands	k a (M -1 s -1 )	k d (s -1 )	K D
					sACE2(WT)-8h	RBD-IgG1	7.1/8.1×10 4	1.1/1.1×10 -2	140/150nM
sACE2-T92Q-8h	RBD-IgG1	1.0×10 5	8.2×10 -3	80nM
					sACE2.v2-8h	RBD-IgG1	1.5×10 5	3.3×10 -4	2.3nM
sACE2.v2.2-8h	RBD-IgG1	1.3×10 5	8.3×10 -4	6.2nM
					sACE2.v2.4-8h	RBD-IgG1	1.4×10 5	5.4×10 -4	3.8nM
sACE2 2 (WT)-8h	RBD-IgG1	9.5×10 4	ND	ND
					sACE2 2 .v2.4-8h	RBD-IgG1	1.5×10 5	ND	ND
sACE2 2 .v2.(CHO-S)	RBD-IgG1	2.0×10 5	ND	ND
					RBD-8h	sACE2 2 (WT)-IgG1	2.8×10 5	6.0×10 -3	22nM
RBD-8h	sACE2 2 .v2-IgG1	5.8×10 5	1.4×10 -4	0.2nM
					RBD-8h	sACE2 2 .v2.4-IgG1	5.7×10 5	3.5×10 -4	0.6nM

All measurements were performed using capture of IgG1 (Fc) fusion proteins to the anti-human IgG Fc biosensor surface.

3-4 analyte concentrations were used for each experiment.

ND, undetermined.

In all experiments, whether ACE2 was purified as an 8his marker protein or used as sfGFP fusion protein in the expression medium, the characterized variant of sa ce2.v2 consistently showed one to two orders of magnitude tighter binding, whether full-length S was expressed on the plasma membrane or the isolated RBD was immobilized on the biosensor surface. These experiments confirm the key finding of deep mutations, i.e. mutations present in human ACE2, enhance binding to SARS-CoV-2S.

In order to solve the problem of decreased expression of sACE2.v2, it was assumed that the mutation load was too high. In the second generation design, each of the four mutations in sace2.v2 recovered to the wild-type state (table 4), and binding to full-length S was found to remain tight at the cell surface (fig. 9A). One of the variants (sACE 2.v2.4 with mutations T27Y, L T and N330Y) was purified, with higher yields than the wild type and showed tight nanomolar binding to RBD (FIG. 9).

The ACE2 construct was extended to include the neck/dimerization domain, resulting in a stable dimer (fig. 10A), referred to herein as sACE2 ₂ It was tightly bound to S on the cell surface or RBD immobilized on the biosensor (fig. 14). Dimer sACE2 compared to wild type ₂ V2.4 competed more effectively with IgG antibodies present in serum of convalescent patients (fig. 10B). By combining sACE ₂ 2-IgG1 (FIG. 15) was immobilized on the biosensor surface and incubated with monomeric RBD-8h as analyte, wild-type sACE ₂ K of RBD of 2 _D Determined to be 22nM (FIG. 10C), and beforeIs very consistent (Wrapp et al, science, eabb2507,2020; shang et al, nature.382,1199, 2020), while sACE2 ₂ V2.4 binding affinity was 600pM (fig. 10D). This is advantageous over recently isolated monoclonal antibodies (Pinto et al, nature doi:10.1038/s41586-020-2349-y,2020;Hansen et al, science, eabd0827,2020; brouwer et al, science, eabc5902,2020; wec et al, science, eabc7424,2020; wang et al, nat Commun.11,2251,2020; wu et al, science, 368,1274,2020; rogers et al, science, eabc7520,2020).

The efficacy of monomer sACE2.v2.4 in neutralizing VeroE6 cells cultured from SARS-CoV-2 infection exceeded that of wild-type protein by nearly two orders of magnitude (FIG. 11), consistent with biochemical binding data. Wild-type dimer sACE2 ₂ Itself two orders of magnitude stronger than the monomer, indicating strong interactions with spikes on the surface of the viral particles, dimer sACE2 ₂ V2.4 is again more efficient, IC ₅₀ In the sub-nanomolar range (fig. 11). Although SARS-CoV-1S structure or sequence was not considered in the engineering process, dimer sACE2 ₂ V2.4 also effectively neutralized SARS-CoV-1 (FIG. 11), and decoy receptors might neutralize a variety of ACE 2-utilizing coronaviruses that have not yet been transmitted to humans.

For improved safety, unlabeled sACE2 ₂ V2.4 was produced in ExpiCHO-S cells (FIG. 16A) and was found to be stable after 6 days incubation at 37℃C (FIG. 16B). The protein and wild type sACE2 ₂ IgG1 competes for S expressed by cells (fig. 16C) and binds tightly to immobilized RBD (fig. 16D). In addition to inhibiting viral entry, recombinant sACE2 may have a second therapeutic mechanism: angiotensin II, an vasoconstrictor peptide hormone, was proteolyzed to alleviate respiratory distress symptoms (Imai et al, nature 436,112-116,2005;Treml et al, crit. Care med 38,596-601,2010). Discovery of soluble ACE2 ₂ V2.4 has catalytic activity, although activity was reduced (fig. 17).

While deep mutations in viral proteins during replication of viruses have been widely studied to understand the escape mechanism of drugs and antibodies, work herein suggests how deep mutations can be directly applied to treatment design when the selection method is separated from viral replication and focused on host factors. The scientific community has established, at a surprising rate, a variety of drug candidates for the treatment of covd-19, particularly monoclonal antibodies with very high affinity for protein S. The studies disclosed herein demonstrate how to engineer comparable affinities into the natural receptor of the virus, while also providing insight into the molecular basis of the initial virus-host interaction.

Example 3

Materials and methods

A plasmid. Mature polypeptide (amino acids 19-805) of human ACE2 (GenBank NM-021804.1) was cloned into the NheI-XhoI site of pCEP4 (Invitrogen), pCEP4 having an N-terminal HA leader sequence (MKTIIALSYIFCLVFA), myc-tag and linker sequence (GSPGGA). Soluble ACE2 fused to superfolder GFP was constructed by ligating the protease domain of ACE2 (amino acids 1-615) to sfGFP (GenBank ASL 68970) via a gly/ser rich linker sequence (GSGGSGSGG) gene and pasting between the NheI-XhoI sites of pcdna3.1 (+) (Invitrogen) (podalactoq et al, nat. Biotechnol.24,79-88,2006). Cloning of equivalent sACE2 construct by GSG linker sequence and 8 histidine tag or GS linker sequence and Fc region of IgG1 (amino acids D221-K447), while dimer sACE2 ₂ The construct comprises amino acids 1-732, otherwise identical. The synthetic human codon optimized gene fragment (Integrated DNA Technologies) for RBD (amino acids 333-529) of SARS-CoV-2S (GenBank YP_ 009724390.1) is fused at the N-terminus to the HA leader sequence and at the C-terminus to the superfolder GFP, fc region of IgG1 or 8 histidine tags. The assembled DNA fragment was ligated to the NheI-XhoI site of pcDNA3.1 (+). Human codon optimized full-length S was subcloned from pUC57-2019-nCoV-S (human) (Molecular cloning), pUC57-2019-nCoV-S was unlabeled (amino acids 1-1273) and had an N-terminal HA leader sequence (MKTIIALSYIFCLVFA), myc-tag and linker sequence (GSPGGA) upstream of the mature polypeptide (amino acids 16-1273).

And (5) tissue culture. Expi293F cells (ThermoFisher) were cultured in Expi293 expression medium (ThermoFisher) at 125rpm, 8% CO ₂ Culturing at 37 ℃. To produce RBD-sfGFP, RBD-IgG1, sACE2-8h and sACE2-IgG1, cells were prepared at a concentration of 2X 10 ⁶ Culture/ml. For each milliliter of the culture,500ng of plasmid and 3. Mu.g of polyethylenimine (MW 25,000; polysciences) were mixed in 100. Mu.l of OptiMEM (Gibco), incubated for 20 minutes at room temperature, and then added to the cells. Transfection enhancers (thermo Fisher) were added 18-23 hours post-transfection and the cells were cultured for 4-5 days. Cells were removed by centrifugation at 800 Xg for 5 min and the medium was stored at-20 ℃. After thawing and immediately before use, the remaining cell debris and sediment were removed by centrifugation at 20,000Xg for 20 minutes. The plasmid for expression of the sACE2-sfGFP protein was transfected into the Expi293F cells using Expifectamine (ThermoFisher) according to the manufacturer's instructions, 22- ¹ / ₂ Transfection enhancer was added in hours and the culture supernatant harvested after 60 hours.

