ENGINEERED RECEPTORS AND MONOCLONAL ANTIBODIES FOR CORONAVIRUSES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/244,669, filed September 15, 2021; and to U.S. Provisional Patent Application No. 63/293,550, filed December 23, 2021, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on September 14, 2022, is named CYR-OOlWO.xml and is 83,277 bytes in size.

FIELD OF THE DISCLOSURE

[0003] This disclosure concerns modified angiotensin-converting enzyme 2 (ACE2) proteins with enhanced folding and increased binding to SARS-CoV-2 and other coronaviruses that use ACE2 as a cell entry receptor. The present disclosure also relates generally to the combination of modified ACE2 proteins with one or more than one monoclonal antibody.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0004] This invention was made with government support under grant number 5R01AI129719-03 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0005] Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of COVID- 19 and responsible for a global pandemic of extraordinary economic, social, and public health impact. Cell attachment and entry of SARS-CoV-2 is mediated by physical association with Angiotensin-Converting Enzyme 2 (ACE2), although other cell surface factors facilitate the process and may act as co-receptors. ACE2 is expressed on the surface of many tissue types and is especially prevalent on epithelia and endothelia of the lung. SARS- CoV-2 infection leads to down-regulation of ACE2 receptor expression, which may in turn adversely affect pathology.

[0006] Coronaviruses (CoV) are pleomorphic RNA viruses that contain crown-shaped peplomers. Like all Coronaviruses, SARS-CoV-2 has a homotrimeric Spike (S) glycoprotein on the viral surface that mediates receptor recognition and undergoes conformational changes to drive fusion of the viral envelope with a host cell membrane. Protein S is proteolytically processed by host proteases, Furin, Cathepsin L, and TMPRSS2, into non-covalently associated SI and S2 subunits. SI is soluble and contains the Receptor Binding Domain (RBD) which associates with ACE2. SI may be shed from the virus surface during the infection process. S2 is membrane-anchored and contains a fusion peptide (FP) motif responsible for fusing viral and host membranes.

[0007] As observed for many viral spikes, SARS-CoV-2 S protein is dynamic and exists in multiple conformations. The RBD binds to the N-terminal, aminopeptidase domain of ACE2 with a dissociation constant (KD) of ~20 nM. Blockade of the ACE2-RBD interaction prevents infection and is a major objective in COVID-19 drug development.

[0008] SARS-CoV-2 variants have emerged with increased transmissibility and virulence due to multiple mutations in S, including changes in immunodominant epitopes that cause partial immune escape 1,2,3 and substitutions within the RBD that can increase ACE2 affinity.

[0009] Recently, a SARS-CoV-2 variant designated omicron by the World Health Organization emerged in southern Africa and is on a trajectory to rapidly replace delta as the dominant variant in human circulation. Omicron is substantially different from original virus isolates, with 32 mutations in the Spike and 10 mutations localized to the ACE2 interaction surface, in addition to mutations in other viral proteins 16. Omicron has markedly increased escape from neutralization by sera from vaccinated and previously infected individuals. Furthermore, monoclonal antibodies in the clinic or in clinical development have substantially reduced neutralization efficacy against omicron, with the notable exception of VIR-7831 which is only moderately impacted. This has immense repercussions for therapy and substantially sets back efforts to mitigate COVID-19 disease.

[0010] Soluble ACE2 (sACE2) is an investigational drug that inhibits virus infection. The sACE2 decoy receptor, containing just the soluble extracellular region of ACE2, sequesters viral spikes and prevents their interaction with native ACE2 receptors. Due to their close resemblance with native receptors, ACE2-based decoys are unlikely to lose effectiveness as SARS-CoV-2 variants evolve; if the virus no longer binds the decoy tightly, it will most likely also have reduced affinity for the native receptor and be attenuated. These decoys may also act as therapeutics against any other CoV that uses ACE2 for entry, such as in a potential SARS-associated betacoronavirus outbreak.

[0011] Monoclonal antibodies (mAbs) have been developed to target specific antigens for the treatment of disease for over 40 years. Antiviral antibodies have been developed against several viruses known to have damaging and often fatal effects in humans, including H7N9, MERS-CoV, Dengue virus, cytomegalovirus, rotavirus, and zika virus NS1(36-41).

[0012] Despite the very recent emergence of SARS-CoV-2, therapeutic progress has been swift and mAb therapies targeting SARS-CoV-2 have been developed. For example, U.S. Food & Drug Administration Emergency Use Authorization (EUA) has been granted to a cocktail containing two mAbs, REGN10987 (imdevimab) and REGN10933 (casirivimab), that bind non-overlapping sites on the SARS-CoV-2 RBD and block ACE2 interactions.

EUA has also been granted to another cocktail containing two mABS, LY-C0VOI6 (etesevimab) and LY-CoV555 (bamlanivimab), but EUA has since been revoked.

SUMMARY OF THE DISCLOSURE

[0013] Described herein are modified human ACE2 polypeptides that exhibit enhanced binding to the S protein of SARS-CoV-2, either through enhanced folding and structural stabilization of ACE2, elimination of a glycan modification, or increased affinity. The modified polypeptides can be used as therapeutic agents in combination with monoclonal antibodies or antigen-binding fragments thereof for the prophylaxis (pre- or post-exposure prophylaxis), or treatment of COVID-19, or disease caused by any coronavirus that utilizes ACE2 as a cellular receptor. The combination of modified polypeptides with monoclonal antibodies prevents the likelihood of viral mutational escape and provides synergistic or additive activity to neutralize infection.

[0014] In certain embodiments, the present disclosure provides a method of inhibiting coronavirus (CoV) replication comprising administering to a subject a therapeutically or prophylactically effective amount of: (i) a modified angiotensin-converting enzyme 2 (ACE2) polypeptide, comprising a 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 increased binding to the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relative to wild-type human ACE2, a fusion protein comprising the modified ACE2 polypeptide, or fragment thereof, fused to a heterologous peptide, or a nucleic acid molecule encoding the modified ACE2 polypeptide or fragment thereof, or the fusion protein or fragment thereof; and (ii) a monoclonal antibody, or antigen-binding fragment thereof, that binds a coronavirus, thereby inhibiting CoV replication in the subject.

[0015] In certain embodiments, the present disclosure provides a method of inhibiting CoV replication in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a bispecific fusion protein comprising: (i) a modified ACE2 polypeptide, comprising a 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 increased binding to the S protein of SARS-CoV-2 relative to wildtype human ACE2, and (ii) a monoclonal antibody, or antigen-binding fragment thereof, that binds a coronavirus, thereby inhibiting CoV replication in the subject.

[0016] In some embodiments the monoclonal antibody, or antigen-binding fragment thereof comprises a single chain variable fragment (scFv) that binds a coronavirus.

[0017] In some embodiments, modified ACE2 polypeptide comprises at least one amino acid substitution at residue position 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, with reference to SEQ ID NO: 1.

[0018] In some embodiments, the modified ACE2 polypeptide comprises at least one amino acid substitution selected from the group consisting of: T27Y, L79T, N330Y, S19P, E23F, Q24T, A25V, K26I, K26A, K26D, T27M, T27L, T27A, T27D, T27K, T27H, T27W, T27F, T27C, L29F, D30I, D30E, K31W, K31Y, N33D, H34V, H34A, H34S, H34P, E35V, E35C, L39K, L39R, F40D, F40R, Y41R, Q42M, Q42L, Q42I, Q42V, Q42K, Q42C, A65W, W69I, W69V, I69T, I69K, F72Y, E75A, E75S, E75T, E75K, E75R, E75W, E75G, Q76M, Q76I, Q76V, Q76T, Q76R, Q76Y, L79I, L79V, L79W, L79Y, L79F, L79P, M82C, Q89I, Q89D, Q89P, N90M, N90L, N90I, N90V, N90A, N90S, N90T, N90Q, N90D, N90E, N90K, N90R, N90H, N90W, N90Y, N90F, N90P, N90G, N90C, L91P, T92M, T92L, T92I, T92V, T92A, T92N, T92Q, T92D, T92E, T92K, T92R, T92H, T92W, T92Y, T92F, T92P, T92G, T92C, T324E, T324P, Q325P, N330L, N330H, N330W, N330F, L351F, A386L, A386I, P389D, R393K and R518G, with reference to SEQ ID NO: 1.

[0019] In some embodiments, the modified ACE2 polypeptide comprises at least one amino acid substitution selected from the group consisting of: T27Y, L79T, N330Y, S19P, A25V, T27M, T27L, T27A, T27D, T27H, T27W, T27F, T27C, D30E, K31W, H34V, H34A, H34P, L39K, L39R, Q42M, Q42L, Q42C, W69V, F72Y, E75K, E75R, Q76V, Q76T, L79I, L79V, L79W, L79Y, L79F, Q89P, N90M, N90L, N90I, N90V, N90A, N90S, N90T, N90Q, N90D, N90E, N90K, N90R, N90H, N90P, N90G, N90C, L91P, T92M, T92L, T92I, T92V, T92A, T92N, T92Q, T92D, T92E, T92K, T92R, T92H, T92W, T92Y, T92F, T92P, T92G, T92C, T324E, T324P, Q325P, N330L, N330H, N330W, N330F, L351F and A386L, with reference to SEQ ID NO: 1.

[0020] In some embodiments, the modified ACE2 polypeptide comprises at least one amino acid substitution selected from the group consisting of: T27Y, L79T, N330Y, A25V, T27M, T27L, K31W, H34V, H34A, H34P, Q42L, Q42C, L79I, L79V, L79W, L79Y, L79F, N90A, N90S, N90T, N90Q, N90E, N90H, L91P, T92M, T92L, T92I, T92V, T92N, T92Q, T92D, T92E, T92R, T92H, T92W, T92Y, T92F, T92G, T92C, T324P, Q325P, N330H, N330W, N330F and A386L, with reference to SEQ ID NO: 1.

[0021] In some embodiments, the modified ACE2 polypeptide comprises at least one amino acid substitution selected from the group consisting of: T27Y, L79T, N330Y, S19P, A25V, K26D, L29F, N33D, L39R, F40D, W69V, F72Y, Q76T, Q89P, L91P, T324P, T324E, Q325P, R518G, L351F, A386L, Q24T, T27H, D30E, K31Y, H34A, Y41R, Q42L, Q42K, E75K, L79V, N90Q, T92Q, N330H and R393K, with reference to SEQ ID NO: 1.

[0022] In some embodiments, the modified ACE2 polypeptide comprises at least one amino acid substitution that removes a glycosylation motif at residues N90, L91, and T92 of human ACE2, with reference to SEQ ID NO: 1. [0023] In some embodiments, the modified ACE2 polypeptide comprises amino acid substitutions selected from: (i) T27Y, L79T, and N330Y; (ii) H34A, T92Q, Q325P, and 386L; (iii) T27Y, L79T, N330Y, and A386L; (iv) L79T, N330Y, and A386L; (v) T27Y, N330Y, and A386L; (vi) T27Y, L79T, and A386L; (vi) A25V, T27Y, T92Q, Q325P, and A386L; (vii) H34A, L79T, N330Y, and A386L; (viii) A25V, T92Q, and A386L; or (ix) T27Y, Q42L, L79T, T92Q, Q325P, N330Y, and A386L, wherein the amino acid substitutions are with reference to SEQ ID NO: 1.

[0024] In some embodiments, the modified ACE2 polypeptide comprises a single amino acid substitution relative to human ACE2 with reference to SEQ ID NO: 1.

[0025] In some embodiments, the modified ACE2 polypeptide comprises full-length human ACE2 with reference to SEQ ID NO: 1.

[0026] In some embodiments, the modified ACE2 polypeptide has an amino acid sequence at least 95%, or at least 99% identical to SEQ ID NO: 1.

[0027] In some embodiments, the modified ACE2 polypeptide consists of a fragment of human ACE2. In some embodiments, the fragment of human ACE2 is an extracellular fragment. In some embodiments, the fragment of human ACE2 corresponds to residues 19 to 615 of human ACE2 with reference to SEQ ID NO: 1. In some embodiments, the fragment of human ACE2 corresponds to residues 20 to 615 of human ACE2 with reference to SEQ ID NO: 1. In some embodiments, the fragment of human ACE2 has an amino acid sequence at least 95%, or at least 99%, identical to residues 19 to 615 SEQ ID NO: 1.

[0028] In some embodiments, the fragment corresponds to residues 1 to 732, 19 to 732, or 19 to 740, of human ACE2 with reference to SEQ ID NO: 1.

[0029] In some embodiments, the fragment has an amino acid sequence consisting of SEQ ID NO: 10.

[0030] In some embodiments, the modified ACE2 polypeptide forms a dimer.

[0031] In some embodiments, the heterologous polypeptide is an Fc protein. In some embodiments, the Fc protein is a human Fc protein. In some embodiments, the human Fc protein is a human IgGl Fc protein. [0032] In some embodiments, the modified ACE2 polypeptide consists of a fragment of human ACE2. In some embodiments, the fragment corresponds to residues 1 to 732, 19 to 732, or 19 to 740, of human ACE2 with reference to SEQ ID NO: 1. In some embodiments, the fragment has an amino acid sequence consisting of SEQ ID NO: 10. In some embodiments, the fusion protein has an amino acid sequence comprising or consisting of SEQ ID NO: 11.

[0033] In some embodiments, the heterologous polypeptide is a fluorescent protein, an enzyme, an antibody or antigen-binding protein, a cytokine, a cellular ligand or receptor, or serum albumin.

[0034] In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, binds to a SARS-CoV-2 spike protein.

[0035] In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, comprises, according the EU numbering scheme, a heavy chain complementarity - determining region 1 (VHCDR1), a heavy chain complementarity-determining region 2 (VHCDR2), a heavy chain complementarity-determining region 3 (VHCDR3), a light chain complementarity-determining region 1 (VLCDR1), a light chain complementaritydetermining region 1 (VLCDR2), and a light chain complementarity-determining region 1 (VLCDR3), each comprising an amino acid sequence corresponding to the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 sequences of a heavy chain variable domain and light chain variable domain listed in Table 1, respectively.