Depth mutation. 117 residues within the ACE2 protease domain were diversified by overlap extension PCR technique (Procko et al, j.mol. Biol.425,3563-3575,2013) using primers with degenerate NNK codons. Plasmid libraries were transfected into Expi293F cells using expicamine under conditions that each cell described previously generally did not produce more than one coding variant (Heredia et al, j.immunol.200, ji1800343-3839,2018;Park et al, J Biol Chem 294,4759-4774,2019); 1ng of the encoding plasmid was isolated from each ml of cell culture (concentration 2X 10 ⁶ Per ml) 1,500ng of pCEP4-. DELTA.CMV vector plasmid was diluted and the medium was changed 2 hours after transfection. After 24 hours, cells were collected, washed with ice-cold PBS supplemented with 0.2% bovine serum albumin (PBS-BSA), and incubated on ice for 30 minutes with PBS-BSA dilutions containing RBD-sfGFP medium diluted 1/20 (repeat 1) or 1/40 (repeat 2). Cells were co-stained with anti-myc Alexa 647 (clone 9B11,1/250 dilution; cell Signaling Technology). Cells were washed twice with PBS-BSA and sorted on BD FACS Aria II at the Roy J.Carver biotechnology center. The main cell population was gated by forward/side scatter to remove debris and doublets (doublets), and DAPI was added to the sample to exclude dead cells. In the myc positive (Alexa 647) population, the first 67% was gated (fig. 1B). Wherein 15% of the cells with highest GFP fluorescence and 20% of the cells with lowest GFP fluorescence were collected in tubes coated overnight with fetal bovine serum and containing an Expi293 expression medium (fig. 1D). Using GThe eneJET RNA purification kit (Thermo Scientific) extracts total RNA from the collected cells and reverse transcribes cDNA with high fidelity Accuscript (Agilent), with primers that are gene specific oligonucleotides. The diverse region of ACE2 was amplified by PCR into 5 fragments. Flanking sequences on the primers add adaptors at the end of the product for annealing to Illumina sequencing primers (unique barcodes) and for binding to the flow cell. Amplicons were sequenced on Illumina NovaSeq 6000 using a 2 x 250nt paired-end protocol. Enrich analytical data (Fowler et al, bioinformation.27, 3430-3431,2011) was used and commands were set in the GEO database. Briefly, the frequency of ACE2 variants in the sorted population transcripts was compared to their frequency in the natural plasmid library to calculate log ₂ The enrichment ratio was then normalized by the same calculation for the wild type. Wild-type sequences were neither significantly enriched nor depleted, and log ₂ The enrichment ratio is-0.2 to +0.2.

Flow cytometry analyzed ACE2-S binding. Using Expifectamine (ThermoFisher), the DNA/ml culture was prepared from pcDNA3-myc-ACE2, pcDNA3-myc-S plasmid or pcDNA3-S plasmid (500 ng of DNA/ml of culture at a concentration of 2X 10 ⁶ /ml) transfected with Expi293F cells. Cells were analyzed by flow cytometry 24 hours after transfection. To analyze RBD-sfGFP binding to full length myc-ACE2, cells were washed with ice-cold PBS-BSA and incubated on ice for 30 min with 1/30 dilution of RBD-sfGFP-containing medium and 1/240 dilution of anti-myc Alexa647 (clone 9B11,Cell Signaling Technology). Cells were washed twice with PBS-BSA and analyzed on BD LSR II. To analyze binding of sACE2-sfGFP to full-length myc-S, cells were washed with PBS-BSA and incubated on ice for 30 min with serial dilutions of medium containing sACE2-sfGFP and 1/240 dilutions of anti-myc Alexa647 (clone 9B11,Cell Signaling Technology). Cells were washed twice with PBS-BSA and analyzed on BD Accuri C6, gating analysis was performed on the entire Alexa647 positive population. To measure binding of sACE2-IgG1 or sACE2-8h, myc-S or S transfected cells were washed with PBS-BSA and incubated with a PBS-BSA solution of purified sACE2 at the indicated concentration for 30 min. Cells were washed twice with secondary antibody (Immunology Consultan diluted 1/100 ts Laboratory chicken anti-HIS-FITC polyclonal antibody; or 1/250 dilution of BioLegend anti-human IgG-APC clone HP 6017) was incubated on ice for 30 min and washed twice more and fluorescence of the total population after debris exclusion by FSC-SSC gating was measured on BD Accuri C6. Data was processed using FCS Express (De Novo software) or BD Accuri C6 software.

The IgG1-Fc fusion protein was purified. The clarified expression medium was incubated with KANEKA KanCapA 3G affinity adsorbent (Pall; equilibrated in PBS) for 90 min at 4 ℃. The resin was collected on a chromatographic column, washed with 12 Column Volumes (CV) PBS, and the protein eluted with 5CV 60mM acetate (pH 3.7). The eluate was immediately neutralized with 1CV of 1M Tris (pH 9.0) and concentrated using a 100kD MWCO centrifuge (Sartorius). Proteins were isolated on a Superdex 200 Increate 10/300GL column (GE Healthcare Life Sciences) running buffer PBS. Peak fractions were pooled, concentrated to-10 mg/ml, and excellent in solubility, snap frozen in liquid nitrogen and stored at-80 ℃. Protein concentration was determined by absorbance at 280nm using the calculated extinction coefficient of the monomeric mature polypeptide sequence.

Purification of 8his marker protein. HisPur Ni-NTA resin equilibrated in PBS (Thermo Scientific) was incubated with clarified expression medium for 90 min at 4 ℃. The resin was collected on a chromatographic column, washed with 12 Column Volumes (CV) PBS and the proteins eluted with PBS step-elution supplemented with 20mM, 50mM and 250mM imidazole (pH 8) (6 CV per fraction). The 50mM and 250mM imidazole fractions were concentrated using a 30kD MWCO centrifuge (MilliporeSigma). Proteins were isolated on a Superdex 200Increase10/300GL column (GE Healthcare Life Sciences) running buffer PBS. Peak fractions were pooled, concentrated to-5 mg/ml, and excellent in solubility, snap frozen in liquid nitrogen and stored at-80 ℃.

Other proteins. Unlabeled sACE2 expressed in ExpiCHO-S cells (ThermoFisher) ₂ V2.4 is produced and provided by Orthogonal Biologics, inc.

Size Exclusion Chromatography (SEC) was analyzed. Proteins (200. Mu.l, 2. Mu.M) were separated on a Superdex 200Increase 10/300GL column (GE Healthcare Life Sciences) equilibrated in PBS. MW standards were from Bio-Rad.

Biological layer interference techniques. The hydrated anti-human IgG Fc biosensor (Molecular Devices) was immersed in RBD-IgG 1-containing expression medium for 60 seconds. The RBD capturing biosensor was washed in assay buffer, immersed in a designated concentration of the sACE2-8h protein, and then returned to assay buffer to measure dissociation. Data was collected on the BLItz instrument and analyzed using the BLItz Pro data analysis software (Molecular Devices) in a 1:1 binding model. Assay buffer was 10mM HEPES (pH 7.6), 150mM NaCl, 3mM EDTA, 0.05% polysorbate 20, 0.5% skimmed milk powder (Bio-Rad).

Reagent and data availability. Plasmids were stored in Addgene under IDs 141183-5, 145145-78, 149268-71, 149663-8 and 154098-106. Raw and processed deep sequencing data are stored in the NCBI's gene expression integrated database (GEO), SEQ ID NO. GSE147194.

ACE2 catalytic activity assay. Activity was measured using a fluorescent ACE2 activity assay kit (BioVision) and proteins were diluted in assay buffer to final concentrations of 22, 7.4 and 2.5nM. Specific activity was reported as pmol MCA (mU) produced per pmol enzyme per minute. Fluorescence values were read on analysis HT (Molecular Devices).

ELISA. anti-RBD IgG titers of human serum samples were measured by indirect ELISA as described by Amant et al (nat. Med.5,562,2020). Wells of the 96-well plates were coated overnight at 4℃with 2. Mu.g/ml RBD-8h protein. After washing, the wells were blocked with 3% skim milk in PBS for 1 hour at room temperature. Next, various diluted heat-inactivated sera (56 ℃,1 hour) were added to the closed wells. After 2 hours at room temperature, the wells were washed and then incubated with goat anti-human IgG-HRP (ThermoFisher) for 1 hour at room temperature. Any unbound HRP conjugated antibody was removed by washing and HRP-added TMB substrate. The colorimetric reaction was carried out for 10 minutes, and then the reaction was terminated by adding 2N sulfuric acid. The absorbance of the product was measured at 450 nm. For competition assays, serum dilutions (equivalent to their titres: 1:5000 for P2, 1:2000 for P3, 1:1000) were premixed with different concentrations of sACE 2. serum-sACE 2 mixtures were added to the blocking plates and the protocol continued as described above.

Non-human subject study (NHSR) determination. The university of Chicago (patients P2 and P3) and commercial suppliers (patient P1; rayBiotech) provide de-identified serum samples of the recovered patient of COVID-19. The university of illinois study subject protection office determines that the samples used in the ELISA study do not meet the human subject study criteria defined in 45CFR46 (d) (f) or 21CFR56.102 (c) (e) and do not require IRB approval.

Virus micro-neutralization assay. Vero E6 cells were cultured and their infection with authentic SARS-CoV-2 was determined as described by Wec et al (Science, eabc7424,2020). Briefly, soluble ACE2 protein WAs serially diluted in culture medium and incubated with SARS-CoV-2 (viral isolate 2019-nCoV/USA-WA1-A12/2020; genBank accession number MT 020880.1) for 1 hour. The mixture was added to VeroE6 cells at a MOI of 0.2 and incubated for 24 hours. Cells were fixed and immunostained with anti-SARS-CoV-2 nucleocapsid antibody (Sino Biological) and Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody. The well plate was imaged on Operetta (PerkinElmer) to determine the number of infected cells and compared to a control well with only virus to calculate the percentage of relative infection.

Example 4

Unlike the ubiquitous human coronaviruses responsible for common respiratory diseases, these zoonotic coronaviruses with pandemic potential can lead to serious and complex diseases, in part because their tissue chemotaxis is driven by receptor utilization. Severe acute respiratory syndrome coronaviruses 1 (SARS-CoV-1) and 2 (SARS-CoV-2) bind to angiotensin converting enzyme 2 (ACE 2) for Cell attachment and entry into cells (Zhou et al, nature.579,270-273,2020;Walls et al, cell, 2020), doi:10.1016/j.cell.2020.02.058; wan et al, SARS.J. Virol, 2020), doi 10.1128/JVI.00127-20; wrapp et al, science, eabb2507,2020; hoffmann et al, cell, 2020), doi 10.1016/j.cell.2020.02.052; li et al, nature.426,450-454,2003; letko et al, nat microbiol.11,1860, 2020). ACE2 is a protease responsible for regulating blood volume and pressure and is expressed on the cell surface of the lung, heart and gastrointestinal tract, among other tissues (Samavati, B.D.Uhal, front.Cell.Infect.Microbiol.10,752,2020; jiang et al, nat Rev cardiol.11,413-426,2014). Continuous transmission of SARS-CoV-2 and its associated disease (COVID-19) has serious implications for the global health care system and economics, and there is a strong need for effective therapies and vaccines.

As SARS-CoV-2 becomes popular among people, it is likely to become mutated and become genetically bleached. As more and more people become infected and develop anti-immune reactions, it is not clear to what extent this will occur, but variants of viral spike protein S (D614G) have emerged rapidly from multiple independent events and affect the stability and kinetics of S protein (Zhang et al, bioRxiv,2020.06.12.148726,2020;Korber et al, cell.182,812-827.e19, 2020). Another S variant (D839Y) is prevalent in the grape teeth, probably due to the founder effect (Borges et al, medRxiv,2020.08.10.20171884,2020). Coronaviruses have medium to high mutation rates (10 per year at each site in HCoV-NL63 ^-4 The individual substitutions were measured (Pyrc et al, j.mol. Biol.364,964-973,2006), HCoV-NL63 is an alpha coronavirus that binds ACE2 despite being shared with only the RBD part of SARS-associated beta coronavirus by a small interface (Wu et al, proc.Natl. Acad.sci.U.S. A.106,19970-19974,2009)), and the coronavirus genome often undergoes significant changes in nature due to recombination events, especially in bats where co-infection levels may be high (Su et al, trends microbiol.24,490-502,2016); boni et al, nat Microbiol 5,1408-1417,2020). There is also a record of MERS-CoV recombination on camelids (Sabir et al science.351,81-84,2016). All this will have profound effects on the current track of pandemics, the likelihood of future coronavirus pandemics and whether resistance to SARS-CoV-2 is prevalent.

Viral spikes are a vulnerable target for neutralizing monoclonal antibodies (entering the clinical stage), but escape mutations in spikes rapidly appear in all antibodies tested in tissue culture (Baum et al Science, eabd0831,2020). Mutations in S are readily identified by deep mutation of the isolated Receptor Binding Domain (RBD) by yeast surface display, which retain high expression levels and ACE2 affinity, but no longer bind to monoclonal antibodies and develop resistance (Greaney et al, bioRxiv,2020.09.10.292078,2020). This has prompted the development of non-competitive monoclonal antibody cocktail therapies (Baum et al, science, eabd0831,2020; totorici et al, science, eabe3354,2020) that inspire from the teachings of treating HIV-1 and ebola viruses, thereby limiting the likelihood of the virus escaping. This has not solved the problem of future coronavirus extravasation from wild animals that may differ in antigenicity. In fact, extensive screening work was required to find antibodies cross-reactive with SARS-CoV-2 from recovered SARS-CoV-1 patients (Pinto et al, nature, 2020), doi:10.1038/s 41586-020-2349-y), which suggests that antibodies have limited ability to interact with variable epitopes on spike surfaces and are unlikely to be widely used for all SARS-associated viruses and are used with their broad specificity.

A protein-based antiviral drug that replaces monoclonal antibodies is to compete for receptor binding sites on viral spikes using soluble ACE2 (sACE 2) as a bait (Li et al, nature.426,450-454,2003;Hofmann et al, biochem. Biophys. Res. Commun.319,1216-1221,2004;Lei et al, nat Commun.11,2070,2020, moneil et al, cell,2020, doi: 10.1016/j.cell.2020.04; chan et al, science.4, eabc0870,2020). In principle, the virus has limited potential to escape the sACE 2-mediated neutralization without simultaneously reducing the affinity for the native ACE2 receptor, thereby attenuating the virulence of the virus. Several groups have now engineered sACE2 to yield SARS-CoV-2 high affinity baits that rival and effectively neutralize infection with mature monoclonal antibodies (Chan et al, science 4, eabc0870,2020; glasgow et al, bioRxiv,2020.07.31.231746,2020;Higuchi et al, bioRxiv,2020.09.16.299891,2020). In the studies disclosed herein, deep mutations were used to identify a number of mutations in ACE2 that enhance affinity for S (Chan et al, science.4, eabc0870,2020). These mutations are distributed at the interface, and also at the distal sites, which are expected to enhance folding of the virus recognition conformation. Combinations of three mutations, termed sACE2 ₂ V2.4, increasing the affinity by a factor of 35 and binding SARS-CoV-2S (K) _D 600pM)(Chan et al.,Science.4, eabc0870, 2020). By strongly binding to trimeric spikes expressed on the membrane, a higher apparent affinity can be achieved. Although engineering process is focused only on SARS-CoV-2 affinity, sACE2 ₂ V2.4 effectively neutralises infection of authentic SARS-CoV-1 and-2 in tissue culture, indicating that its high similarity to wild-type receptors makes it generally broadly active against the beta coronavirus using ACE 2. Soluble ACE2 ₂ V2.4 is a dimer and a monodispersed, free of aggregation, catalytically active, highly soluble, stable after storage for several days at 37 ℃, and preferably expressed at a higher level than the wild-type protein. sACE2 ₂ V2.4 combines the high activity with the required production characteristics perfectly, and is a truly candidate drug for preclinical development.

The engineered high affinity decoy receptor, while very similar to native ACE2, has mutations at or near the interaction surface. Thus, it is possible for the viral spike mutant to distinguish between engineered decoys and wild-type receptors, thereby creating a resistance pathway. Disclosed herein are engineered decoy sACE2 ₂ V2.4 RBD of a number of SARS-associated beta coronaviruses using ACE2 into cells are widely and tightly combined. Although many mutations that favor binding to engineering baits were found in competition binding assays, no mutations were found in RBDs that were in direct contact with ACE2 and may have escape mutations, which would specifically shift to wild-type receptors. The results indicate that resistance to engineered decoy receptors will be rare and that sACE2 ₂ V2.4 is directed against a common attribute of affinity for S in SARS-associated virus.

Results

RBD of engineering decoy receptor widely binding SARS related CoV with close affinity

Determination of the bait receptor sACE2 ₂ V2.4 affinity for RBD purified from five coronaviruses (isolates LYRa11, rs4231, rs7327, rs4084 and RsSHC 014) and two human coronaviruses (SARS-CoV-1 and SARS-CoV-2) S proteins of the species bats of the genus Jupiter (Rhinophytus). These viruses belong to the co-clade of the beta coronavirus using ACE2 as an entry receptor (Letko et al, nat microbiol.11,1860, 2020). They have close sequence identity within the RBD coreThe highest variation in functional ACE2 binding sites (fig. 18 and 19) may be due to a "army competition" co-evolving with polymorphic ACE2 sequences in ecologically diverse bat species (Frank et al, biorxiv.5,562, 2020). Affinity was measured by Biological Layer Interferometry (BLI) wherein sACE2 ₂ (amino acids S19-G732) fused at the C-terminus to the Fc portion of human IgG1 immobilized on the sensor surface, and the monomer 8 his-labeled RBD was used as a soluble analyte. This arrangement precludes affinity effects, otherwise when dimeric sACE2 is in solution ₂ Binding to the immobilized RBD that modifies the interacting surface results in an artificially high (picomolar) apparent affinity. Wild type sACE2 ₂ Binding to all RBDs, the affinity for SARS-CoV-2 was 16nM, the affinity for LYRa11 was 91nM, and the median affinity was 60nM (Table 6). The measured affinities for RBD of SARS-CoV-1 and SARS-CoV-2 were comparable to those of published data (Wrapp et al, science, eabb2507,2020; chan et al, science.4, eabc0870,2020; shang et al, nature.382,1199,2020; kirchdoerfer et al, sci Rep.8,15701,2018; li et al, EMBO J.24,1634-1643,2005). Engineering sACE2 ₂ V2.4 affinity for all RBDs was significantly increased for K of SARS-CoV-2 _D 0.4nM, K for isolate Rs4231 _D At 3.5nM, the median affinity was less than 2nM (Table 6). The affinity of engineering baits is increased by about 35-fold, which is generally applicable to coronaviruses in test groups, so the molecular basis for affinity enhancement must be based on the common property of RBD/ACE2 recognition.