[0036] In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain having an amino acid sequence at least 90% identical to a heavy chain variable domain sequence listed in Table 1, and a light chain variable domain having an amino acid sequence at least 90% identical to a light chain variable domain sequence listed in Table 1.

[0037] In some embodiments, the monoclonal antibody comprises a heavy chain having an amino acid sequence at least 90% identical to a heavy chain sequence listed in Table 1, and a light chain having an amino acid sequence at least 90% identical to a light chain sequence listed in Table 1. [0038] In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, is multispecific. In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, is bispecific.

[0039] In some embodiments, the modified ACE2 polypeptide, the fusion protein, the bispecific fusion protein, or the nucleic acid molecule, is administered to the subject as a single dose, or as two or more doses.

[0040] In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, is administered to the subject as a single dose or as two or more doses.

[0041] In some embodiments, the modified ACE2 polypeptide, the fusion protein, or the nucleic acid molecule, is administered to the subject concurrently with the monoclonal antibody, or antigen-binding fragment thereof.

[0042] In some embodiments, the modified ACE2 polypeptide, the fusion protein, or the nucleic acid molecule, is administered to the subject at least 1 hour before the monoclonal antibody, or antigen-binding fragment thereof, is administered to the subject.

[0043] In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, is administered to the subject at least 1 hour before the modified ACE2 polypeptide, the fusion protein, or the nucleic acid molecule, is administered to the subject.

[0044] In some embodiments, the modified ACE2 polypeptide, the fusion protein, the bispecific fusion protein, or the nucleic acid molecule, is administered to the subject intravenously, intratracheally, or by inhalation. In some embodiments, the modified ACE2 polypeptide, the fusion protein, the bispecific fusion protein, or the nucleic acid molecule, is administered by inhalation using a nebulizer.

[0045] In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, is administered to the subject subcutaneously, intravenously, or intramuscularly.

[0046] In some embodiments, the coronavirus is a human coronavirus. In some embodiments, the human coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus HKU1 (HKUl-CoV), human coronavirus OC43 (OC43-CoV), human coronavirus 229E (229E-CoV), or human coronavirus NL63 (NL63-CoV). [0047] In some embodiments, the coronavirus is a zoonotic coronavirus. In some embodiments, is a bat coronavirus or a rodent coronavirus. In some embodiments, the bat coronavirus is LYRal l, Rs4231, Rs7327, Rs4084 or RsSHC014.

[0048] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising: (i) a modified ACE2 polypeptide, comprising a 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 increased binding to the S protein of SARS-CoV-2 relative to wild-type human ACE2, a fusion protein comprising the modified ACE2 polypeptide, or fragment thereof, fused to a heterologous peptide, or a nucleic acid molecule encoding the modified ACE2 polypeptide or the fusion protein; (ii) a monoclonal antibody, or antigen-binding fragment thereof, that binds SARS-CoV-2; and (iii) a pharmaceutically acceptable carrier.

[0049] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising: (i) a bispecific protein comprising a modified angiotensinconverting enzyme 2 (ACE2) polypeptide, comprising a 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 increased binding to the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relative to wild-type human ACE2, and a monoclonal antibody, or antigen-binding fragment thereof, that binds a coronavirus, and (ii) a pharmaceutically acceptable carrier.

[0050] The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

[0051] Other embodiments and details of the disclosure are presented herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIGS. 1A-1D: A selection strategy for ACE2 variants with high binding to the RBD of SARS-CoV-2 S. (FIG. 1 A) Media from Expi293F cells secreting the SARS- CoV-2 S-RBD fused to sfGFP was collected and incubated at different dilutions with Expi293F cells expressing myc-tagged ACE2. Bound S-RBD-sfGFP was measured by flow cytometry. The dilutions of S-RBD-sfGFP-containing medium used for FACS selections are indicated by arrows. (FIGS. 1B-1C) Expi293F cells were transiently transfected with wild type ACE2 plasmid diluted with a large excess of carrier DNA. Under these conditions, cells typically acquire no more than one coding plasmid and most cells are negative. Cells were incubated with S-RBD-sfGFP-containing medium and co-stained with fluorescent anti-myc to detect surface ACE2 by flow cytometry. During analysis, the top 67% were chosen from the ACE2-positive population (FIG. IB). Bound S-RBD was subsequently measured relative to surface ACE2 expression (FIG. 1C). (FIG. ID) Expi293F cells were transfected with an ACE2 single site-saturation mutagenesis library and analyzed as in FIG. IB. During FACS, the top 15% of cells with bound S-RBD relative to ACE2 expression were collected (nCoV- 15 S-High sort) and the bottom 20% were collected separately (nCoV-S-Low sort).

[0053] FIG. 2: A mutational landscape of ACE2 for high binding signal to the RBD of SARS-CoV-2 S. Log2 enrichment ratios from the nCoV-S-High sorts are plotted from < -3 (i.e. depleted/deleterious) to neutral to > +3 (i.e. enriched). ACE2 primary structure is on the vertical axis, amino acid substitutions are on the horizontal axis. *, stop codon.

[0054] FIGS. 3A-3F: Data from independent replicates show close agreement.

(FIGS. 3A-3B) Log2 enrichment ratios for ACE2 mutations in the nCoV-S-High (FIG. 3 A) and nCoV-S-Low (FIG. 3B) sorts closely agree between two independent FACS experiments. Replicate 1 used a 1/40 dilution and replicate 2 used a 1/20 dilution of S-RBD-sfGFP- containing medium. R ² values are for nonsynonymous mutations. (FIG. 3C) Average log2 enrichment ratios tend to be anticorrelated between the nCoV-S-High and nCoV-S-Low sorts. Nonsense mutations and a small number of nonsynonymous mutations are not expressed at the plasma membrane and are depleted from both sort populations (i.e. fall below the diagonal). (FIGS. 3D-3F) Correlation plots of residue conservation scores from replicate nCoV-S-High (FIG. 3D) and nCoV-S-Low (FIG. 3E) sorts, and from the averaged data from both nCoV-S-High sorts compared to both nCoV-S-Low sorts (FIG. 3F). Conservation scores are calculated from the mean of the log2 enrichment ratios for all amino acid substitutions at each residue position.

[0055] FIGS. 4A-4C: Sequence preferences of ACE2 residues for high binding to the RBD of SARS-CoV-2 S. (FIG. 4A) Conservation scores from the nCoV-S-High sorts are mapped to the cryo-EM structure (PDB 6M17) of S-RBD bound ACE2 (surface). The view at left is looking down the substrate-binding cavity, and only a single protease domain is shown for clarity. (FIG. 4B) Average hydrophobicity-weighted enrichment ratios are mapped to the RBD-bound ACE2 structure. (FIG. 4C) A magnified view of part of the ACE2.

Accompanying heatmap plots log2 enrichment ratios from the nCoV-S-High sort for substitutions of ACE2-T27, D30 and K31 from < -3 (depleted) to > +3 (enriched).

[0056] FIGS. 5A-5C: Single amino acid substitutions in ACE2 predicted from the deep mutational scan to increase RBD binding have small effects. (FIG. 5 A) Expi293F cells expressing full length ACE2 were stained with RBD-sfGFP-containing medium and analyzed by flow cytometry. Data are compared between wild type ACE2 and a single mutant (L79T). Increased RBD binding is most discernable in cells expressing low levels of ACE2 (smaller gate). In this experiment, ACE2 has an extracellular N-terminal myc tag upstream of residue S19 that is used to detect surface expression. (FIG. 5B) RBD-sfGFP binding was measured for 30 amino acid substitutions in ACE2. Data are the relative change in GFP mean fluorescence in the low expression gate with background fluorescence subtracted. Mean of n = 2, error bars represent range. (FIG. 5C) Relative RBD-sfGFP binding measured for the total ACE2-positive population (larger gate in FIG. 5 A) is shown in the upper graph, while the lower graph plots relative ACE2 expression measured by detection of the extracellular myc tag. RBD-sfGFP binding to the total positive population correlates with total ACE2 expression, and differences in binding between the mutants are therefore most apparent only after controlling for expression levels as in FIG. 5B.

[0057] FIGS 6A-6B: Engineered sACE2 with enhanced binding to S. (FIG. 6A) Expression of sACE2-sfGFP mutants was qualitatively evaluated by fluorescence of the transfected cell cultures. (FIG. 6B) Cells expressing full length S were stained with dilutions of sACE2-sfGFP-containing media and binding was analyzed by flow cytometry.

[0058] FIGS. 7A-7D: Analytical size exclusion chromatography (SEC) of purified sACE2 proteins. (FIG. 7A) Purified sACE2 proteins (10 pg) were separated on a 4-20% SDS-polyacrylamide gel and stained with Coomassie. (FIG. 7B) Analytical SEC of IgGl- fused wild type sACE2 and sACE2.v2. Molecular weights (MW) of standards are indicated in kD above the peaks. Absorbance of the MW standards is scaled for clarity. (FIG. 7C) Analytical SEC of 8his-tagged proteins. The major peak corresponds to the expected MW of a monomer. A dimer peak is also observed, although its abundance differs between independent protein preparations (compare to FIG. 9D). (FIG. 7D) Soluble ACE2-8h proteins were incubated at 37 °C for 40 h and analyzed by SEC. [0059] FIGS. 8A-8E: A variant of sACE2 with high affinity for S. (FIG. 8 A)

Expi293F cells expressing full length S were incubated with purified wild type sACE2 or sACE2.v2 fused to 8his (solid lines) or IgGl-Fc (broken lines). After washing, bound protein was detected by flow cytometry. (FIG. 8B) Binding of 100 nM wild type sACE2-IgGl (broken lines) was competed with wild type sACE2-8h or sACE2.v2-8h. The competing proteins were added simultaneously to cells expressing full length S, and bound proteins were detected by flow cytometry. (FIG. 8C) Biolayer interferometry (BLI) kinetics of wild type sACE2-8h association (t = 0 to 120 s) and dissociation (t > 120 s) with immobilized RBD- IgGl. (FIG. 8D) Kinetics of sACE2.v2-8h binding to immobilized RBD-IgGl measured by BLI. (FIG. 8E) Competition for binding to immobilized RBD in an ELISA between serum IgG from recovered COVID-19 patients versus wild type sACE2-8h or sACE2.v2-8h. Three different patient sera were tested (Pl to P3 in light to dark shades).

[0060] FIGS. 9A-9G: Optimization of a high affinity sACE2 variant for improved yield. (FIG. 9A) Dilutions of sACE2-sfGFP-containing media were incubated with Expi293F cells expressing full length S. After washing, bound sACE2-sfGFP was analyzed by flow cytometry. (FIG. 9B) Coomassie-stained SDS-polyacrylamide gel compares the yield of sACE2-IgGl variants purified from expression medium by protein A resin. (FIG. 9C) Coomassie-stained gel of purified sACE2-8h variants (10 pg per lane). (FIG. 9D) By analytical SEC, sACE2.v2.4-8h is indistinguishable from wild type sACE2-8h. The absorbance of MW standards is scaled for clarity, with MW indicated above the elution peaks in kD. (FIG. 9E) Analytical SEC after storage at 37 °C for 60 h. Variant sACE2.v2.2 has a more hydrophobic surface and higher propensity to partially aggregate compared to sACE2.v2.4, and therefore the partial storage instability may be intrinsically linked to increased hydrophobicity. (FIG. 9F) Wild type sACE2-8h association (t = 0 to 120 s) and dissociation (t > 120 s) with immobilized RBD-IgGl measured by BLI. Data are comparable to a second independent preparation of sACE2-8h shown in FIG. 8C. (FIG. 9G) BLI kinetics of sACE2.v2.4-8h with immobilized RBD-IgGl.

[0061] FIGS. 10A-10D: A dimeric sACE2 variant with improved properties for binding viral spike. (FIG. 10A) Analytical SEC of wild type sACE22-8h and sACE22.v2.4- 8h after incubation at 37 °C for 62 h. (FIG. 10B) ELISA analysis of serum IgG from recovered patients (Pl to P3 in light to dark shades) binding to RBD. Dimeric sACE22(WT)- 8h or sACE22.v2.4-8h are added to compete with antibodies recognizing the receptor binding site. Concentrations are based on monomeric subunits. (FIG. IOC) RBD-8h association (t = 0 to 120 s) and dissociation (t > 120 s) with immobilized sACE22(WT)-IgGl measured by BLI. (FIG. 10D) BLI kinetics of RBD-8h binding to immobilized sACE22.v2.4-IgGl.

[0062] FIGS. 11A-11B: Enhanced neutralization of SARS-CoV-2 and SARS-CoV-1 by engineered receptors. In a microneutralization assay, monomeric (solid lines) or dimeric (broken lines) sACE2(WT)-8h or sACE2.v2.4-8h were preincubated with SARS-CoV-2 (FIG. 11 A) or SARS-CoV-1 (FIG. 11B) virus before adding to VeroE6 cells. Concentrations are based on monomeric subunits. Data are mean ± SEM of n = 4 (2 independent experiments with 2 technical replicates).

[0063] FIGS. 12A-12C: Binding of a sACE2 glycosylation mutant to the RBD of SARS-CoV-2. (FIG. 12A) The protease domain of soluble ACE2 carrying mutation T92Q was purified as a 8his-tagged fusion. Six pg was separated on a Coomassie-stained 4-20% SDS-polyacrylamide gel to assess purity. (FIG. 12B) Analytical SEC shows a major peak eluting as monomer, with a smaller fraction eluting at the expected MW of dimer. (FIG. 12C) In biolayer interferometry (BLI) experiments, RBD-IgGl was immobilized to a sensor surface and association and dissociation of sACE2-T92Q-8h was measured. The reported affinity (80 nM) is tighter than that of the equivalent wild type protein (140-150 nM, FIGS. 8C and 9F).