Deep mutation scans RBD in the case of full length S, residues in the ACE2 binding site were found to be mutation tolerant

To explore the potential sequence diversity in SARS-CoV-2S that might act as a "repository" of drug resistance, one goes through depth Mutation the mutation tolerance of RBD was assessed (Fowler and Fields, nat. Methods.11,801-807,2014). The saturation mutation focused on RBD (amino acids C336-L517) of the full-length S, which was labeled with a C-myc epitope at the extracellular N-terminus, for detection of surface expression. A spike library comprising 3,640 single amino acid substitutions was transfected into human Expi293F cells under conditions where the cells typically obtained no more than single sequence variants (Heredia et al, J. Immunol.200, ji1800343-3839,2018;Park et al, J Biol chem.294,4759-4774,2019). Dimer sACE2 of cultures with wild-type 8his marker ₂ Incubated together at a sub-saturation concentration (2.5 nM). Bound sACE2 ₂ 8h and surface expressed S were stained with fluorescent antibody for flow cytometry analysis (FIG. 20A). The library expressed poorly compared to cells expressing wild-type S, indicating that many mutations were detrimental to folding and expression. Cell populations expressing S variants with reduced binding affinity to ACE2 can be clearly identified (fig. 20B). Following gating of c-myc positive cells expressing S, cells with high and low levels of binding to sACE2 were collected by Fluorescence Activated Cell Sorting (FACS) ₂ Is referred to as ACE2-High population and ACE2-Low population, respectively (FIG. 20C). During sorting, expression and sACE2 ₂ The binding signal decreased within minutes to hours, probably due to the shedding of the S1 subunit. Thus, cells were collected and pooled from three separate FACS experiments, and the sorting time was 8 hours total.

Transcripts from the sorted cells were Illumina sequenced and compared to a natural plasmid library to determine the enrichment ratio for each amino acid substitution (Fowler et al, bioenformats.27, 3430-3431,2011). Mutations expressed in S and closely binding to ACE2 were selectively enriched in ACE2-High scores (fig. 21); mutations that express but have reduced ACE2 binding are selectively enriched in ACE2-Low sorting; poorly expressed mutations were depleted from both sorted populations. By log of each possible amino acid at the residue position ₂ The enrichment ratios were averaged to calculate a position conservation score. By adding a conservation score for ACE2-High and ACE2-Low sorting, a score for surface expression was derived, indicating that the hydrophobic RBD check is strictly conserved in folding and transporting viral spikes (FIG.22A) A. The invention relates to a method for producing a fibre-reinforced plastic composite In contrast, mutation of residues on the exposed RBD surface enables S-surface expression. This is generally matched to the mutation tolerance of the protein.

For tight binding of ACE2 (e.g., S variants in the ACE2-High population), conservation of RBD residues at the ACE2 interface is enhanced, but mutation tolerance is still High (fig. 22C). Thus, sequence diversity was observed in native β coronaviruses that exhibit a high degree of diversity in ACE2 binding sites, replicated in deep mutation scans, which predicts that SARS-CoV-2 spike can tolerate a large genetic diversity of receptor binding sites to function. From this available sequence diversity, SARS-CoV-2 may be mutated to gain resistance to monoclonal antibodies or engineered decoy receptors targeting the ACE2 binding site.

Comparison of deep mutation scans on isolated RBDs displayed by Yeast surface

Two deep mutation scans were reported for isolated RBDs displayed on yeast surfaces (Starr et al, bioRxiv,2020.06.17.157982,2020;Linsky et al, bioRxiv,2020.08.03.231340,2020). The data described herein are selected from full-length S expressed in human cells, compared to the publicly available Starr et al dataset. Important residues in RBD for surface expression of human cell full length spikes are closely related to data of yeast surface display isolated RBD (fig. 22B), except for one significant region. The RBD surface opposite to ACE2 binding sites (e.g., V362, Y365 and C391) can be freely mutated for yeast surface display, but its sequence is limited in this experiment; this region of the RBD is buried by the global folding of the S subunit by linking the structural element to a closed conformation (which is the primary conformation of the S subunit and cannot bind to the receptor) (Walls et al, cell, 2020), doi 10.1016/j.cell.2020.02.058; wrapp et al, science, eabb2507,2020; cai et al, science.369,1586,2020; yao et al, cell 183 (3), 730-738.e13, 2020). The targeted mutations were used to test alanine substitutions for all cysteines in RBD alone (fig. 23). All cysteine to alanine mutations severely reduced S surface expression in the Expi293F cells, including C391A and C525A on the RBD "back side", which were neutral in yeast display scans. These differences indicate that in the case of intact spike expression on human cell membranes, there are more stringent sequence restrictions on RBD, but overall, these two data sets are very consistent.

For the dimer sACE2 ₂ In combination, the interfacial residues were more conserved in the Starr et al dataset (FIG. 22D) probably due to the three differences between the deep mutation experiments. First, ACE2 binding selection of S variants on plasma membranes appears to reflect mainly the effect of mutations on surface expression, which can almost certainly be more stringent in human cells. Yeast allows many poorly folded proteins to leak to the cell surface (Rocklin et al, science.357,168-175,2017). Second, yeast selection was performed at various sACE2 concentrations, from which apparent K was calculated _D A change; the data in this regard are very comprehensive. Since the human cell library requires a longer sorting time, and only a small fraction of the cells express spikes, sorting is performed on a single sACE2 ₂ At concentrations, a range of different binding affinities cannot be accurately and quantitatively recorded. Third, dimer sACE2 ₂ Can be geometrically complementary to trimers S densely packed on human cell membranes, such that affinity masks the effects of affinity reducing mutations. Nevertheless, it is widely believed that ACE2 binding persists after mutations at the RBD surface, and these data only indicate that mutation tolerance may be greater than that already observed by Starr et al.

Screening for S variants that preferentially bind wild-type ACE2 over engineering decoys

After proving that the ACE2 binding site of SARS-CoV-2 protein S can tolerate a number of mutations, a study was made as to whether it was possible to find a target for engineering decoy sACE2 ₂ V2.4 generates a mutation of resistance. Resistance mutations are expected to lose pair sACE2 while maintaining binding to wild-type receptors ₂ V2.4 and most likely in RBD where physical contact is made. Similar reasoning lays the foundation for deep mutation selection of isolated RBDs by yeast surface display, thereby finding escape mutations of monoclonal antibodies, and as a result predicting escape mutations in pseudovirus growth selection (Greaney et al, bioRxiv,2020.09.10.292078,2020).

To address whether escape mutations of engineering baits are likely to be found in RBDs, the S protein library was reused for specific selection. Cells expressing the library (encoding all possible substitutions in RBD) and wild type sACE2 fused to the Fc region of IgG1 ₂ And 8 his-tagged sACE2 ₂ V2.4 co-incubated at concentrations where the two proteins compete for binding (Chan et al science.4, eabc0870,2020). Immediately evident by flow cytometry on an Expi293F culture expressing an S library, the presence of cells expressing the S variant turned to preferentially bind to sACE2 ₂ V2.4, but no significant population preferentially bound to wild-type receptors (FIGS. 24A-24B). Expression may be preferential to binding to sACE2 ₂ (WT) -IgG1 or sACE2 ₂ V2.4 cells of the S variant were gated and collected by FACS (fig. 24C) and then the S transcripts were depth sequenced to determine the enrichment ratio. Two independent replicates were very identical (fig. 24D-24G). Most RBD mutations were depleted after sorting, consistent with deleterious effects on S folding and expression.

Soluble ACE2 ₂ V2.4 has three mutations of wild type ACE 2: T27Y buried within the RBD interface, and L79T and N330Y at the periphery of the interface (fig. 25A). A large number of mutations in RBD of S are selectively enriched to preferentially bind sACE2 ₂ V2.4 (fig. 25B, upper left quadrant). Although sACE2 can be found in ACE2 in close proximity to engineered mutation sites ₂ V2.4 specific mutations (in particular the S-F486 mutation adjacent to ACE2-L79 and the S-T500 mutation adjacent to ACE 2-N330), sACE2 ₂ The major hot spot for v 2.4-specific mutations was also determined in RBD loop 498-506, which contacts the region where the ACE2- α1 helix stacks with the β -hairpin motif (fig. 25A). In contrast, there is no sACE2 in RBD ₂ (WT) hot spot of specific mutation. Indeed, only a few mutations were selectively enriched to preferentially bind wild-type receptors (fig. 25B), and these putative wild-type specific mutations were found to be in abundance barely exceeding the expected noise level in the deep mutation data. Thus, in this competition assay, S and wild-type sACE2 ₂ Binding to RBD mutation ratio S and engineered sACE2 ₂ V2.4 binding is more sensitive.

To determine discovery by deep mutationWhether or not the potential wild-type ACE 2-specific mutations were authentic, rather than mispredicted due to data noise, 24S mutants selectively enriched in wild-type specific gates by targeted mutations were tested (blue data points in fig. 25B). Only binding to wild-type sACE2 was observed ₂ Is shown (fig. 26). At sACE2 ₂ Two S mutants, N501W and N501Y, which all maintained high receptor binding and showed a high binding to wild-type sACE2 in competition experiments were further studied in titration experiments ₂ Is a small movement of (a). N501 of S is located in the 498-506 ring, and substitution thereof with a bulky aromatic side chain may alter the ring conformation, resulting in interaction with sACE2 ₂ Spatial tension adjacent to ACE2 mutation N330Y in v 2.4. 8 his-labeled sACE2 at different concentrations by titration ₂ (WT) and sACE2 ₂ V2.4 and measuring binding proteins to S-expressing cells by flow cytometry, it was found that S-N501W and S-N501Y indeed show a binding to wild-type sACE2 ₂ But with a weak effect and sACE2 ₂ V2.4 is still a stronger conjugate (fig. 25C); thus, these mutations do not render the virus resistant to the engineered bait.

Dimer sACE2 ₂ Tightly combined with S protein on the surface of the membrane; in the infection assay, sACE2 was also observed ₂ Tight interactions with spikes on authentic SARS-CoV-2 (Chan et al science 4, eabc0870,2020). BLI kinetic measurement, wherein immobilized sACE2 ₂ IgG1 interactions with monomeric RBD for determining observed sACE2 ₂ How the change in tight binding to S-expressing cells translates into a change in monovalent affinity. Both the N501W and N501Y mutants of SARS-CoV-2RBD showed an increased affinity for wild-type ACE2 and engineered ACE2.V2.4, more for wild-type receptors (table 6). This is consistent with flow cytometry data, indicating that there was a small change in the specificity for wild-type ACE2, but insufficient to escape the engineering bait. In contrast, multiple independent escape mutations are readily found in S of SARS-CoV-2, which reduce the efficacy of monoclonal antibodies by orders of magnitude (Baum et al, science, eabd0831,2020; great et al, bioRxiv,2020.09.10.292078,2020).

Finally, representative mutations of 8S predicted from depth mutation scan were cloned to enhance the gene for sACE2 ₂ V2.4 (FIG. 25B), and 7 mutations were found to shift preferentially to bind sACE2 in a competition assay ₂ V2.4 (fig. 27). These S mutations are Y449K/Q/S, L455G/R/Y and G504K. Why mutation would enhance the expression of engineered sACE2 ₂ V2.4 specificity, which is not yet defined, because the RBD residues Y449, L455 and G504 are not in direct contact with the engineering site of the receptor. For immobilized sACE2 ₂ BLI kinetics between IgG1 and monomeric RBD as analyte, showing that the representative mutant RBD-Y449K was specific for wild-type and engineered sACE2 ₂ Affinity decrease of (Table 6). However, sACE2 ₂ V2.4 changes in affinity in the picomolar range were hidden during tight cell surface binding to full-length S-Y449K, whereas wild-type sACE2 ₂ The tight binding to S-Y449K (affinity measured by BLI in the medium millimolar range) is significantly reduced. This finding may explain why competing choices have found many ways to shift specificity to engineered sACE2 ₂ V2.4 mutations, because mutations that result in a small decrease in affinity may have a greater impact on tightly binding to the weak binding wild-type receptor.

Overall, this selection confirmed by verification of the targeted mutation that the specifically altered mutation in S could be successfully found. Mutations with high specificity for wild-type receptors cannot be found in RBD, which means that such mutations are rare, and may not even be present, at least within the receptor binding domain where direct physical contact with the receptor occurs. Mutations that have long Cheng Gou image effects elsewhere cannot be excluded. Thus, engineered soluble decoy receptors can be a broad therapeutic candidate for which viruses cannot easily escape.

Discussion of the invention

The attractive force of soluble decoy receptors is that viruses cannot easily mutate to escape neutralization. Mutations that reduce soluble decoy affinity may also reduce affinity for wild-type receptors on host cells at the expense of reduced infectivity and virulence. However, this assumption has not been strictly tested and is due to engineering mutagenesisUnlike its wild-type counterpart, the bait receptor, even through only a small number of mutations, has the potential to evolve to distinguish between the two. It was demonstrated herein that the engineered decoy receptor for SARS-CoV-2 is at low nanomolar K despite the high sequence diversity of the ACE2 binding site _D The spike of SARS-associated beta coronavirus, which enters cells using ACE2, is widely combined. In a comprehensive screening of all substitutions within RBD, no mutation in S was found to give high specificity for wild-type ACE 2. Thus, the engineered decoy receptor is widely resistant to human and livestock co-suffering coronaviruses that utilize ACE2 (which may spill from animal hosts in the future), as well as SARS-CoV-2 variants that may occur with the current blast of the covd-19 pandemic. Decoy receptors are unlikely to need to be combined in cocktail formulations as required by monoclonal antibodies or engineered miniprotein conjugates to prevent rapid emergence of resistance (Baum et al, science, eabd0831,2020; cao et al, science, eabd9909,2020).

Soluble decoy receptors have proven clinically effective, particularly in modulating immune responses. Etanercept (trade name)Soluble TNF receptor), abelmoschus (, A/B)>Soluble chimeras of VEGF receptors 1 and 2) and Abelip (/ i>Soluble CTLA-4) is only three examples of soluble receptors that have profound effects on treatment of human diseases (useni et al, PLoS one.12, e0181748,2017), but soluble receptors against viral pathogens have been approved as drugs. There are two main reasons for this. First, the affinity of the entry receptor for viral glycoproteins is typically moderate to low, which reduces neutralization efficacy compared to affinity matured monoclonal antibodies. For SARS-CoV-2, this problem has been solved by engineering ACE2 to have picomolar affinity for virus S (Chan et al, science.4, eabc0870,2020; glasgow et albioRxiv,2020.07.31.231746,2020; higuchi et al, bioRxiv,2020.09.16.299891,2020). Second, viral entry receptors have endogenous functions of normal physiology, and their soluble counterparts may affect such normal physiology, thereby producing unacceptable toxicity. For example, the human cytomegalovirus entry receptor is a growth factor receptor that must be knocked out for growth factor interaction to produce a virus-specific bait suitable for in vivo administration (Park et al, PLoS Pathog.16, e1008647,2020). ACE2, however, is different in this regard, its endogenous activity-catalytic conversion of vasoconstrictor and inflammatory peptides of the renin-angiotensin system and kinin system-may be directly beneficial in alleviating the symptoms of covd-19. During infection, ACE2 activity is down-regulated and the renin-angiotensin system becomes unbalanced, potentially leading to Acute Respiratory Distress Syndrome (ARDS), such that the patient requires mechanical ventilation (Imai et al, nature 436,112-116,2005;Treml et al, crit. Care med 38,596-601,2010;Verdecchia et al, eur J international med 76,14-20,2020). Administration of recombinant soluble ACE2 can restore lost biochemical activity and has potential protective properties for the lung and cardiovascular system due to proteolytic conversion of angiotensin and bradykinin, including decreasing lung elasticity, increasing blood oxygenation, decreasing hypertension, and decreasing fluid accumulation in the lung (Imai et al, nature 436,112-116,2005;Treml et al, crit. Care med.38,596-601,2010;Wang et al, pulm Pharmacol ter.58, 101833,2019; chung et al, ebiomedicine.58,102907-102907,2020;Johnson et al, PLoS one.6, e20828,2011; liu et al, kidney int.94,114-125,2018;Garvin et al, elife.9, e59177,2020). Soluble wild type ACE2 ₂ Medicaments that have been developed as ARDS are acceptable for human safety (Haschke et al, clin pharmacokinet.52,783-792,2013;Khan et al, critcae.21, 234, 2017), and are currently being evaluated by aperon in clinical trials. Engineered high affinity sACE2 ₂ Decoy, most likely fused to immunoglobulin Fc to improve serum stability (Lei et al, nat Commun.11,2070,2020; liu et al, kidney int.94,114-125,2018;Iwanaga et al, bioRxiv,2020.06.15.152157,2020), represents a dual mechanism of actionA first generation therapy: (i) Potent viral neutralization due to high affinity blocking of viral spikes, and (ii) proteolytic switching peptide hormones to directly alleviate the symptoms of covd-19.

Example 5

This example evaluates sACE ₂ V2.4 Pharmacokinetic (PK) in mice. The results indicate that sACE can be prolonged after intravenous administration by fusion with the Fc portion of human IgG1 ₂ V2.4 serum half-life. The fusion protein is proteolytically produced long-standing IgG1 fragments for longer than 7 days, while the ACE2 moiety is rapidly depleted within hours. sACE2 by Intratracheal (IT) administration or nebulization ₂ V2.4-IgG1 was delivered directly to the lung, and the protein was maintained at high levels in lung tissue for at least 4 hours with minimal proteolytic degradation. These results indicate that delivering high affinity sACE2 derivatives directly to the lungs is a viable alternative to intravenous infusion and brings potential benefits to outpatient clinical care.

Wild type human sACE2 ₂ The serum half-life was 8.5 hours when administered intraperitoneally (Wysocki et al, hypertension 55,90-98,2010), but this was affected by the kinetics of blood absorption, i.e. for macromolecules, it was usually delayed for several hours (Shoyaib et al, pharmacut res.37,12,2020). When sACE2 ₂ V2.4 (0.5 mg/kg) was injected into the tail vein of male and female mice without fusion partner, protein was cleared rapidly, serum half-life was estimated to be less than 10 minutes, as measured by ACE2 ELISA (fig. 28A) and ACE2 catalytic activity in serum (fig. 28B). This is compared to wild-type sACE2 ₂ Serum half-life in humans (2 to 3 hours) is much shorter (Haschke et al, clin Pharmacokinet 52,783-792,2013). When sACE2 ₂ V2.4 at 0.5mg/kg no toxicity was observed when administered intravenously twice daily for 5 days (days 0, 1, 2, 3 and 4). Mice were euthanized on day 7, and blood chemistry, hematology and histopathology indicated no difference from mock-treated mice.