[0064] FIGS. 13A-13C: Flow cytometry measurements of sACE2 binding to myc- tagged S expressed at the plasma membrane. (FIG. 13 A) Expi293F cells expressing full length S, either untagged (FIG. 8A) or with an extracellular myc epitope tag, were gated by forward-side scattering properties for the main cell population (gated area). (FIG. 13B) Histograms showing representative raw data from flow cytometry analysis of myc-S- expressing cells incubated with 200 nM wild type sACE2-8h or sACE2.v2. After washing, bound protein was detected with a fluorescent anti-HIS-FITC secondary. Fluorescence of myc-S-expressing cells treated without sACE2 is black. (FIG. 13C) Binding of purified wild type sACE2 or sACE2.v2 fused to 8his (solid lines) or IgGl-Fc (broken lines) to cells expressing myc-S.

[0065] FIGS. 14A-14D: Dimeric sACE22 binds avidly to RBD. (FIG 14A) SDS- PAGE of purified dimeric sACE22-8h proteins (10 pg per lane, stained with Coomassie). (FIG. 14B) Preparative SEC of sACE22-8h proteins. The eluate fromNiNTA affinity chromatography was concentrated and injected on the gel filtration column. Absorbance of MW standards is scaled and kD is indicated above the elution peaks. (FIG. 14C) Expi293F cells expressing full length S were incubated with wild type and v2.4 sACE22-8h, washed and stained with fluorescent anti -his. Mean of n = 2, error bars represent range. Binding resembles that observed for IgGl-fusions of sACE2 (FIGS. 8A and 13C), indicating avid interactions between dimeric sACE22-8h and trimeric spike on the cell surface. (FIG. 14D) BLI kinetics for dimeric sACE22(WT)-8h and sACE22.v2.4-8h binding avidly to dimeric RBD-IgGl immobilized on the sensor surface.

[0066] FIGS. 15A-15B: Purified sACE22-IgGl is a dimer. (FIG. 15 A) Coomassie- stained gel of purified sACE22-IgGl proteins (10 pg per lane). (FIG. 15B) Analytical SEC of purified sACE22-IgGl, overlaid with scaled absorbance of MW standards (kD indicated above elution peaks). Note the absence of high MW peaks that might correspond to concatemers mediated by sACE22 and IgGl dimerization between different subunits.

[0067] FIGS. 16A-16D: Untagged sACE22.v2.4 expressed in nonhuman cells binds

S tightly. (FIG. 16A) SDS-PAGE comparison of sACE22.v2.4 purified from human Expi293F cells with a 8h tag and untagged protein manufactured in the nonhuman ExpiCHO- S line. 10 pg per lane. (FIG. 16B) Analytical SEC of sACE22.v2.4 before and after incubation at 37 °C for 146 h. Absorbance of MW standards is scaled and kD is indicated. (FIG. 16C) S-expressing Expi293F cells were co-incubated with 100 nM wild type sACE22- IgGl and increasing concentrations of human cell-derived sACE22(WT)-8h, human cell- derived sACE22.v2.4-8h or ExpiCHO-S-derived sACE22.v2.4. Bound his-tagged proteins (solid lines) and sACE22(WT)-IgGl (broken lines) were measured by flow cytometry. (FIG. 16D) Avid binding of untagged sACE22.v2.4 to immobilized RBD-IgGl measured by BLI.

[0068] FIGS. 17A-17C: The engineered receptor has reduced catalytic activity.

Cleavage of a peptide substrate to release a fluorescent product was measured for human cell- derived sACE22(WT)-8h (FIG. 17A), human cell-derived sACE22.v2.4-8h (FIG. 17B) and ExpiCHO-S-derived sACE22.v2.4 (FIG. 17C). Specific activity was determined across initial time points where product release was linear, and data for the highest concentration of wild type protein (22 nM) was excluded. Time = 0 min indicates when fluorescence recording began. [0069] FIG. 18: SARS-associated coronaviruses have high sequence diversity at the ACE2-binding site. The RBD of SARS-CoV-2 (PDB 6M17) is colored by diversity between 7 SARS-associated CoV strains.

[0070] FIG. 19: The ACE2-binding site of SARS-associated betacoronaviruses is a region of high sequence diversity. RBD sequences from 2 human and 5 bat betacoronaviruses that use ACE2 as an entry receptor are aligned (SEQ ID NOs: 3-9). Numbering is based on SARS-CoV-2 protein S. Asterisks indicate residues of SARS-CoV-2 RBD that are within 6.0 A of ACE2 in PDB 6M17.

[0071] FIGS. 20A-20C: FACS selection for variants of S with high or low binding signal to ACE2. (FIG. 20 A) Flow cytometry analysis of Expi293F cells expressing full- length S of SARS-CoV-2 with an N-terminal c-myc tag. Staining for the myc-epitope is on the x-axis while the detection of bound sACE22-8h (2.5 nM) is on the y-axis. S plasmid was diluted 1500-fold by weight with carrier DNA so that cells typically express no more than one coding variant; under these conditions most cells are negative. (FIG. 20B) Flow cytometry of cells transfected with the RBD single site-saturation mutagenesis (SSM) library shows cells expressing S variants with reduced sACE22-8h binding. (FIG. 20C) Gating strategy for FACS. S-expressing cells positive for the c-myc epitope were gated and the highest ("ACE2-High") and lowest ("ACE2-Low") 20% of cells with bound sACE22-8h relative to myc-S expression were collected.

[0072] FIG. 21: The mutational landscape across the RBD of full-length S from SARS-CoV-2 for binding to soluble ACE22. Log2 enrichment ratios from the deep mutational scan of the RBD in full-length S are plotted from < -3 (depleted/deleterious) to 0 (neutral) to > +3 (enriched). Wild type amino acids are black. RBD sequence is on the vertical axis and amino acid substitutions are on the horizontal axis. *, stop codons.

[0073] FIGS. 22A-22D: Deep mutagenesis reveals that the ACE2-binding site of SARS-CoV-2 tolerates many mutations. (FIG. 22A) Positional scores for surface expression are mapped to the structure of the SARS-CoV-2 RBD (PDB 6M17, oriented as in FIG. 18). Several residues in the protein core are highly conserved in the FACS selection for surface S expression (judged by depletion of mutations from the AC E2 -High and ACE2-Low gates), while some surface residues tolerate mutations. (FIG. 22B) Correlation plot of expression scores from mutant selection in human cells of full-length S (x-axis) versus the conservation scores (mean of the log2 enrichment ratios at a residue position) from mutant selection in the isolated RBD by yeast display (y-axis). Notable outliers are indicated. (FIG. 22C) Conservation scores from the ACE2-High gated cell population are mapped to the RBD structure. (FIG. 22D) Correlation plot of RBD conservation scores for high ACE2 binding from deep mutagenesis of S in human cells (x-axis) versus deep mutagenesis of the RBD on the yeast surface (mean of AKD app; y-axis).

[0074] FIGS. 23A-23C: Alanine substitutions of disulfide-bonded cysteines in the RBD diminish S surface expression in human cells. (FIG. 23 A) The RBD, colored by expression score from deep mutagenesis (conserved or mutationally tolerant), forms a continuous hydrophobic core with the rest of the SI subunit in a closed-down conformation (PDB 6VSB chain B). (FIG. 23B) Based on surface immuno-staining and flow cytometry analysis, Expi293F cells transfected with myc-S cysteine mutants displayed decreases in both the percent of myc-positive cells (gated area) and in mean fluorescence of the positive population. To capture both effects in a single number, the change in mean fluorescence units (AMFU) compared to vector-transfected control cells was calculated for the entire cell population, after first gating by scattering for viable cells. (FIG. 23C) Surface expression of myc-S cysteine mutants relative to wild type myc-S. Data are mean ± range, n = 2 independent replicates.

[0075] FIGS. 24A-24G: A competition-based selection to identify RBD mutations within S of SARS-CoV-2 that preferentially bind wild type or engineered ACE2 receptors. (FIG. 24A) Expi293F cells were transfected with wild type myc-S and incubated with competing sACE22(WT)-IgGl (25 nM) and sACE22.v2.4-8h (20 nM). Bound protein was detected by flow cytometry after immuno-staining for the respective epitope tags. (FIG. 24B) As in FIG. 24A, except cells were transfected with the RBD SSM library. A population of cells expressing S variants with increased specificity towards sACE22.v2.4 is apparent (cells shifted to the upper-left of the main population). (FIG. 24C) Gates used for FACS of cells expressing the RBD SSM library. After excluding cells without bound protein, the top 20% of cells for bound sACE22.v2.4-8h (upper gate) and for bound sACE22(WT)-IgGl (lower gate) were collected. (FIGS. 24D-24E) Agreement between log2 enrichment ratios from two independent FACS selections for cells expressing S variants with increased specificity for sACE22(WT) (FIG. 24D) or sACE22.v2.4 (FIG. 24E). R ² values are calculated for nonsynonymous mutations. (FIGS. 24F-24G) Conservation scores are calculated from the mean of the log2 enrichment ratios for all nonsynonymous substitutions at a given residue position. Correlation plots show agreement between conservation scores for two independent selections for cells within the sACE22(WT) (FIG. 24D) or sACE22.v2.4 (FIG. 24E) specific gates.

[0076] FIGS. 25A-25C: Mutations within the RBD that confer specificity towards wild type ACE2 are rare. (FIG. 25 A) The SARS-CoV-2 RBD is colored by specificity score (the difference between the conservation scores for cells collected in the sACE22(WT) and SACE22.V2.4 specific gates). Some residues are hot spots for mutations with increased specificity towards sACE22(WT) or towards sACE22.v2.4. The contacting surface of ACE2 is shown as a ribbon, with sites of mutations in sACE22.v2.4 labeled and shown as spheres. (FIG. 25B) Log2 enrichment ratios for mutations in S expressed by cell populations collected in the sACE22(WT) (x-axis) and sACE22.v2.4 (y-axis) specific gates. Data are the mean from two independent sorting experiments. S mutants predicted to have increased specificity for SACE22(WT) were tested by targeted mutagenesis in FIG. 26. S mutants predicted to have increased specificity for sACE22.v2.4 were tested by targeted mutagenesis in FIG. 27. (FIG. 25C) Wild type myc-S and two variants, N501W and N501Y, were expressed in Expi293F cells and tested by flow cytometry for binding to sACE22(WT)-8h (dashed lines) or sACE22.v2.4-8h (solid lines). Minor increases in specific binding for wild type sACE22 are observed.

[0077] FIGS. 26A-26B: Screening mutations of SARS-CoV-2 S predicted by deep mutagenesis to have enhanced specificity towards wild type sACE22 over sACE22.v2.4. (FIG. 26A) Relative surface expression of myc-S mutants, determined as described in FIG. 23. Data are mean ± range, n = 2 independent replicates. (FIG. 26B) Competition binding between sACE22(WT)-IgGl (x-axis) and sACE22.v2.4-8h (y-axis) on Expi293F cells expressing the indicated myc-tagged S proteins. Cells expressing S variants with increased specificity towards wild type receptor will be shifted to the lower-right; only minor shifts are observed. Cells expressing S variants with reduced surface expression and/or ACE2 affinity have a lower percentage in the gated area. Results are representative of 2 replicates.

[0078] FIGS. 27A-27B: Screening mutations of SARS-CoV-2 S predicted by deep mutagenesis to have enhanced specificity towards sACE22.v2.4 over wild type sACE22. (FIG. 27A) Relative surface expression of myc-S mutants measured by flow cytometry. Data are mean ± range, n = 2. (FIG. 28B) Flow cytometry analysis of cells expressing myc-S variants bound to competing sACE22(WT)-IgGl (x-axis) and sACE22.v2.4-8h (y-axis). Cells expressing S with increased specificity towards sACE22.v2.4 will be shifted to the upper-left. Results are representative of 2 replicates.

[0079] FIGS. 28A-28B: Serum half-life of sACE2 peptides following IV administration. Unfused sACE22.v2.4 was injected in the tail veins of mice (3 male and 3 female per time point; 0.5 mg/kg). Serum was collected and analyzed by ACE2 ELISA (FIG. 28A) and for proteolytic activity towards a fluorogenic substrate (FIG. 28B). Data are mean ± S.E.

[0080] FIG. 29: Pharmacokinetics of sACE2 fused to human IgGl Fc following IV administration. IV administration of 2.0 mg/kg wild type sACE22-IgGl (open circles) or sACE22.v2.4-IgGl (filled circles) in 3 male mice per time point. Protein in serum was quantified by human IgGl ELISA. Data are mean ± S.E.

[0081] FIGS. 30A-30D: Pharmacokinetics of sACE22.v2.4-IgGl in serum following IV administration. sACE22.v2.4-IgGl was IV administered to mice (3 male and 3 female per time point; 2.0 mg/kg). Serum was collected and analyzed by human IgGl ELISA (FIG. 30A), by ACE2 ELISA (FIG. 30B), and for 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 electrophoretic gel and probed with anti -human IgGl. The standard is 10 ng of purified sACE22.v2.4-IgGl. The predicted molecular weight (excluding glycans) is 216 kD.

[0082] FIGS. 31A-31F: PK of ACE2 proteins delivered directly to the lungs. (FIGS. 31A and 31B) Wild type sACE22-IgGl (open circles) and sACE22.v2.4-IgGl (filled circles) were administered IT at a dose of 1.0 mg/kg. Lung tissue was collected and proteins were extracted and analyzed by human IgGl ELISA (FIG. 31 A) and ACE2 ELISA (FIG. 3 IB). Data are mean ± S.E., n = 3 males per time point. (FIG. 31C) Lung extracts from representative mice IT administered sACE22.v2.4-IgGl were analyzed under non-reducing conditions by anti-human IgGl immunoblot. (FIGS. 3 ID and 3 IE) Mice inhaled nebulized sACE22.v2.4-IgGl. Extracts from lung tissue were analyzed by ACE2 ELISA (FIG. 3 ID) and human IgGl ELISA (FIG. 3 IE). Data are mean ± S.E., n = 3 males per time point. (FIG.