To extend serum half-life, sACE2 was tested ₂ Fusion to IgG1 Fc. Although other groups have studied sACE2 ₂ With IgG1 mutants (Iwanaga et al, biorxiv, in press,doi 10.1101/2020.06.15.152157) or IgG4 Fc (Svilenov et al, biorxiv, in press, doi 10.1101/2020.12.06.413443) to inhibit interaction with pro-inflammatory FcgammaR, but the present study uses unmodified IgG1 (allotype nG1m 1) to recruit effector functions that have been demonstrated in the anti-SARS-CoV-2 mAb to be necessary for optimal protection J Exp Med.218 (2020), doi: 10.1084/jem.20201993. Regarding sACE2 ₂ Fusion to IgG1, published PK data is divergent. Although there is clear evidence that murine sACE2 ₂ IgG1 fusion in mice for several days (Liu et al, kidney int.94,114-125,2018), but human sACE2 ₂ The results of the IgG1 fusion are conflicting. Two reports of detection of human IgG1 fraction using ELISA indicate that sACE2 ₂ Serum half-life of IgG1 for several days (Iwanaga et al, biorxiv, in press, doi:10.1101/2020.06.15.152157;Lei et al., nat Commun.11,2070, 2020), but another study report using ELISA to detect ACE2 moieties was reported to be rapid clearance within several hours (Higuchi et al, biorxiv, in press, doi: 10.1101/2020.09.16.). Only one published report detected two parts of the fusion protein, and the long serum half-life of the human fusion protein in mice was measured using an anti-ACE 2 capture antibody and an anti-IgG 1 detection antibody (Liu et al, int J Biol macromol 165,1626-1633,2020). The reasons for the differences are not clear, but may indicate that cleavage of the fusion protein results in fragments with different serum stabilities.

Detection of human IgG1 using ELISA wild-type sACE2 after intravenous administration (2.0 mg/kg) in male mice ₂ IgG1 and sACE2 ₂ V2.4-IgG1 (SEQ ID NO: 11) showed that serum PK was identical and that protein was present for more than 7 days (FIG. 29). It was therefore concluded that high affinity sACE2 ₂ Three mutations in the v2.4 variant (T27Y, L79T and N330Y) did not significantly alter PK, consistent with previous studies on another modified sACE2 derivative (Higuchi et al, biorxiv, in press, doi: 10.1101/2020.09.16.299891). Because of the lack of materials, serum components could not be further characterized, another PK study was performed in male and female miceTo more thoroughly track sACE2 in serum ₂ How v2.4-IgG1 changes over time. Likewise, human IgG1 protein persisted in serum for several days (fig. 30A), but according to the ACE2 ELISA (fig. 30B), ACE2 fraction cleared rapidly within 24 hours. Measurement of ACE2 catalytic activity showed a faster decay (fig. 30C). Immunoblots of human IgG1 demonstrated that the fusion protein was hydrolyzed in mouse blood to release long-standing IgG1 fragments (fig. 30D). Overall, sACE2 ₂ Fusion of v2.4 with IgG1 Fc only moderately enhanced serum stability. Intravenous administration of sACE2 to mice ₂ V2.4-IgG1 (2.0 mg/kg), no toxicity was observed after 7 days for blood chemistry, hematology and histopathology analysis.

In order to improve the serum PK observed after intravenous administration, a study was conducted to deliver the protein directly to the respiratory tract, which is the major site of SARS-CoV-2 infection. Following intratracheal delivery (1.0 mg/kg), sACE2 was found by ACE2 ELISA, human IgG1ELISA and anti-human IgG1 immunoblotting ₂ V2.4-IgG1 was elevated in the lung for at least 4 hours (FIGS. 31A-31C). sACE2 absorbed into blood ₂ V2.4-IgG1 levels were too low to detect. Wild-type sACE2 as observed upon intravenous administration ₂ IgG1 and sACE2 ₂ V2.4-IgG1 the PK in the lung after intratracheal administration was identical (within experimental error). Further study of administration of sACE2 by inhalation ₂ V2.4-IgG1. In this study, the protein was nebulized into a chamber containing mice for 30 minutes. Although the dose in the nebulizer receiving chamber was lower than that achieved by intratracheal administration, it was observed that sACE2 ₂ V2.4-IgG1 remained at higher levels and relatively constant over 4 hours as measured by ACE2 ELISA, human IgG1ELISA and immunoblots (FIGS. 31D-31F). Direct delivery to the respiratory tract can achieve high protein levels in lung tissue with minimal degradation in more than 4 hours. Based on the different PK characteristics of the route of administration (e.g., protein delivered directly to the lung is present for several hours but does not reach detectable levels in plasma, whereas protein delivered intravenously reaches high but transient plasma concentrations) the following clinical opportunities are made possible: treatment of patients (possibly) Depending on the extent of disease progression or whether the infection is systemic or not), or the patient is treated using intravenous and intratracheal or inhalation routes of administration.

Example 6

This example illustrates experiments performed using SARS-CoV-2 pseudovirus to assess whether modified ACE2 polypeptides are capable of preventing viral entry into cells.

Human A549 lung epithelial cells, human A549 lung epithelial cells and human lung endothelial cells over-expressing ACE2 receptor and VSV-SARS-CoV-2-luciferase-pseudotype virus and wild-type sACE2 ₂ IgG1 or engineered sACE2 ₂ V2.4-IgG1 peptides were incubated at concentrations of 0, 5 or 25. Mu.g/ml. Each experiment contained a virus-free control; all other samples contained virus with an MOI of 0.01. Cells were harvested and the extent of viral entry was quantified based on expression of the luciferase reporter gene (fig. 32). Engineering sACE2 ₂ V.2.4-IgG1 has excellent effect of preventing SARS-CoV-2 pseudovirus from entering human lung epithelial cells and human endothelial cells.

In a second study, K18-hACE2 transgenic mice, expressing human ACE2 receptor in epithelial cells, were intravenously injected with wild type sACE2 ₂ IgG1 or sACE22.v2.4-IgG1, and intraperitoneal injection of VSV-SARS-CoV-2-luciferase pseudotype virus. Lungs and livers were harvested at 24 hours and the extent of viral entry was quantified by luciferase activity (fig. 33). Engineering sACE2 ₂ V.2.4-IgG1 has excellent effect of preventing SARS-CoV-2 pseudotyped virus from entering the lung and liver of mice expressing human ACE 2.

Taken together, these results indicate that v2.4 derivatives of soluble ACE2 more effectively prevent SARS-CoV-2 pseudovirus from entering human ACE2 expressing cells in tissue culture and animal models.

Example 7

This example describes a study aimed at studying sACE2 ₂ V2.4-IgG1 exhibits protective and/or therapeutic effects on SARS-CoV-2-induced pulmonary vascular leakage in a mouse model of COVID-19. Although specific methods are provided, those skilled in the art will recognize that the methods used may deviate from these specific methods, including the addition or omission of one or more steps。

The records of this study relate to pulmonary vascular leakage and pulmonary edema formation. The following animal groups were used for this study:

group 1 (control), 4 mice (2 males and 2 females, 2 months old).

Group 2 (SARS-CoV-2, 5x 10) ⁴ pfu/mouse for 7 days), 4 mice (2 males and 2 females, 2 months old).

Group 3 (intravenous administration (10 mg/kg) or intratracheal administration (2 mg/kg) or inhalation administration of sACE2 hours prior to SARS-CoV-2 infection) ₂ .v2.4-IgG1，5x 10 ⁴ pfu/mouse for 7 days), 4 mice (2 males and 2 females, 2 months old). The panel evaluates pre-exposure prophylaxis.

Group 4 (intravenous administration (10 mg/kg) or intratracheal administration (2 mg/kg) or inhalation administration of sACE2 after SARS-CoV-2 infection) ₂ .v2.4-IgG1，5x 10 ⁴ pfu/mouse for 7 days), 4 mice (2 males and 2 females, 2 months old). The panel assessed treatment after infection.

Administration of sACE2 to mice by one of several methods (e.g., intravenous, intratracheal, inhalation) ₂ V2.4-IgG1 polypeptide and infection of SARS-CoV-2 through the airways to mimic human pulmonary infection.

Expected sACE2 ₂ V2.4-IgG1 will alleviate SARS-CoV-2 induced pulmonary vascular leakage and slow down edema formation, a major cause of respiratory failure and death in COVID-19 patients.

Example 8

This example describes a study aimed at studying sACE2 ₂ V2.4-IgG1 exhibits protective and/or therapeutic effects on SARS-CoV-2-induced pulmonary vascular injury and long-term fibrosis in a mouse model of COVID-19. Although specific methods are provided, one of ordinary skill in the art will recognize that the methods used may deviate from these specific methods, including adding or omitting one or more steps.

The recordings of this study were made on H & E staining, masson trichromatic and sirius red staining, MPO assay and protein lysates to assess signal transfer and inflammatory pathology. The following animal groups were used for this study:

Group 1 (control), 4 mice (2 males and 2 females, 2 months old).

Group 2 (SARS-CoV-2, 5x 10) ⁴ pfu/mouse for 7 days), 4 mice (2 males and 2 females, 2 months old).

Group 3 (intravenous administration (10 mg/kg) or intratracheal administration (2 mg/kg) or inhalation administration of sACE2 prior to SARS-CoV-2 infection) ₂ .v2.4-IgG1，5x 10 ⁴ pfu/mouse for 7 days), 4 mice (2 males and 2 females, 2 months old). The panel evaluates pre-exposure prophylaxis.