3 IF) Representative extracts from lung tissue of mice receiving nebulized sACE22.v2.4-IgGl were analyzed by anti-human IgGl immunoblot. [0083] FIG. 32: Neutralization of pseudovirus entry into human lung cells by sACE22-IgGl. Human A549 lung epithelial cells over-expressing the ACE2 receptor, human A549 lung epithelial cells, and human lung endothelial cells were incubated with the VSV- SARS-CoV-2-luciferase-pseudotype virus and with wild-type sACE22-IgGl or engineered sACE22.v2.4-IgGl at the indicated concentrations. Each experiment contained ano virus control (left-most bar in each graph), all other samples contained the virus dose at the indicated MOI. The extent of viral entry was quantified based on luciferase activity.

[0084] FIG. 33: Efficacy of sACE22-IgGl to inhibit pseudovirus entry into the lung and liver in an in vivo infection model. K18-hACE2 mice, which express the human ACE2 receptor in epithelial cells, were injected IV with sACE22-IgGl (wild-type, middle bar; engineered v2.4, right bar) and intraperitoneally with the VSV-SARS-CoV-2-luciferase- pseudotype virus. The lung and the liver were harvested at 24 hours and the extent of viral entry was quantified based on luciferase expression.

[0085] FIG. 34: ACE2 decoys carrying v2.4 mutations bind S proteins from multiple highly transmissible SARS-CoV-2 VOCs. Human Expi293F cells expressing myc-tagged S from four SARS-CoV-2 variants (Wuhan, B.1.1.7/alpha, P.l/gamma, and B1.351/beta) were incubated with monomeric sACE2-8h (black) or dimeric sACE22-IgGl (grey) and bound protein was detected by flow cytometry. N=2.

[0086] FIG. 35: Decoy receptors diminish SI at a membrane surface. Human Expi293F cells expressing myc-S from the B.1.617.2 (FIGs. 35A-C), B.1.427/B.1.429 (FIGs. 35 D-F), or C.37 (FIGs. 35 G-I) lineages were incubated with monomeric sACE2-8h (black) or dimeric sACE22-IgGl (grey), washed, and then stained for flow cytometry (N = 2). (FIGs. 35 A, D, G) Bound wild type (dashed lines, open circles) and v2.4 (solid lines, filled circles) sACE2 proteins. (FIGs. 35 B-C, E-F, H-I) Relative surface SI protein based on detection of the myc tag, after incubating cells with sACE2-8h (FIGs. 35B, E, H) or sACE22-IgGl (FIGs. 35C, F, I). The ratio of bound decoy receptor to surface SI is shown in magenta.

[0087] FIG. 36: Monoclonal antibodies display a range of binding strengths to the S proteins of SARS-CoV-2 variants. Binding of mAbs versus sACE22.v2.4-IgGl to S proteins of four VOCs (B.1.1.7/alpha, P.l/gamma, B1.351/beta, and B.1.617.2/delta) measured by flow cytometry. N=2 [0088] FIG. 37: Anti-RBD monoclonals and sACE22.v2.4-IgGl can together bind Spike at sub-saturating concentrations. Human Expi293F cells expressing full-length S (Delta variant) were co-incubated with 9 or 1 nM mAh and sACE22.v2.4-8h. Bound mAh was detected by flow cytometry.

[0089] FIG. 38: Competitive and non-competitive binding to the RBD by anti- SARS-CoV-2 monoclonals and sACE22.v2.4. Monoclonal antibodies were immobilized to BLI sensors and bound (time = 100 to 220 s) to 100 nM RBD from the (FIG. 38 A) Delta and (FIG. 38B) Gamma VOCs. Sensors were then transferred to 200 nM sACE22.v2.4 (time = 220 to 340 s).

[0090] FIGS. 39A-39D: ACE2 decoys carrying v2.4 mutations bind S proteins from multiple highly transmissible SARS-CoV-2 VOCs and SARS-CoV-1. Human Expi293F cells expressing myc-tagged S from 4 SARS-CoV-2 variants (Wuhan, B.1.1.7/alpha, B1.351/beta, and P.l/gamma) were incubated with monomeric sACE2-8h (black) or dimeric sACE22-IgGl (grey) and bound protein was detected by flow cytometry. WT ACE2 proteins are in broken lines, v2.4 proteins are in solid lines. N=3 independent replicates, mean ± SEM. FIGS. 39E-39I: Comparison of ACE2 decoys carrying v2.4 mutations to mAbs. Binding of mAbs (VIR-7831 in green, LY-CoV555 in grey, REGN10933 in yellow, REGN10987 in orange) versus sACE22.v2.4-IgGl (blue) to S proteins of SARS-CoV-2 VOCs (B.1.1.7/alpha, B1.351/beta, B.1.617.2/delta, and P.l/gamma) and S of SARS-CoV-1, as measured by flow cytometry. N=3 independent replicates, mean ± SEM.

[0091] FIG. 40: Soluble ACE2 decoys have increased binding to cells expressing S from highly transmissible SARS-CoV-2 variants. Expi293F cells expressing S proteins from the Wuhan, Alpha, Beta, and Gamma SARS-CoV-2 variants were incubated with 11 nM monomeric sACE2-8h (cyan) and sACE2.v2.4-8h (orange) and bound protein was detected by flow cytometry. Fluorescence signals for bound proteins are shown relative to sACE2.v2.4-8h binding Alpha, which generally had the highest signals. N=3 independent replicates, mean ± SEM.

[0092] FIGS. 41A-41I: Decoy receptors diminish SI at a membrane surface. Human Expi293F cells expressing myc-S from the B.l.617.2 (FIGs 41A-41C), B.1.427/B.1.429 (FIGS. 41D- 41F), or C.37 (FIGS. 41G-41I) lineages were incubated with monomeric sACE2-8h (black) or dimeric sACE2 ₂ -IgGl (grey), washed, and then stained for flow cytometry (N = 2). (FIGS. 41 A, 41D, and 41G) Bound wild type (dashed lines, open circles) and v2.4 (solid lines, filled circles) sACE2 proteins. (FIGS .41B, 41C, 41E, 41F, 41H, and 411) Relative surface SI protein based on detection of the myc tag, after incubating cells with sACE2-8h (FIGS. 41B, 41E, and 41H) or sACE2 ₂ -IgGl (FIGS. 41C, 41F, and 411). The ratio of bound decoy receptor to surface SI is shown in magenta.

[0093] FIG. 42: BLI binding kinetics of delta and gamma RBDs for sACE22.v2.4-

IgGl versus monoclonal antibodies. Sensors displaying immobilized sACE22.v2.4-IgGl or mAbs were transferred to solutions of monomeric RBD (time = 0 to 60 s) to measure association and then returned to buffer to measure dissociation (time = 60 to 360 s) using biolayer interferometry. RBD concentrations are indicated on the rightmost sensorgrams. Upper panels show binding to delta RBD and lower panels show binding to gamma RBD. Measured responses are colored while fitted curves (1:1 binding model) are black.

[0094] FIGS. 43A and 43B: Engineered receptor decoys tightly bind S of omicron.

Monomeric (ACE2 residues 19-615) 8his tagged sACE2 proteins were incubated with Expi293F cells expressing S of delta (FIG. 43A). Binding was measured by flow cytometry, N=2. Monoclonal antibodies and dimeric (ACE2 residues 19-732) sACE2 ₂ -IgGl proteins were incubated with Expi293F cells expressing omicron S and bound protein was detected by flow cytometry (FIG. 43B). Mean ± SEM, N=4.

SEQUENCE LISTING

[0095] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

[0096] SEQ ID NO: 1 is the amino acid sequence of human ACE2 (also called peptidyl-dipeptidase A; deposited under GenBank Accession No. NP_068576.1):

MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEE NVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDK SKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWR SEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLI EDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYS LTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTD PGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQP FLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVG TLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPAS LFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLR LGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYA DQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDV RVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPT LGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENPYASIDISKGE NNPGFQNTDDVQTSF

[0097] 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 No. YP_009724390.1):

[0098] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHS TQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGT

TLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSAN NCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSA LEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFG EVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNV YADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY LYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYR VVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFG RDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAI HADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPR RARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYI CGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGF NFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLT VLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVL YENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAIS SVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSEC VLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAH FPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELD SFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG KYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDS

EPVLKGVKLHYT

[0099] SEQ ID NOs: 3-9 are amino acid sequences of RBD sequences from human and bat betacoronaviruses (see FIG. 19).

[0100] SEQ ID NO: 10 is the amino acid sequence of sACE22.v2.4, comprised of residues 19-732 of human ACE2 (including the protease and dimerization domains) with three amino acid substitutions relative to human ACE2: T27Y, L79T, and N330Y.

STIEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLK EQSTTAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGK VCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLK NEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHA YVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMV DQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLG KGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEI MSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVF KGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTL YQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGA

KNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKA YEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAP KNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLG

[0101] SEQ ID NO: 11 is the amino acid sequence of sACE22.v2.4-IgGl, comprised of SACE22.V2.4 fused to human IgGl Fc.

STIEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLK EQSTTAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGK VCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLK NEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHA YVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMV DQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLG KGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEI MSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVF KGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTL YQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGA

KNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKA

YEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAP

KNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGSDKTHTCPPCPAP ELL

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0102] SEQ ID NO: 12 is the amino acid sequence of SARS-CoV2 spike protein

(omicron).

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP F

FSNVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSL LI

VNNATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMD

LEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFSALEPLVDLPIGI NIT

RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA

LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFAS VY

AWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVR

QIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFE

RDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPA

TVCGPKKSTNLVKNKCVNFNFNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRD

PQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRV Y STGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAY TMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLL

QYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSK P

SKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFKGLTVLPPLLTDEM I

AQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQ

FNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDIFSR L

DKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVD

FCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVS NGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKY FKNHTSPDVDLGDIS GINAS VVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWP WYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKL

HYT

[0103] SEQ ID NO: 13 is the amino acid sequence of SARS-CoV2 spike protein

(delta).

MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP F FSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVS QPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSVLEPLVDLP IGINITRFQTLL ALHRS YLTPGDS S S GWTAGAAAYYVGYLQPRTFLLKYNENGTITD A VDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRF ASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRG DEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSN LKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL HAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTD AVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPT WRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSRRRARSVAS QSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTEC SNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILP DPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLT DEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLI ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDIL SRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKR VDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVF VSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELD KYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK WPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGV

KLHYT

[0104] SEQ ID NO: 14 is the amino acid sequence of SARS-CoV2 RBD-8h (Delta).

MKTIIALSYIFCLVFATNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS F STFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNC

YFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKGSGHHHHHHHH

[0105] SEQ ID NO: 15 is the amino acid sequence of SARS-CoV2 RBD-8h (Gamma).

MKTIIALSYIFCLVFATNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS F STFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGTIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNC YFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKGSGHHHHHHHH

[0106] SEQ ID NO: 16 is the amino acid sequence of sACE2.v2.4.2-8h (ACE2 residues 1-615).

MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEE NVQNMNNAGDKWSAFLKEVSTTAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDK SKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWR SEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLI EDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYS LTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTD

PGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQP FLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVG TLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPAS LFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLR LGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYA DGSGHHHHHHHH

[0107] SEQ ID NO: 17 is the amino acid sequence of sACE22.v2.4.2-IgGl (ACE2 residues 1-732).

MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEE NVQNMNNAGDKWSAFLKEVSTTAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDK SKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWR SEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLI EDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYS LTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTD

PGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQP FLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVG TLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPAS LFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLR LGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYA DQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDV RVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPT LGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

[0108] SEQ ID NO: 18 is the amino acid sequence of sACE2.v2.4.10-8h (ACE2 residues 1-615).

MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDYFNHEAEDLFYQSSLASWNYNTNITEE NVQNMNNAGDKWSAFLKEQSTTAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDK SKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWR SEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLI EDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYS LTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTD

PGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQP FLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVG TLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPAS LFHVSNDYSFIRYYTGTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLR LGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYA DGSGHHHHHHHH

[0109] SEQ ID NO: 19 is the amino acid sequence of sACE22.v2.4.10-IgGl (ACE2 residues 1-732).

MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDYFNHEAEDLFYQSSLASWNYNTNITEE NVQNMNNAGDKWSAFLKEQSTTAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDK SKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWR SEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLI EDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYS LTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTD PGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQP FLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVG TLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPAS LFHVSNDYSFIRYYTGTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLR LGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYA DQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDV RVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPT LGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

[0110] SEQ ID NO: 20 is the amino acid sequence of sACE2.v2.4.2 (ACE2 residues 1-

615).

MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEE NVQNMNNAGDKWSAFLKEVSTTAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDK SKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWR SEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLI EDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYS LTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTD PGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQP FLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVG TLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPAS LFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLR LGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYA DGSG

[0111] SEQ ID NO: 21 is the amino acid sequence of sACE2.v2.4.10 (ACE2 residues 1-615). MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDYFNHEAEDLFYQSSLASWNYNTNITEE NVQNMNNAGDKWSAFLKEQSTTAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDK SKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWR SEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLI EDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYS LTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTD

PGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQP FLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVG TLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPAS LFHVSNDYSFIRYYTGTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLR LGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYA DGSG

DETAILED DESCRIPTION

[0112] The present application provides modified human ACE2 polypeptides, or fragments thereof, that exhibit enhanced binding to the S protein of SARS-CoV-2, or increased affinity, that can be used as therapeutic agents in combination with monoclonal antibodies, or antigen-binding fragments thereof, for the prophylaxis (pre- or post-exposure prophylaxis), or treatment of COVID-19, or disease caused by any coronavirus that utilizes ACE2 as a cellular receptor. Various aspects of the ACE2 polypeptides, or fragments thereof, and combinations with monoclonal antibodies, or antigen-binding fragments thereof, described in the present application are set forth below in sections; however, aspects of the proteins and/or monoclonal antibodies described in one particular section are not to be limited to any particular section.

I. Abbreviations

[0113] ACE2 angiotensin-converting enzyme 2

[0114] BLI biolayer interferometry

[0115] CoV coronavirus [0116] COVID-19 coronavirus disease 2019

[0117] IT intratracheal

[0118] IV intravenous

[0119] MOI multiplicity of infection

[0120] RBD receptor binding domain

[0121] sACE2 soluble angiotensin-converting enzyme 2

[0122] SARS severe acute respiratory syndrome

[0123] SARS-CoV-2 SARS coronavirus 2

[0124] SEC size exclusion chromatography

[0125] sfGFP superfolder green fluorescent protein

II. Terms and Methods

[0126] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes X. published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley -V CH in 16 volumes, 2008; and other similar references.

[0127] As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes single or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:

[0128] Aerosol: A suspension of fine solid particles or liquid droplets in a gas (such as air).

[0129] Administration: To provide or give a subject an agent, such as a modified human ACE2 polypeptide, by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous), transdermal, intranasal, intratracheal and inhalation routes.

[0130] Biological sample: A sample obtained from a subject (such as a human or veterinary subject). Biological samples include, for example, fluid, cell and/or tissue samples. In some embodiments herein, the biological sample is a fluid sample. Fluid sample include, but are not limited to, serum, blood, plasma, urine, feces, saliva, cerebral spinal fluid (CSF), bronchoalveolar lavage (BAL), nasal swab, or other bodily fluid. Biological samples can also refer to cells or tissue samples, such as biopsy samples or tissue sections.

[0131] Contacting: Placement in direct physical association; includes both in solid and liquid form.

[0132] Coronavirus: A large family of positive-sense, single-stranded RNA viruses that can infect humans and non-human animals. Coronaviruses get their name from the crown-like spikes on their surface. The viral envelope is comprised of a lipid bilayer containing the viral membrane (M), envelope (E) and spike (S) proteins. Most coronaviruses cause mild to moderate upper respiratory tract illness, such as the common cold. However, three coronaviruses have emerged that can cause more serious illness and death in humans: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Other coronaviruses that infect humans include human coronavirus HKU1 (HKUl-CoV), human coronavirus OC43 (OC43-CoV), human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV). In some embodiments of the present disclosure, “coronavirus” includes any human coronavirus or zoonotic coronavirus that utilizes ACE2 as a cellular receptor, including known and emerging strains of coronavirus. Zoonotic coronaviruses include, but are not limited to, bat and rodent coronaviruses. [0133] Fusion protein: A protein comprising at least a portion of two different (heterologous) proteins. In some embodiments, the fusion is comprised of a modified ACE2 polypeptide and an Fc protein, such as an Fc from human IgGl.

[0134] Heterologous: Originating from a separate genetic source or species.

[0135] Inhibiting or Inhibition: Reduction in an amount or activity.

[0136] Isolated: An “isolated” biological component, such as a nucleic acid or protein, has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component naturally occurs, for example other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0137] Nebulizer: A device for converting a therapeutic agent (such as a polypeptide) in liquid form into a mist or fine spray (an aerosol) that can be inhaled into the respiratory system, such as the lungs. A nebulizer is also known as an “atomizer.”

[0138] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the polypeptides and other compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can 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. [0139] Polypeptide, peptide and protein: Refer to polymers of amino acids 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 terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

[0140] Preventing, treating or ameliorating a disease: “Preventing” a disease refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. The prophylactic treatment can be pre-exposure or post-exposure.

[0141] Prophylaxis: The use of a medical treatment for preventing (or reducing the risk of developing) a disease or infection, such as a CoV infection or COVID-19. In the context of a viral infection, pre-exposure prophylaxis refers to treatment that is administered before a subject has been exposed to the virus, while post-exposure prophylaxis refers to treatment administered immediately or shortly after exposure to the virus, but before signs or symptoms of infection occur.

[0142] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified polypeptide preparation is one in which the polypeptide is more enriched than the polypeptide is in its natural environment, such as within a cell. In one embodiment, a preparation is purified such that the polypeptide represents at least 50% of the total peptide or protein content of the preparation. Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components. [0143] Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide or nucleic acid molecule will possess a relatively high degree of sequence identity when aligned using standard methods.

[0144] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: 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 51, 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, presents a detailed consideration of sequence alignment methods and homology calculations.

[0145] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

[0146] Homologs and variants of a polypeptide, such as a modified human ACE2 polypeptide or a monoclonal antibody, are typically characterized by possession of at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of the polypeptide or antibody using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

[0147] Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.

[0148] Therapeutically effective amount: A quantity of a specific substance (such as a modified human ACE2 polypeptide) sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit CoV replication or reduce CoV titer in a subject. In one embodiment, a therapeutically effective amount is the amount necessary 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 the absence of treatment). In another embodiment, a therapeutically effective amount is the amount necessary 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 the absence of treatment). The therapeutically effective amount can also be the amount necessary to reduce or eliminate one of more symptoms of CoV infection, such as the amount necessary reduce or eliminate fever, cough or shortness of breath. Similarly, in some embodiments, a prophylactically effect amount is the amount necessary to reduce the risk of becoming infected with a CoV or developing disease, such as COVID- 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 the absence of treatment).

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A 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 virus vector, such as a lentivirus vector. III. Modified ACE2 Polypeptides and Methods of Use

[0149] The spike (S) glycoprotein of SARS-CoV-2 binds angiotensin-converting enzyme 2 (ACE2) on host cells. S is a trimeric class I viral fusion protein that is proteolytically processed into SI and S2 subunits that remain noncovalently associated in a prefusion state (Walls et al., Cell. 2020 Mar 6; 181(2):281-292.e6; Hoffmann et al., Cell.

2020 Mar 4; 181(2)271-280.e8; Tortorici and Veesler. A/v Virus Res. Elsevier; 2019;105:93- 116). Upon engagement of ACE2 by a receptor binding domain (RBD) in SI (Wong et al., J Biol Chenr, 2004 Jan 30;279(5):3197-3201), conformational rearrangements occur that cause SI shedding, cleavage of S2 by host proteases, and exposure of a fusion peptide adjacent to the S2' proteolysis site (Tortorici and Veesler. Virus Res. Elsevier; 2019;105:93-116; Madu et al., J Virol,' 2009 Aug;83(15):7411-7421; Walls et al., Proc Natl Acad Sci USA,' 2017 Oct 17;114(42): 11157-11162; Millet and Whittaker, Proc Natl Aca d Sci USA,' 2014 Oct 21; 111(42): 15214-15219). Favorable folding of S to a post-fusion conformation is coupled to host cell/virus membrane fusion and cytosolic release of viral RNA. Atomic contacts with the RBD are restricted to the protease domain of ACE2 (Yan et al., Science. 2020 Mar 4;:eabb2762; Li et al., Science. 2005 Sep 16;309(5742): 1864-1868), and soluble ACE2 (sACE2) in which the neck and transmembrane domains are removed, is sufficient for binding S and neutralizing infection (Li et al., Nature. 2003 Nov 27;426(6965):450-454; Hofmann et al., Biochem Biophys Res Commun. 2004 Jul 9;319(4): 1216-1221; Lei et al., bioRxiv. 2020 Jan l;:2020.02.01.929976; Moore et al., J Virol' 2004 Oct;78(19):10628- 10635). In principle, the virus has limited potential to escape sACE2-mediated neutralization without simultaneously decreasing affinity for native ACE2 receptors, thereby attenuating virulence. Furthermore, fusion of sACE2 to the Fc region of human immunoglobulin can provide an avidity boost while recruiting immune effector functions and increasing serum stability, an especially desirable quality if intended for prophylaxis (Moore et al., J Virol,' 2004 Oct;78(19): 10628-10635; Liu e/ al., Kidney Int. 2018 Jul;94(l): 114-125), and recombinant sACE2 has proven safe in healthy human subjects (Haschke et al., Clin Pharmacokinet . 2013 Sep;52(9):783-792) and patients with lung disease (Khan et al., Crit Care. 2017 Sep 7;21(1):234).

[0150] The rapid and escalating spread of SARS coronavirus 2 (SARS-CoV-2) poses an immediate public health emergency, and no approved therapeutics or vaccines are currently available. The viral spike protein S binds membrane-tethered ACE2 on host cells in the lungs to initiate molecular events that ultimately release the viral genome intracellularly. The extracellular protease domain of ACE2 inhibits cell entry of both SARS and SARS-2 coronaviruses by acting as a soluble decoy for receptor binding sites on S, and is a leading candidate for therapeutic and prophylactic development. Mutations are found across the protein-protein interface and also at buried sites where they can enhance folding and presentation of the interaction epitope. In some embodiments herein, the N90-glycan on ACE2 is removed because it hinders association with S. The mutational landscape offers a blueprint for engineering high affinity ACE2 receptors to meet this unprecedented challenge. The disclosed ACE2 polypeptides are advantageous because there is very little risk of SARS- CoV-2, or any other coronavirus that binds ACE2, to develop resistance to these receptor decoys.

[0151] In certain aspects, modified ACE2 polypeptides of the present invention bind to S proteins from one or more SARS-CoV-2 variants selected from Wuhan, alpha, beta, gamma, delta, and omicron.

[0152] Described herein are ACE2 polypeptides (such as human ACE2 polypeptides) that have improved properties for binding CoV S protein. In particular, provided herein are modified ACE2 polypeptides that include a human ACE2 or a fragment thereof, such as an extracellular fragment thereof. The polypeptides include at least one amino acid substitution relative to wild-type human ACE2 (SEQ ID NO: 1). Modified ACE2 polypeptides of the present disclosure can be used in combination with monoclonal antibodies, or antigenbinding fragments thereof, for the treatment and/or prophylaxis of SARS-CoV-2 infection or infection by another coronavirus that utilizes ACE2 as a cellular receptor.

[0153] 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.

[0154] 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.

[0155] 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 4. [0156] 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.

[0157] In some embodiments, the modified polypeptides contain only a single amino acid substitution relative to a wild-type human ACE2 (SEQ ID NO: 1), such as one amino acid substitution listed in Table 2. In other examples, the modified polypeptides include two, three, four, five or more amino acid substitutions, such as two, three, four, five or more amino acid substitutions listed in Table 2. In specific examples, the modified polypeptide includes only a single substitution 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 human ACE2 of SEQ ID NO: 1. In other specific examples, the modified polypeptide includes 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 polypeptide includes a combination of substitutions listed in Table 5.

[0158] In some embodiments, the modified polypeptides are full-length human ACE2 polypeptides. 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 includes at least one amino acid substitution disclosed herein.

[0159] In other embodiments, the modified polypeptides consist of an extracellular fragment of human ACE2. For example, the modified polypeptide can consist of the complete extracellular protease domain of human ACE2, for example amino acid residues 19-615 of SEQ ID NO: 1, or the modified polypeptides can consist of a portion of the extracellular domain, such as 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.

[0160] In some embodiments, the modified polypeptides consist of a fragment of human ACE2. In some examples, the modified polypeptides are 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 of SEQ ID NO: 1 and include at least one amino acid substitution disclosed herein. In particular 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, such as residues 1-732, 19-732 or 19-740 of SEQ ID NO: 1, and includes at least one amino acid substitution disclosed herein. In specific examples, the modified polypeptide consists of amino acid residues 1-615, 1-732, 19-732 or 19-740 of SEQ ID NO: 1 and includes at least one amino acid substitution disclosed herein.

[0161] In some examples, the modified polypeptide comprises: T27Y, L79T, and N330Y amino acid substitutions; H34A, T92Q, Q325P, and A386L amino acid substitutions; T27Y, L79T, N330Y, and A386L amino acid substitutions; L79T, N330Y, and A386L amino acid substitutions; T27Y, N330Y, and A386L amino acid substitutions; T27Y, L79T, and A386L amino acid substitutions; A25V, T27Y, T92Q, Q325P, and A386L amino acid substitutions; H34A, L79T, N330Y, and A386L amino acid substitutions; A25V, T92Q, and A386L amino acid substitutions; or T27Y, Q42L, L79T, T92Q, Q325P, N330Y, and A386L amino acid substitutions, wherein the amino acid substitutions are with reference to SEQ ID NO: 1.

[0162] In certain aspects, the modified polypeptide comprises one or more amino acid substitution selected fromN90Q, T27Y, L79T, N330Y, L91P, Q76V, K31Y, Q76V, S19P, R518G, K31M, E35K, S47A, and L79F, wherein the amino acid substitutions are with reference to SEQ ID NO: 1. In certain aspects, the modified polypeptide comprises one or more amino acid substitutions selected from: (i) N90Q; (ii) T27Y, L79T, and N330Y; (iii) T27Y, L79T, N330Y, and L91P; (iv) T27Y, L79T, N330Y, Q76V, and L91P; (v) T27Y, L79T, N330Y, K31Y, and Q76V; (vi) T27Y, L79T, N330Y, S19P, and L91P; (vii) T27Y, L79T, N330Y, K31Y, and N90Q; (viii) T27Y, L79T, N330Y, S19P, Q76V, and N90Q; (ix) T27Y, L79T, N330Y, K31Y, Q76V, and L91P; (x) T27Y, L79T, N330Y, K31Y, Q76V, and N90Q; (xi) T27Y, L79T, N330Y, K31Y, L91P, and R518G; and (xii) N330Y, K31M, E35K, S47A, L79F, and L91P, wherein the amino acid substitutions are with reference to SEQ ID NO: 1.

[0163] In certain aspects, the modified polypeptide comprises residues 1-615 or 1-732 of SEQ ID NO: 1, and at least one amino acid substitution disclosed herein, such as one, two, three, four or five amino acid substitutions. In certain aspects, the modified polypeptide comprises an ACE2 variant having the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21.

[0164] Dimers of the modified polypeptides disclosed herein are also provided. In some embodiments, the dimeric polypeptide includes residues 1-732 or 19-732 of SEQ ID NO: 1, and at least one amino acid substitution disclosed herein, such as one, two, three, four or five amino acid substitutions. In some examples, the dimer is a dimer of the sACE2v.2.4 variant having the amino acid sequence of SEQ ID NO: 10.