Group 4 (intravenous administration (10 mg/kg) or intratracheal administration (2 mg/kg) or inhalation administration of sACE2 after SARS-CoV-2 infection) ₂ .v2.4-IgG1，5x 10 ⁴ pfu/mouse for 7 days), 4 mice (2 males and 2 females, 2 months old). The panel assessed treatment after infection.

Expected sACE2 ₂ V2.4-IgG1 reduces inflammatory injury and fibrosis in such a mouse model of COVID-19.

Example 9

This example describes a study, namely study sACE2 ₂ V2.4 (fused or unfused to IgG1 Fc) blocks spike protein of highly infectious SARS-CoV-2 variants. SARS-CoV-2 mutants have emerged, showing enhanced transmissibility and possibly toxicity. By month 3 of 2021, interesting virus variants were B.1.351 (Tegaly et al, midRxiv, in press, doi: 10.1101/2020.12.21.20248640) from south Africa, P.1 from Brazil and B.1.1.7 (Leung et al, euroscurvell area 26,2021, doi:10.2807/1560-7917.ES.2020.26.1.2002106;Volz et al, midRxiv, in press, doi: 10.1101/2020.12.30.20249034). All three virus variants have an N501Y mutation of S, increasing the monovalent affinity for wild-type ACE2 by a factor of 20 (example 4-table 6). High affinity v2.4 ACE2 derivatives also bind with enhanced affinity (example 4-table 6). The present study tested dimer sACE2 ₂ Apparent monovalent affinity and tight binding of IgG1 (wild-type and v 2.4) to full-length S variants of p.1, b.1.1.7 and b.1.351 lineages.

S proteins were expressed in human Expi293F cells, with an N-terminal c-myc tag, for surface expression measurement using fluorescent anti-myc antibodies and flow cytometry. Cells were incubated with a series of dilutions of sACE2-8his and sACE2.v2.4-8his (monomers: ACE2 residues 19-615), washed, and binding proteins were measured by flow cytometry using anti-his fluorescent antibody staining. Cells were also combined with a series of dilutions of sACE2 ₂ IgG1 and sACE2 ₂ V2.4-IgG1 (dimer: ACE2 residues 19-732) and binding proteins were measured by flow cytometry using an anti-human IgG1 fluorescent antibody. Based on the previously described deep mutations (example 4), the expected results will confirm that highly infectious viral variants remain easily tightly bound to the sACE2 engineered v2.4 derivative.

In view of the many possible embodiments to which the principles of the disclosed subject matter may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the present disclosure and should not be taken as limiting the scope of the present disclosure. Rather, the scope of the present disclosure is defined by the following claims. Accordingly, we claim all that comes within the scope and spirit of these claims.

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Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

1160 1165 1170

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

1175 1180 1185

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu

1205 1210 1215

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met

1220 1225 1230

Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro

1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 3

<211> 196

<212> PRT

<213> SARS-CoV-2 Wuhan

<400> 3

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala

1 5 10 15

Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

20 25 30

Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr

35 40 45

Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr

50 55 60

Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro

65 70 75 80

Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

85 90 95

Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys

100 105 110

Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn

115 120 125

Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly

130 135 140

Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu

145 150 155 160

Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr

165 170 175

Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val

180 185 190

Cys Gly Pro Lys

195

<210> 4

<211> 195

<212> PRT

<213> SARS-CoV-1 Wu Erba Ni

<400> 4

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro

1 5 10 15

Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp

20 25 30

Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr

35 40 45

Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr

50 55 60

Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro

65 70 75 80

Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

85 90 95

Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr

100 105 110

Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys

115 120 125

Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp

130 135 140

Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn

145 150 155 160

Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg

165 170 175

Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys

180 185 190

Gly Pro Lys

195

<210> 5

<211> 195

<212> PRT

<213> Rs4084-CoV

<400> 5

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Thr Phe Pro

1 5 10 15

Ser Val Tyr Ala Trp Glu Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

20 25 30

Tyr Ser Ile Leu Tyr Asn Ser Thr Ser Phe Ser Thr Phe Lys Cys Tyr

35 40 45

Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr

50 55 60

Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro

65 70 75 80

Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

85 90 95

Phe Leu Gly Cys Val Leu Ala Trp Asn Thr Asn Ser Lys Asp Ser Ser

100 105 110

Thr Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Trp Val Arg Arg Ser Lys

115 120 125

Leu Asn Pro Tyr Glu Arg Asp Leu Ser Asn Asp Ile Tyr Ser Pro Gly

130 135 140

Gly Gln Ser Cys Ser Ala Val Gly Pro Asn Cys Tyr Asn Pro Leu Arg

145 150 155 160

Pro Tyr Gly Phe Phe Thr Thr Ala Gly Val Gly His Gln Pro Tyr Arg

165 170 175

Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys

180 185 190

Gly Pro Lys

195

<210> 6

<211> 195

<212> PRT

<213> RsSHC014-CoV

<400> 6

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Thr Phe Pro

1 5 10 15

Ser Val Tyr Ala Trp Glu Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

20 25 30

Tyr Ser Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr Phe Lys Cys Tyr

35 40 45

Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr

50 55 60

Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro

65 70 75 80

Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

85 90 95

Phe Leu Gly Cys Val Leu Ala Trp Asn Thr Asn Ser Lys Asp Ser Ser

100 105 110

Thr Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Trp Val Arg Arg Ser Lys

115 120 125

Leu Asn Pro Tyr Glu Arg Asp Leu Ser Asn Asp Ile Tyr Ser Pro Gly

130 135 140

Gly Gln Ser Cys Ser Ala Val Gly Pro Asn Cys Tyr Asn Pro Leu Arg

145 150 155 160

Pro Tyr Gly Phe Phe Thr Thr Ala Gly Val Gly His Gln Pro Tyr Arg

165 170 175

Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys

180 185 190

Gly Pro Lys

195

<210> 7

<211> 195

<212> PRT

<213> Rs4231-CoV

<400> 7

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Thr Phe Pro

1 5 10 15

Ser Val Tyr Ala Trp Glu Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

20 25 30

Tyr Ser Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr Phe Lys Cys Tyr

35 40 45

Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr

50 55 60

Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro

65 70 75 80

Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

85 90 95

Phe Leu Gly Cys Val Leu Ala Trp Asn Thr Asn Ser Lys Asp Ser Ser

100 105 110

Thr Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Trp Val Arg Arg Ser Lys

115 120 125

Leu Asn Pro Tyr Glu Arg Asp Leu Ser Asn Asp Ile Tyr Ser Pro Gly

130 135 140

Gly Gln Ser Cys Ser Ala Ile Gly Pro Asn Cys Tyr Asn Pro Leu Arg

145 150 155 160

Pro Tyr Gly Phe Phe Thr Thr Ala Gly Val Gly His Gln Pro Tyr Arg

165 170 175

Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys

180 185 190

Gly Pro Lys

195

<210> 8

<211> 195

<212> PRT

<213> LYRa11-CoV

<400> 8

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Thr Phe Pro

1 5 10 15

Ser Val Tyr Ala Trp Glu Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

20 25 30

Tyr Ser Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr Phe Lys Cys Tyr

35 40 45

Gly Val Ser Ala Ile Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr

50 55 60

Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro

65 70 75 80

Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

85 90 95

Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr

100 105 110

Ser Ser Gly Asn Phe Asn Tyr Lys Tyr Arg Ser Leu Arg His Gly Lys

115 120 125

Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp

130 135 140

Gly Lys Pro Cys Thr Pro Pro Ala Phe Asn Cys Tyr Trp Pro Leu Asn

145 150 155 160

Asp Tyr Gly Phe Tyr Thr Thr Asn Gly Ile Gly Tyr Gln Pro Tyr Arg

165 170 175

Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys

180 185 190

Gly Pro Lys

195

<210> 9

<211> 195

<212> PRT

<213> Rs7327-CoV

<400> 9

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Thr Phe Pro

1 5 10 15

Ser Val Tyr Ala Trp Glu Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

20 25 30

Tyr Ser Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr Phe Lys Cys Tyr

35 40 45

Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr

50 55 60

Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro

65 70 75 80

Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

85 90 95

Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr

100 105 110

Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Ser Leu Arg His Gly Lys

115 120 125

Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp

130 135 140

Gly Lys Pro Cys Thr Pro Pro Ala Phe Asn Cys Tyr Trp Pro Leu Asn

145 150 155 160

Asp Tyr Gly Phe Phe Thr Thr Asn Gly Ile Gly Tyr Gln Pro Tyr Arg

165 170 175

Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys

180 185 190

Gly Pro Lys

195

<210> 10

<211> 714

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 10

Ser Thr Ile Glu Glu Gln Ala Lys Tyr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Thr Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg Pro

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Tyr Ser Met Leu Thr Asp Pro Gly Asn

305 310 315 320

Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys Asn

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys Ser

595 600 605

Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr Leu

610 615 620

Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys Val

625 630 635 640

Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala Asn

645 650 655

Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys Asn

660 665 670

Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg Met

675 680 685

Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser Leu

690 695 700

Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly

705 710

<210> 11

<211> 942

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 11

Ser Thr Ile Glu Glu Gln Ala Lys Tyr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Thr Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg Pro

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Tyr Ser Met Leu Thr Asp Pro Gly Asn

305 310 315 320

Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys Asn

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys Ser

595 600 605

Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr Leu

610 615 620

Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys Val

625 630 635 640

Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala Asn

645 650 655

Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys Asn

660 665 670

Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg Met

675 680 685

Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser Leu

690 695 700

Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Ser Asp Lys Thr His Thr

705 710 715 720

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

725 730 735

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

740 745 750

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

755 760 765

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

770 775 780

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

785 790 795 800

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

805 810 815

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

820 825 830

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

835 840 845

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

850 855 860

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

865 870 875 880

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

885 890 895

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

900 905 910

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

915 920 925

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

930 935 940

Claims (48)

1. A modified angiotensin converting enzyme 2 (ACE 2) polypeptide comprising human ACE2 or a fragment thereof, wherein the polypeptide comprises at least one amino acid substitution relative to wild type human ACE2 of SEQ ID No. 1 and has enhanced S protein binding to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relative to wild type human ACE 2.