[0165] Also provided are fusion proteins that include a modified ACE2 polypeptide disclosed herein and a heterologous polypeptide. In some embodiments, the heterologous polypeptide is an Fc protein, such as a human Fc protein, for example the Fc from human IgGl . In specific non-limiting examples, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 11. In certain aspects, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 17. In certain aspects, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 18. In other embodiments, the heterologous polypeptide is a protein that can be used as a diagnostic/detection reagent, such as a fluorescent protein (for example, GFP) or an enzyme (for example, alkaline phosphatase, HRP or luciferase). In some embodiments, the heterologous polypeptide is an antibody or antigen-binding protein for avid binding to a second CoV antigen. In some embodiments, the heterologous polypeptide is an antibody or antigen-binding protein for tethering to cells or cellular surroundings (for example, to recruit immune cells). In some embodiments, the heterologous polypeptide is a cytokine, ligand or receptor for evoking a biological response. In some embodiments, the heterologous polypeptide is a protein that increases the serum half-life (for example, antibody Fc or serum albumin). Fusion proteins of the present disclosure, or fragments thereof, can be used in combination with monoclonal antibodies, or antigen-binding fragments thereof, for the treatment and/or prophylaxis of SARS-CoV-2 infection or infection by another coronavirus that utilizes ACE2 as a cellular receptor.

[0166] Compositions that include a modified ACE2 polypeptide or fusion protein thereof and a pharmaceutically acceptable carrier are also provided. In some embodiments, the modified ACE2 polypeptide or fusion protein is formulated for intratracheal or inhalation administration. Intratracheal or inhalation preparations can be liquid (e.g., solutions or suspensions) and include mists, sprays, aerosols and 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. Compositions that include a modified ACE2 polypeptide, fragment thereof, or fusion protein of the present disclosure can be used in combination with monoclonal antibodies, or antigenbinding fragments thereof, for the treatment and/or prophylaxis of SARS-CoV-2 infection or infection by another coronavirus that utilizes ACE2 as a cellular receptor..

[0167] Methods of inhibiting CoV replication in a subject are also provided. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a modified ACE2 polypeptide, fragment thereof, fusion protein or composition disclosed herein. Also provided is a method of treating a CoV infection (e.g. COVID-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 embodiments, the method comprises administering to the subject a therapeutically effective amount of a modified ACE2 polypeptide, fragment thereof, fusion protein or composition disclosed herein, in combination with a monoclonal antibody, or antigen-binding fragment thereof, for the treatment and/or prophylaxis of SARS-CoV-2 infection or infection by another coronavirus that utilizes ACE2 as a cellular receptor..

[0168] In some examples, the subject is elderly or has an underlying medical condition (such as heart disease, lung disease, obesity, or diabetes). In some examples, the subject has COVID- 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 via inhalation (such as by using a nebulizer). In specific non-limiting examples, the modified ACE2 polypeptide, fusion protein or composition is administered via 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 inhalatory routes, such as direct instillation in the nasotracheal or endotracheal tubes in an intubated patient. In specific non-limiting examples, the amino acid sequence of the modified ACE2 polypeptide comprises of consists of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 19.

[0169] Also provided is a method of prophylactically treating (e.g. preventing) 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 both pre-exposure prophylaxis and post-exposure prophylaxis. In some embodiments, the prophylactic treatment method comprises administering to the subject a therapeutically effective amount of a modified ACE2 polypeptide, fragment thereof, fusion protein or composition disclosed herein, in combination with a monoclonal antibody, or antigen-binding fragment thereof, for the treatment and/or prophylaxis of SARS-CoV-2 infection or infection by another coronavirus that utilizes ACE2 as a cellular receptor. . In some examples, the subject is elderly or has an underlying medical condition. In some examples, the underlying condition is cardiac disease, lung disease, obesity, or diabetes. In some examples, the subject has been exposed to patients with COVID- 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 via inhalation (such as by using a nebulizer). Other routes of administration to the lungs or respiratory tract include bronchial, intranasal, or other inhalatory routes, such as direct instillation in the nasotracheal or endotracheal tubes in an intubated patient. In specific non-limiting examples, the amino acid sequence of the modified ACE2 polypeptide comprises of consists of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 19.

[0170] In some examples of the prophylactic treatment, the treatment comprises preexposure prophylaxis. For example, a subject exposed to a high-risk environment, such as a health care worker or essential worker, can be administered a modified ACE polypeptide, fusion protein or composition thereof to reduce their risk of SARS-CoV-2 infection and/or development of COVID- 19. In some embodiments, the pre-exposure prophylactic method comprises administering to the subject a therapeutically effective amount of a modified ACE2 polypeptide, fragment thereof, fusion protein or composition disclosed herein, in combination with a monoclonal antibody, antigen-binding fragment thereof, or coronavirus antiviral for the treatment and/or prophylaxis of SARS-CoV-2 infection or infection by another coronavirus that utilizes ACE2 as a cellular receptor. In particular non-limiting examples, the pre-exposure prophylactic treatment comprises administration of the polypeptide, fusion protein or composition intratracheally or by inhalation (such as by using a nebulizer).

[0171] In some examples of the prophylactic treatment, the treatment comprises postexposure prophylaxis. In some embodiments, the post-exposure prophylactic method comprises administering to the subject a therapeutically effective amount of a modified ACE2 polypeptide, fragment thereof, fusion protein or composition disclosed herein, in combination with a monoclonal antibody, or antigen-binding fragment thereof, for the treatment and/or prophylaxis of SARS-CoV-2 infection or infection by another coronavirus that utilizes ACE2 as a cellular receptor. In this type of method, the subject is administered the modified ACE polypeptide, fusion protein or composition thereof immediately or shorter after exposure to SARS-CoV-2, such as 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 administration of the polypeptide, fusion protein or composition intratracheally or by inhalation (such as by using a nebulizer).

[0172] Also provided are nucleic acid molecules and vectors that encode a modified ACE2 polypeptide or fusion protein disclosed herein. In some examples, the nucleic acid molecules and vectors have different codon usage or may be codon optimized for expression in specific cell types, such as mammalian cells. In some examples, the nucleic acid molecules and vectors carry natural human polymorphisms.

[0173] Further provided are compositions that include a nucleic acid molecule or vector disclosed herein and a pharmaceutically acceptable carrier. [0174] Methods of inhibiting CoV replication in a subject by administering a therapeutically effective amount (or a prophylactically effective amount for pre- or postexposure prophylactic methods) of a nucleic acid molecule, vector or composition disclosed herein are further provided. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a nucleic acid molecule, vector or composition disclosed herein, in combination with a monoclonal antibody, or antigen-binding fragment thereof, for the treatment and/or prophylaxis of SARS-CoV-2 infection or infection by another coronavirus that utilizes ACE2 as a cellular receptor. Further provided are methods of treating a 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 via inhalation (such as by using a nebulizer). In specific 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 inhalatory routes, such as direct instillation in the nasotracheal or endotracheal tubes in an intubated patient. In some examples, the subject is elderly or has an underlying medical condition (such as heart disease, lung disease, obesity, or diabetes). In some examples, the subject has COVID- 19. In some examples, the subject is a healthcare worker.

[0175] In some embodiments, the subject is administered one or more doses of a modified ACE2 polypeptide, fusion protein, nucleic acid, or composition disclosed herein. For example, the subject may be administered one or more, two or more, three or more, four or more, or five or more doses, such as twice daily, once daily, every other day, twice per week, once per week, or monthly. One of ordinary skill in the art can select an appropriate number of doses and timing of administration based on factors such as the subject being treated, condition of the subject, and underlying conditions.

[0176] 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 coronavirus strains. 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 (HKUl-CoV), human coronavirus OC43 (OC43-CoV), human coronavirus 229E (229E-CoV), or human coronavirus NL63 (NL63-CoV). In other examples, the coronavirus is a zoonotic coronavirus, such as a zoonotic coronavirus that has the potential to cross over to infect humans. In specific examples, the coronavirus is a bat coronavirus or a rodent coronavirus. In specific non-limiting examples, the bat coronavirus is LYRal l, Rs4231, Rs7327, Rs4084 or RsSHC014.

[0177] Further provided are kits that include a modified polypeptide or fusion protein disclosed herein bound to a solid support.

IV. Combinations of Modified ACE2 Polypeptides and Monoclonal Antibodies

[0178] In some embodiments, modified ACE2 polypeptides as described herein, or fragments thereof, are used in combination with monoclonal antibodies, or antigen-binding fragments thereof, to inhibit coronavirus replication in a subject. In some embodiments, fusion proteins of the present disclosure comprising a modified ACE2 polypeptide, or fragment thereof, and a heterologous protein (e.g., human IgGl Fc), are used in combination with monoclonal antibodies, or antigen-binding fragments thereof, to inhibit coronavirus replication in a subject. In some embodiments, nucleic acids encoding a modified ACE2 polypeptide of the present disclosure or fragment thereof, or fusion protein of the present disclosure or fragment thereof, and a heterologous protein (e.g., human IgGl Fc), are used in combination with monoclonal antibodies, or antigen-binding fragments thereof, to inhibit coronavirus replication in a subject.

[0179] As used herein, the term “monoclonal antibody” or “monoclonal antibodies” refers to a preparation or preparations of antibody molecules having a single molecular composition. A monoclonal antibody displays a unique antigen-binding site having a unique binding specificity and affinity for particular epitopes. In some embodiments, monoclonal antibodies of the present application can be of any isotype, for example, IgG, IgM, IgA, IgD, or IgE. In some embodiments, monoclonal antibodies can be IgGl, IgG2, IgG3, IgG4. In some embodiments, monoclonal antibodies can be human, or from another animal such as a mouse, rat, rabbit, cat, dog, goat, horse, or camel.

[0180] As used herein, the term “antigen-binding fragment” refers to a part of an immunoglobulin (Ig) molecule comprising an antigen-binding site and is capable of antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues of the N-terminal variable (“V”) domains of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as “hypervariable regions” which are interposed between more conserved flanking stretches known as “framework regions,” or “FR.” Thus, the term “FR” refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. In a human antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of an antigen to which the antigen-binding site specifically binds, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” In certain animals, such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain providing a “single domain antibody.” Antigen-binding sites can exist in an intact antibody, in an antigen-binding fragment of an antibody that retains the antigenbinding surface (for example. Fab, Fab’, F(ab’)2, or in a recombinant polypeptide such as a single chain variable fragment (scFv), using a peptide linker to connect the heavy chain variable domain to the light chain variable domain in a single polypeptide, a minibody, or a nanobody (VHH).

[0181] As used herein, the term “fragment thereof’ refers to a portion of a protein or polypeptide that maintains the ability to perform a biological function of the whole protein or polypeptide. For example, a functional fragment of a polypeptide or protein of the present application maintains its ability to bind its cognate binding partner or ligand.

[0182] In some embodiments, monoclonal antibodies, or antigen-binding fragments thereof, used in combination with: (i) modified ACE2 polypeptides of the present disclosure, or fragments thereof; (ii) fusion proteins of the present disclosure, or fragments thereof; or (iii) nucleic acid molecules encoding modified ACE2 polypeptides of the present disclosure or fragments thereof, or fusion proteins of the present disclosure or fragments thereof, bind to a SARS-CoV-2 spike protein.

[0183] In some embodiments, a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds SARS-CoV-2 spike protein comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). Monoclonal antibodies, or antigenbinding fragments thereof, of the present disclosure comprise VH and VL domains, and/or HC CDRs and LC CDRs that specifically bind SARS-CoV-2 spike protein, such as the VH and VL domains and/or HC CDRs and LC CDRs disclosed in, but not limited to, International Patent Publication WO2021045836, U.S. Patent Publication US 2021/0261650, the disclosures of which are incorporated herein in their entirety. Exemplary VH and VL domains, and/or HC CDRs and LC CDRs capable of binding SARS-CoV-2 spike protein also include the VH and VL domain, and/or HC CDR and LC CDR sequences of LY-CoV555 (Bamlanivimab, Eh Lilly), and LY-C0VOI6 (Etesevimab, Eh Lilly).

[0184] Table 1 lists VH and VL CDRs that, in combination, can specifically bind to SARS-CoV-2 spike protein. In some embodiments, a monoclonal antibody, or antigenbinding fragment thereof, that specifically binds SARS-CoV-2 spike protein comprises VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 sequences selected from the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 sequences listed in Table 1, determined under the EU numbering scheme, IMGT unique numbering scheme, Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk A M, (1987), J. Mol. Biol. 196: 901-917), MacCallum (see MacCallum R M et al., (1996) J. Mol. Biol. 262: 732-745), or any other CDR determination method known in the art.

[0001] Table 1 additionally lists amino acid sequences of exemplary VH and VL domains that, in combination, can specifically bind to SARS-CoV-2 spike protein. In some embodiments, monoclonal antibodies, or antigen-binding fragments thereof, of the present disclosure comprise VH and VL having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to VH domain and VL sequences listed in Table 1.

[0185] Table 1 also lists amino acid sequences of heavy chains (HC) and light chains (LC) that, in combination, can specifically bind SARS-CoV-2 spike protein. In some embodiments, the heavy chain and the light chains have sequences at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to HC and LC sequences listed in Table 1.

[0186] Unless indicated otherwise, the CDR sequences provided in Table 1 are determined under the EU numbering scheme.

[0187] TABLE 1. Exemplary Sequences for monoclonal antibodies, or antigenbinding fragments thereof, that bind SARS-CoV-2

[0188] As used herein “multispecific,” refers to the ability of an antibody, or fragment thereof, to bind more than one epitope. For example, an antibody, or fragment thereof, may bind to two epitopes (i.e. bispecific), three epitopes (z.e. trispecific), or more epitopes. In some embodiments, monoclonal antibodies, or antigen-binding fragments thereof, used in combination with (i) modified ACE2 polypeptides of the present disclosure, or fragments thereof; (ii) fusion proteins of the present disclosure, or fragments thereof; or (iii) nucleic acid molecules encoding modified ACE2 polypeptides of the present disclosure or fragments thereof, or fusion proteins of the present disclosure or fragments thereof, are multispecific. In some embodiments, monoclonal antibodies, or antigen-binding fragments thereof, used in combination with (i) modified ACE2 polypeptides of the present disclosure, or fragments thereof; (ii) fusion proteins of the present disclosure, or fragments thereof; or (iii) nucleic acid molecules encoding modified ACE2 polypeptides of the present disclosure or fragments thereof, or fusion proteins of the present disclosure or fragments thereof, are bispecific.

[0189] In some embodiments, when used in combination with monoclonal antibodies, or antigen-binding fragments thereof, (i) modified ACE2 polypeptides of the present disclosure, or fragments thereof; (ii) fusion proteins of the present disclosure, or fragments thereof; or (iii) nucleic acid molecules encoding modified ACE2 polypeptides of the present disclosure or fragments thereof, or fusion proteins of the present disclosure or fragments thereof, are administered to a subject as a single dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, ten doses, or more. In some embodiments (i) modified ACE2 polypeptides of the present disclosure, or fragments thereof; (ii) fusion proteins of the present disclosure, or fragments thereof; or (iii) nucleic acid molecules encoding modified ACE2 polypeptides of the present disclosure or fragments thereof, or fusion proteins of the present disclosure or fragments thereof, are administered one or more times daily.

[0190] In some embodiments, when used in combination with (i) modified ACE2 polypeptides of the present disclosure, or fragments thereof; (ii) fusion proteins of the present disclosure, or fragments thereof; or (iii) nucleic acid molecules encoding modified ACE2 polypeptides of the present disclosure or fragments thereof, or fusion proteins of the present disclosure or fragments thereof, monoclonal antibodies, or antigen-binding fragments thereof, are administered to a subject as a single dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, ten doses, or more. In some embodiments, monoclonal antibodies, or antigen-binding fragments thereof, are administered one or more times daily.

[0191] In some embodiments, when used in combination monoclonal antibodies, or antigen-binding fragments thereof, and (i) modified ACE2 polypeptides of the present disclosure, or fragments thereof; (ii) fusion proteins of the present disclosure, or fragments thereof; or (iii) nucleic acid molecules encoding modified ACE2 polypeptides of the present disclosure or fragments thereof, or fusion proteins of the present disclosure or fragments thereof, are administered concurrently.

[0192] In some embodiments, when used in combination (i) modified ACE2 polypeptides of the present disclosure, or fragments thereof; (ii) fusion proteins of the present disclosure, or fragments thereof; or (iii) nucleic acid molecules encoding modified ACE2 polypeptides of the present disclosure or fragments thereof, or fusion proteins of the present disclosure or fragments thereof, are administered before administration of monoclonal antibodies, or antigen-binding fragments thereof. For example, (i) modified ACE2 polypeptides of the present disclosure, or fragments thereof; (ii) fusion proteins of the present disclosure, or fragments thereof; or (iii) nucleic acid molecules encoding modified ACE2 polypeptides of the present disclosure or fragments thereof, or fusion proteins of the present disclosure or fragments thereof, are administered at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks at least 4 weeks, at least 8 weeks, at 12 weeks, or at least 1 year before administration of monoclonal antibodies, or antigenbinding fragments thereof.

[0193] In other embodiments, when used in combination monoclonal antibodies, or antigen-binding fragments thereof, are administered before administration of (i) modified ACE2 polypeptides of the present disclosure, or fragments thereof; (ii) fusion proteins of the present disclosure, or fragments thereof; or (iii) nucleic acid molecules encoding modified ACE2 polypeptides of the present disclosure or fragments thereof, or fusion proteins of the present disclosure or fragments thereof. For example, monoclonal antibodies, or antigenbinding fragments thereof, are administered at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks at least 4 weeks, at least 8 weeks, at 12 weeks, or at least 1 year before administration of (i) modified ACE2 polypeptides of the present disclosure, or fragments thereof; (ii) fusion proteins of the present disclosure, or fragments thereof; or (iii) nucleic acid molecules encoding modified ACE2 polypeptides of the present disclosure or fragments thereof, or fusion proteins of the present disclosure or fragments thereof. [0194] In some embodiments, when used in combination with (i) modified ACE2 polypeptides of the present disclosure, or fragments thereof; (ii) fusion proteins of the present disclosure, or fragments thereof; or (iii) nucleic acid molecules encoding modified ACE2 polypeptides of the present disclosure or fragments thereof, or fusion proteins of the present disclosure or fragments thereof, monoclonal antibodies, or antigen-binding fragments thereof are administered to a subject subcutaneously, intravenously, intramuscularly, intratracheally, by inhalation, intraarterially, intraperitoneally, intrapleurally, or intrathecally.

[0195] In some embodiments, the present disclosure provides a bispecific fusion protein comprising: (i) a modified ACE2 polypeptide as described herein, or fragments thereof, and (ii) a monoclonal antibody, or antigen-binding fragment thereof, that binds a coronavirus. In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, is fused to the N-terminus of a modified ACE2 polypeptide of the present disclosure, or fragment thereof. In other embodiments, the monoclonal antibody, or antigen-binding fragment thereof, is fused to the C-terminus of a modified ACE2 polypeptide of the present disclosure, or fragment thereof. In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, is fused to a modified ACE2 polypeptide of the present disclosure, or fragment thereof, via a polypeptide linker. In some embodiments of the bispecific fusion proteins of the present disclosure, the antigen binding fragments comprise one or more antigen binding sites selected from, but not limited to, an scFv, Fab, Fab’, or F(ab’)2,. In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof binds SARS-CoV-2 S. In some embodiments, the monoclonal antibody comprises an Fc domain that is capable of binding the Fc-receptor (FcR) on the cell surface of immune effector cells. In some embodiments, the monoclonal antibody comprises one or more than one mutation that silences Fc effector functions.

[0196] In some embodiments, methods for the treatment and/or prophylaxis of a coronavirus infection are provided, comprising administering to the subject a therapeutically effective amount of a nucleic acid molecule, vector or composition disclosed herein, wherein the nucleic acid, or vector encodes a bispecific fusion protein as disclosed herein. In some examples, the nucleic acid or vector is administered intravenously.

[0197] In some embodiments bispecific fusion proteins of the present disclosure, or nucleic acids or vectors encoding the same, are administered to a subject in need thereof subcutaneously, intravenously, intramuscularly, intratracheally, by inhalation (e.g., by nebulizer), intraarterially, intraperitoneally, intrapleurally, or intrathecally. In some embodiments, bispecific fusion proteins of the present disclosure, or nucleic acids or vectors encoding the same, are administered to a subject in need thereof as a single dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, ten doses, or more. In some embodiments, bispecific fusion proteins of the present disclosure, or nucleic acids or vectors encoding the same, are administered one or more times daily.

[0198] In some embodiments, the present disclosure provides pharmaceutical compositions comprising: (i) a modified ACE2 polypeptide, comprising a 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 increased binding to the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relative to wildtype human ACE2, a fusion protein comprising the modified ACE2 polypeptide, or fragment thereof, fused to a heterologous peptide, or a nucleic acid molecule encoding the modified ACE2 polypeptide or the fusion protein; (ii) a monoclonal antibody, or antigen-binding fragment thereof, that binds SARS-CoV-2; and (iii) a pharmaceutically acceptable carrier.

[0199] In some embodiments, the present disclosure provides pharmaceutical compositions comprising: (i) a bispecific protein comprising a modified angiotensinconverting enzyme 2 (ACE2) polypeptide, comprising a 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 increased binding to the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relative to wild-type human ACE2, and a monoclonal antibody, or antigen-binding fragment thereof, that binds a coronavirus, and (ii) a pharmaceutically acceptable carrier.

[0200] The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

EXAMPLES

[0002] The disclosure now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the scope of the disclosure in any way.

EXAMPLE 1:

[0201] Since human ACE2 has not evolved to recognize SARS-CoV-2 S, it was hypothesized that mutations may be found that increase affinity for therapeutic and diagnostic applications. The coding sequence of full length ACE2 with an N-terminal c-myc epitope tag was diversified by introduction of degenerate codons to create a library containing all possible single amino acid substitutions at 117 sites spanning the entire interface with S and lining the substrate-binding cavity. S binding is independent of ACE2 catalytic activity (Moore et al., J Virol,' 2004 Oct;78(19): 10628-10635) and occurs on the outer surface of ACE2 (Yan et al., Science. 2020 Mar 4;:eabb2762; Li et al., Science. 2005 Sep 16;309(5742): 1864-1868), whereas angiotensin substrates bind within a deep cleft that houses the active site (Towler et al., J Biol Chenr, 2004 Apr 23;279(17): 17996-18007). Substitutions within the substrate-binding cleft of ACE2 therefore act as controls that are anticipated to have minimal impact on S interactions, yet may be useful for engineering out substrate affinity to enhance in vivo safety. However, the benefits of catalytically inactive sACE2 for treating COVID-19 have been questioned (Kruse, FlOOORes; 2020;9(72):72).

[0202] The ACE2 library was transiently expressed in human Expi293F cells under conditions that typically yield no more than one coding variant per cell, providing a tight link between genotype and phenotype (Heredia et al., J Immunol,' 2018 Apr 20;200(ll):jil800343-3839; Park etal., J Biol Chem; 2019;294(13):4759-4774). Cells were then incubated with a subsaturating dilution of medium containing the RBD (a.a. 333-529 of SEQ ID NO: 2) of SARS-CoV-2 fused C-terminally to superfolder GFP (sfGFP: (Pedelacq et al., Nat Biotechnol. 2006 Jan;24(l):79-88)) (FIG. 1A). Levels of bound S-RBD-sfGFP correlate with surface expression levels of myc-tagged ACE2 measured by dual color flow cytometry. Compared to cells expressing wild type ACE2 (FIG. 1C), many variants in the ACE2 library failed to bind S-RBD, while there appeared to be a smaller number of ACE2 variants with higher binding signals (FIG. ID). Cells expressing ACE2 variants with high or low binding to S-RBD were collected by fluorescence-activated cell sorting (FACS), referred to as "nCoV-S-High" and "nCoV-S-Low" sorted populations, respectively. During FACS, fluorescence signal for bound S-RBD-sfGFP continuously declined, requiring the collection gates to be regularly updated to 'chase' the relevant populations. This is consistent with S- RBD dissociating over hours during the experiment. Reported affinities of S-RBD for ACE2 range from 1 to 15 nM (Walls etal., Cell. 2020 Mar 6; 181(2):281-292.e6; Wrapp et al., Science. 2020 Feb 19;:eabb2507).

[0203] Transcripts in the sorted populations were deep sequenced, and frequencies of variants were compared to the naive plasmid library to calculate the enrichment or depletion of all 2,340 coding mutations in the library (FIG. 2). This approach of tracking an in vitro selection or evolution by deep sequencing is known as deep mutagenesis (Fowler and Fields, Nat Methods. 2014 Aug;ll(8):801-807). Enrichment ratios (FIGS. 3A and 3B) and residue conservation scores (FIGS. 3D and 3E) closely agree between two independent sort experiments, giving confidence in the data. For the most part, enrichment ratios (FIG. 3C) and conservation scores (FIG. 3F) in the nCoV-S-High sorts were anticorrelated with the nCoV-S-Low sorts, with the exception of nonsense mutations which were appropriately depleted from both gates. This indicated that most, but not all, nonsynonymous mutations in ACE2 did not eliminate surface expression. The library was biased towards solvent-exposed residues and has few substitutions of buried hydrophobics that might have bigger effects on plasma membrane trafficking (Park et al., J Biol Chenr, 2019;294(13):4759-4774).

[0204] Mapping the experimental conservation scores from the nCoV-S-High sorts to the structure of S-RBD-bound ACE2 (Yan et al., Science. 2020 Mar 4;:eabb2762) showed that residues buried in the interface tend to be conserved, whereas residues at the interface periphery or in the substrate-binding cleft were mutationally tolerant (FIG. 4A). The region of ACE2 surrounding the C-terminal end of the ACE2 al helix and P3-|34 strands has a weak tolerance of polar residues, while amino acids at the N-terminal end of al and the C-terminal end of a2 prefer hydrophobics (FIG. 4B), likely in part to preserve hydrophobic packing between al-a2. These discrete patches contact the globular RBD fold and a long protruding loop of the RBD, respectively.

[0205] Two ACE2 residues, N90 and T92 that together form a consensus N- glycosylation motif, are notable hot spots for enriched mutations (FIGS. 2 and 4 A). Indeed, all substitutions of N90 and T92, with the exception of T92S which maintains the N-glycan, are highly favorable for S-RBD binding, and the N90-glycan is thus predicted to partially hinder S/ACE2 interaction. [0206] Mining the data identified many ACE2 mutations that were enriched for S-RBD binding. For instance, there were 122 mutations to 35 positions in the library that had log2 enrichment ratios >1.5 in the nCoV-S-High sort. Table 2 lists these mutations. Table 3 lists mutations with log2 enrichment ratios >2.0. Table 4 list mutations with log2 enrichment ratios >2.5.

[0207] At least a dozen ACE2 mutations at the structurally characterized interface enhance S-RBD binding, and may be useful for engineering highly specific and tight binders of SARS-CoV-2 S, especially for point-of-care diagnostics. The molecular basis for how some of these mutations enhance S-RBD binding can be rationalized from the S-RBD-bound cryo-EM structure (FIG. 4C): hydrophobic substitutions of ACE2-T27 increase hydrophobic packing with aromatic residues of S-RBD, ACE2-D30E extends an acidic side chain to reach S-RBD-K417, and aromatic substitutions of ACE2-K31 contribute to an interfacial cluster of aromatics. However, engineered ACE2 receptors with mutations at the interface may present binding epitopes that are sufficiently different from native ACE2 that virus escape mutants can emerge, or they may be strain specific and lack breadth. Instead, attention was drawn to mutations in the second shell and farther that do not directly contact the S-RBD but instead have putative structural roles. For example, proline substitutions were enriched at five library positions (S19, L91, T92, T324 and Q325) where they might entropically stabilize the first turns of helices. Proline was also enriched at H34, where it may enforce the central bulge in al. Multiple mutations were also enriched at buried positions where they will change local packing (e.g. A25V, L29F, W69V, F72Y and L351F). The selection of ACE2 variants for high binding signal therefore not only reports on affinity, but also on presentation at the membrane of folded structure recognized by SARS-CoV-2 S. The presence of enriched structural mutations in the sequence landscape is especially notable considering the ACE2 library was biased towards solvent-exposed positions.