2. The modified polypeptide of claim 1, wherein the at least one amino acid substitution is a substitution selected from the group consisting of: t27 79 79 330 19 24 26 26 27 27 27 27 27 27 30 26 27 27 30 30 30 27 34 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 79 79 82 89 89 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 3 communication 3.3 communication 3 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386 386.

3. The modified polypeptide of claim 1, wherein the at least one amino acid substitution is a substitution selected from the group consisting of: 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 92 90 90 330 330 330 330F of the method of T27 79 79 and 330 of the method of 92 92 92 92 92 92 92 92 92 324 324.

4. The modified polypeptide of claim 1, wherein the at least one amino acid substitution is a substitution selected from the group consisting of: t27 79 330 27 27 34 34 79 79 79 79 79 90 90 90 90 90 90 90 90 92 92 92 92 92 92 92 92 92 92 92 92 92 92 92 92 92 92 92 92 92 92 92 92 324 330F and a386L.

5. The modified polypeptide of claim 1, wherein the at least one amino acid substitution is at residue 19, 23, 24, 25, 26, 27, 29, 30, 31, 33, 34, 35, 39, 40, 41, 42, 65, 69, 72, 75, 76, 79, 82, 89, 90, 91, 92, 324, 325, 330, 351, 386, 389, 393, or 518 of SEQ ID No. 1 human ACE 2.

6. The modified polypeptide of claim 5, wherein the at least one amino acid substitution is a substitution selected from the group consisting of: T27Y, L79Y, L330Y, L19Y, L25Y, L29Y, L33Y, L39Y, L40Y, L69Y, L72Y, L76Y, L89Y, L5297Y, L324Y, L324Y, L325Y, L518Y, L351Y, L386Y, L24Y, L27Y, L30Y, L31Y, L34Y, L41Y, L42Y, L42Y, L75Y, L79Y, L5297Y, L H92Y, L H and R393K.

7. The modified polypeptide of any one of claims 1-6, wherein the at least one amino acid substitution removes the glycosylation motifs at the human ACE2 residues N90, L91, and T92 of SEQ ID No. 1.

8. The modified polypeptide of any one of claims 1-6, comprising:

T27Y, L T and N330Y amino acid substitutions;

amino acid substitutions H34A, T92Q, Q P and a 386L;

T27Y, L79T, N Y and a386L amino acid substitutions;

L79T, N amino acid substitutions of 330Y and A386L;

T27Y, N Y and a386L amino acid substitutions;

T27Y, L T and a386L amino acid substitutions;

amino acid substitutions a25V, T27Y, T Q, Q325P and a 386L;

amino acid substitutions H34A, L79T, N Y and a 386L;

amino acid substitutions a25V, T92Q and a 386L; or alternatively

Amino acid substitutions T27Y, Q42L, L T, T92Q, Q P, N Y and A386L,

wherein said amino acid substitution is referred to SEQ ID NO. 1.

9. The modified polypeptide of any one of claims 1-6 having a single amino acid substitution relative to human ACE2 of SEQ ID No. 1.

10. The modified polypeptide of any one of claims 1-9, comprising full length human ACE2 and comprising at least one amino acid substitution relative to wild type human ACE 2.

11. The modified polypeptide of claim 10, wherein the amino acid sequence of the polypeptide is at least 95% identical to SEQ ID No. 1.

12. The modified polypeptide of claim 10, wherein the amino acid sequence of the polypeptide is at least 99% identical to SEQ ID No. 1.

13. The modified polypeptide of any one of claims 1-9, wherein the polypeptide consists of a fragment of human ACE 2.

14. The modified polypeptide of claim 13, wherein the fragment of human ACE2 is an extracellular fragment.

15. The modified polypeptide of claim 14, wherein the extracellular fragment corresponds to residues 19 to 615 of human ACE2 of SEQ ID No. 1.

16. The modified polypeptide of claim 14, wherein the extracellular fragment corresponds to human ACE2 residues 20 to 615 of SEQ ID No. 1.

17. The modified polypeptide of any one of claims 14-16, wherein the amino acid sequence of the extracellular fragment is at least 95% identical to residues 19 to 615 of SEQ ID No. 1.

18. The modified polypeptide of any one of claims 14-17, wherein the amino acid sequence of the extracellular fragment is at least 99% identical to residues 19 to 615 of SEQ ID No. 1.

19. The modified polypeptide of claim 13, wherein the fragment corresponds to residues 1-732, 19-732 or 19-740 of human ACE2 of SEQ ID No. 1.

20. The modified polypeptide of claim 19, wherein the fragment corresponds to residues 19-732 of human ACE2 of SEQ ID No. 1.

21. The modified polypeptide of claim 20, wherein the amino acid sequence of the fragment consists of SEQ ID No. 10.

22. The modified polypeptide of any one of claims 1-21, wherein the polypeptide forms a dimer.

23. A fusion protein comprising the modified polypeptide of any one of claims 1-22 and a heterologous polypeptide.

24. The fusion protein of claim 23, wherein the heterologous polypeptide is an Fc protein.

25. The fusion protein of claim 24, wherein the Fc protein is a human Fc protein.

26. The fusion protein of claim 25, wherein the human Fc protein is human IgG1 Fc.

27. The fusion protein according to any one of claims 23-26, wherein the amino acid sequence of the fusion protein comprises or consists of SEQ ID No. 11.

28. The fusion protein of claim 23, wherein the heterologous polypeptide is a fluorescent protein, an enzyme, an antibody or antigen binding protein, a cytokine, a cell ligand or receptor, or serum albumin.

29. A composition comprising the modified polypeptide of any one of claims 1-22, or the fusion protein of any one of claims 23-28, and a pharmaceutically acceptable carrier.

30. The composition of claim 29, formulated for intratracheal or inhalation administration.

31. An in vitro method of inhibiting replication of a coronavirus (CoV), comprising contacting the CoV with the modified polypeptide of any one of claims 1-22 or the fusion protein of any one of claims 23-28.

32. A method of inhibiting replication and/or transmission of a coronavirus (CoV) in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the modified polypeptide of any one of claims 1-22, the fusion protein of any one of claims 23-28, or the composition of claim 29 or 30, thereby inhibiting replication and/or transmission of CoV in the subject.

33. The method of claim 32, comprising administering the modified polypeptide or fusion protein intravenously, intratracheally, or by inhalation.

34. The method of claim 33, wherein the modified polypeptide or fusion protein is administered by inhalation using a nebulizer.

35. A nucleic acid molecule encoding the modified polypeptide of any one of claims 1-22 or the fusion protein of any one of claims 23-28.

36. A vector comprising the nucleic acid molecule of claim 35.

37. A composition comprising the nucleic acid molecule of claim 35 or the vector of claim 36, and a pharmaceutically acceptable carrier.

38. A method of inhibiting replication and/or transmission of a coronavirus (CoV) in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the nucleic acid molecule of claim 35, the vector of claim 36, or the composition of claim 37, thereby inhibiting replication and/or transmission of CoV in the subject.

39. The method of any one of claims 32-34 and 38, wherein the subject is (i) a healthcare worker; (ii) a patient positive for coronavirus; (iii) a patient with covd-19; (iv) an aged or potentially diseased subject; (iv) a subject exposed to coronavirus.

40. The method of claim 38 or 39, wherein the nucleic acid molecule, vector or composition is administered intravenously, intratracheally or by inhalation.

41. A method of detecting coronavirus (CoV) in a biological sample, comprising:

contacting the biological sample with the modified polypeptide of any one of claims 1-22 or the fusion protein of any one of claims 23-28; and

detecting binding of the modified polypeptide or fusion protein to the biological sample, thereby detecting coronavirus in the biological sample.

42. The method of claim 41, wherein the biological sample is blood, saliva, sputum, nasal swab, or bronchoalveolar lavage sample.

43. The method of any one of claims 31-34 and 38-42, wherein the coronavirus is a human coronavirus.

44. The method of claim 43, wherein the human coronavirus is Severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, middle east respiratory syndrome coronavirus (MERS-CoV), human coronavirus HKU1 (HKU 1-CoV), human coronavirus OC43 (OC 43-CoV), human coronavirus 229E (229E-CoV), or human coronavirus NL63 (NL 63-CoV).

45. The method of any one of claims 31-34 and 38-42, wherein the coronavirus is a human-animal co-patient coronavirus.

46. The method of claim 45, wherein the human-animal co-patient coronavirus is a bat coronavirus or a rodent coronavirus.

47. The method of claim 46, wherein said baton coronavirus is LYRa11, rs4231, rs7327, rs4084, or RsSHC014.

48. A kit comprising the modified polypeptide of any one of claims 1-22, or the fusion protein of any one of claims 23-28, bound to a solid support.