[0208] TABLE 2. ACE2 mutations with enhanced binding signals to SARS-CoV-2

S. Log2 enrichment ratios in selection > 1.5

S19P H34A E75T N90I T92Q R393K

E23F H34S E75K N90V T92D R518G

Q24T H34P E75R N90A T92E

A25V E35V E75W N90S T92K

K26I E35C E75G N90T T92R

K26A L39K Q76M N90Q T92H

K26D L39R Q76I N90D T92W T27M F40D Q76V N90E T92Y

T27L F40R Q76T N90K T92F

T27A Y41R Q76R N90R T92P

T27D Q42M Q76Y N90H T92G

T27K Q42L L79I N90W T92C

T27H Q42I L79V N90Y T324E

T27W Q42V L79T N90F T324P

T27Y Q42K L79W N90P Q325P

T27F Q42C L79Y N90G N330L

T27C A65W L79F N90C N330H

L29F W69I L79P L91P N330W

D30I W69V M82C T92M N330Y

D30E I69T Q89I T92L N330F

K31W I69K Q89D T92I L351F

K31Y F72Y Q89P T92V A386L

N33D E75A N90M T92A A386I

H34V E75S N90L T92N P389D

[0209] TABLE 3. ACE2 mutations with enhanced binding signals to SARS-CoV-2

S. Log2 enrichment ratios in selection > 2.0

S19P L39K L79F N90G T92Y

A25V L39R Q89P N90C T92F

T27M Q42M N90M L91P T92P

T27L Q42L N90L T92M T92G

T27A Q42C N90I T92L T92C

T27D W69V N90V T92I T324E

T27H F72Y N90A T92V T324P

T27W E75K N90S T92A Q325P

T27Y E75R N90T T92N N330L

T27F Q76V N90Q T92Q N330H

T27C Q76T N90D T92D N330W

D30E L79I N90E T92E N330Y

K31W L79V N90K T92K N330F

H34V L79T N90R T92R L351F

H34A L79W N90H T92H A386L

H34P L79Y N90P T92W [0210] TABLE 4. ACE2 mutations with enhanced binding signals to SARS-CoV-2

S. Log2 enrichment ratios in selection > 2.5

A25V L79I N90E T92E N330H

T27M L79V N90H T92R N330W

T27L L79T L91P T92H N330Y

T27Y L79W T92M T92W N330F

K31W L79Y T92L T92Y A386L H34V L79F T92I T92F

H34A N90A T92V T92G

H34P N90S T92N T92C

Q42L N90T T92Q T324P

Q42C N90Q T92D Q325P

EXAMPLE 2:

[0211] Thirty single substitutions highly enriched in the nCoV-S-High sort were validated by targeted mutagenesis (FIG. 5). Binding of RBD-sfGFP to full length ACE2 mutants increased compared to wild type, yet improvements were small and were most apparent on cells expressing low ACE2 levels (FIG. 5 A). Differences in ACE2 expression between the mutants also correlated with total levels of bound RBD-sfGFP (FIG. 5C), demonstrating how one must use caution in interpreting deep mutational scan data as mutations may impact both activity and expression. To rapidly assess mutations in a format more relevant to therapeutic development, the soluble ACE2 protease domain was fused to sfGFP. Expression levels of sACE2-sfGFP were qualitatively evaluated 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). A single substitution (T92Q) that eliminates the N90 glycan gave a small increase in binding signal (FIG. 6B), which was confirmed by analysis of purified protein (FIG. 12). Focusing on the most highly enriched substitutions in the selection for S binding that were also spatially segregated to minimize negative epistasis (Heredia et al., J Virol 93(ll):e00219-19, 2019), combinations of mutations in sACE2 gave large increases in S binding (Table 5 and FIG. 6B). While this assay only provides relative differences, the combinatorial mutants have enhanced binding by at least an order of magnitude. Unexplored combinations of mutations may have even greater effects.

[0212] TABLE 5. Combinatorial mutants of sACE2

[0213] A single variant, sACE2.v2, was chosen for purification and further characterized (FIG. 7). This variant was selected because it was well expressed fused to sfGFP and maintains the N90-glycan, and will therefore present a surface that more closely matches native sACE2 to minimize immunogenicity. The yield of sACE2.v2 was lower than the wild type protein when purified as an 8his-tagged protein (20% lower) or as an IgGl-Fc fusion (60% lower), and by analytical size exclusion chromatography (SEC) a small fraction of sACE2.v2 was found to aggregate after incubation at 37°C for 40 h (FIG. 7D). Otherwise, sACE2.v2 was indistinguishable from wild type by SEC (FIG. 7C).

[0214] In flow cytometry experiments using purified 8his-tagged sACE2, only sACE2.v2-8h was found to bind strongly to full length S at the cell surface, suggestive that wild type sACE2 has a high off-rate that causes dissociation during sample washing (FIG. 8A and FIG. 13). Differences between wild type and the variant were less pronounced in the context of an IgGl-Fc fusion (FIG. 8A and FIG. 13), indicating that avidity masks gains in binding of the mutant, again suggestive that there are off-rate differences between wild type and variant sACE2. Soluble ACE2.v2-8h outcompetes wild type sACE2-IgGl for binding to S-expressing cells, yet wild type sACE2-8h does not outcompete sACE2-IgGl even at 10- fold higher concentrations (FIG. 8B). Furthermore, only engineered sACE2.v2-8h effectively competed with anti-RBD IgG in the serum of three recovered COVID-19 patients when tested by ELISA (FIG. 8E). This aligns with studies showing that while sACE2 is highly effective at inhibiting SARS-CoV-2 replication in cell lines and organoids, extremely high concentrations are required (Monteil et al., Cell DOI: 10.1016/j. cell.2020.04.004: 1-28, 2020). Using biolayer interferometry (BLI), sACE2.v2 was found to have 65-fold tighter affinity than the wild type protein for immobilized RBD, almost entirely due to a slower off- rate (Table 6 and FIGS. 8C and 8D).

[0215] TABLE 6. Summary of BLI kinetics data

All measurements were performed using capture of IgGl (Fc)-fused proteins to an antihuman IgG Fc biosensor surface.

3-4 analyte concentrations were used for each experiment.

ND, not determined.

[0216] Across all experiments, whether ACE2 was purified as a 8his-tagged protein or used as a sfGFP-fusion in expression medium, and whether full-length S was expressed on the plasma membrane or the isolated RBD was immobilized on a biosensor surface, the characterized sACE2.v2 variant consistently showed one to two orders of magnitude tighter binding. These experiments support the key discovery from deep mutagenesis that mutations in human ACE2 exist that increase binding to S of SARS-CoV-2.

[0217] To address the decreased expression of sACE2.v2, it was hypothesized that the mutational load is too high. In second-generation designs, each of the four mutations in sACE2.v2 was reverted back to the wild type identity (Table 5) and binding to full length S at the cell surface was found to remain tight (FIG. 9A). One of the variants (sACE2.v2.4 with mutations T27Y, L79T and N330Y) was purified with even higher yields than wild type and displayed tight nanomolar binding to the RBD (FIG. 9).

[0218] The ACE2 construct was lengthened to include the neck/dimerization domain, yielding a stable dimer (FIG. 10 A) referred to here as sACE22, which binds with tight avidity to S on the cell surface or immobilized RBD on a biosensor (FIG. 14). Compared to the wild type, dimeric sACE22.v2.4 more effectively competes with IgG antibodies present in serum of recovered patients (FIG. 10B). By immobilizing sACE22-IgGl (FIG. 15) to a biosensor surface and incubating with monomeric RBD-8h as the analyte, the KD of RBD for wild type SACE22 was determined to be 22 nM (FIG. 10C) in close agreement to previous reports (Wrapp et al., Science, eabb2507, 2020; Shang et al., Nature. 382, 1199, 2020), whereas SACE22.V2.4 bound with 600 pM affinity (FIG. 10D). This compares favorably with 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; Wee 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).

[0219] The efficacy of monomeric sACE2.v2.4 to neutralize SARS-CoV-2 infection of cultured VeroE6 cells exceeded the wild type protein by nearly two orders of magnitude (FIG. 11), consistent with the biochemical binding data. Wild type, dimeric sACE22 is itself two orders of magnitude more potent than monomer, indicating strong avid interactions with spike on the virion surface, and dimeric sACE22.v2.4 is yet again more potent with a subnanomolar ICso (FIG. 11). Dimeric sACE22.v2.4 also potently neutralizes SARS-CoV-1 (FIG. 11), despite no consideration of SARS-CoV-1 S structure or sequence during the engineering process, and it is possible that the decoy receptor will neutralize diverse ACE2- utilizing coronaviruses that have yet to cross over to humans.

[0220] To improve safety, untagged sACE22.v2.4 was manufactured in ExpiCHO-S cells (FIG. 16A) and found to be stable after incubation at 37 °C for 6 days (FIG. 16B). The protein competes with wild type sACE22-IgGl for cell-expressed S (FIG. 16C) and binds with tight avidity to immobilized RBD (FIG. 16D). In addition to inhibiting virus entry, recombinant sACE2 may have a second therapeutic mechanism: proteolysis of angiotensin II (a vasoconstrictive peptide hormone) to relieve symptoms of respiratory distress (Imai et al., Nature. 436, 112-116, 2005; Treml et al., Crit. Care Med. 38, 596-601, 2010). Soluble ACE22.V2.4 is found to be catalytically active, albeit with reduced activity (FIG. 17).

[0221] While deep mutagenesis of viral proteins in replicating viruses has been extensively pursued to understand escape mechanisms from drugs and antibodies, the work here shows how deep mutagenesis can be directly applicable to therapeutic design when the selection method is decoupled from virus replication and focused on host factors. With astonishing speed, the scientific community has identified multiple candidates for the treatment of COVID-19, especially monoclonal antibodies with exceptional affinity for protein S. The studies disclosed herein show how comparable affinity can be engineered into the virus' natural receptor, while also providing insights in to the molecular basis for initial virus-host interactions. EXAMPLE 3:

Materials and Methods

[0222] Plasmids The mature polypeptide (a. a. 19-805) of human ACE2 (GenBank NM_021804.1) was cloned in to the Nhel-Xhol sites of pCEP4 (Invitrogen) with a N- terminal HA leader (MKTIIALSYIFCLVFA), myc-tag, and linker (GSPGGA). Soluble ACE2 fused to superfolder GFP (Pedelacq et al., Nat. Biotechnol. 24, 79-88, 2006) was constructed by genetically joining the protease domain (a.a. 1-615) of ACE2 to sfGFP (GenBank ASL68970) via a gly/ser-rich linker (GSGGSGSGG; SEQ ID NO: 77), and pasting between the Nhel-Xhol sites of pcDNA3.1(+) (Invitrogen). Equivalent sACE2 constructs were cloned with a GSG linker and 8 histidine tag or a GS linker and the Fc region of IgGl (a.a. D221-K447), while dimeric sACE22 constructs encompassed a.a. 1-732 and were otherwise identical. A synthetic human codon-optimized gene fragment (Integrated DNA Technologies) for the RBD (a.a. 333-529) of SARS-CoV-2 S (GenBank YP_009724390.1) was N-terminally fused to a HA leader and C-terminally fused to either superfolder GFP, the Fc region of IgGl or a 8 histidine tag. Assembled DNA fragments were ligated in to the Nhel-Xhol sites of pcDNA3.1(+). Human codon-optimized full length S was subcloned from pUC57-2019-nCoV-S(Human) (Molecular Cloud), both untagged (a.a. 1- 1273) and with a N-terminal HA leader (MKTIIALSYIFCLVFA; SEQ ID NO: 78), myc-tag and linker (GSPGGA; SEQ ID NO: 79) upstream of the mature polypeptide (a.a. 16-1273).

[0223] Tissue Culture. Expi293F cells (ThermoFisher) were cultured in Expi293 Expression Medium (ThermoFisher) at 125 rpm, 8 % CO2, 37 °C. For production of RBD- sfGFP, RBD-IgGl, sACE2-8h and sACE2-IgGl, cells were prepared to 2 x 10 ⁶ / ml. Per ml of culture, 500 ng of plasmid and 3 pg of polyethylenimine (MW 25,000; Polysciences) were mixed in 100 pl of OptiMEM (Gibco), incubated for 20 minutes at room temperature, and added to cells. Transfection Enhancers (ThermoFisher) were added 18-23 h post-transfection, and cells were cultured for 4-5 days. Cells were removed by centrifugation at 800 x g for 5 minutes and medium was stored at -20 °C. After thawing and immediately prior to use, remaining cell debris and precipitates were removed by centrifugation at 20,000 x g for 20 minutes. Plasmids for expression of sACE2-sfGFP protein were transfected in to Expi293F cells using Expifectamine (ThermoFisher) according to the manufacturer's directions, with Transfection Enhancers added 22-1/2 h post-transfection, and medium supernatant harvested after 60 h.

[0224] Deep mutagenesis. 117 residues within the protease domain of ACE2 were diversified by overlap extension PCR (Procko et al., J. Mol. Biol. 425, 3563-3575, 2013) using primers with degenerate NNK codons. The plasmid library was transfected in to Expi293F cells using Expifectamine under conditions previously shown to typically give no more than a single coding variant per cell (Heredia et al., J. Immunol. 200, jil 800343-3839, 2018; Park et al., J Biol Chem 294, 4759-4774, 2019); 1 ng coding plasmid was diluted with 1,500 ng pCEP4-ACMV carrier plasmid per ml of cell culture at 2 x 10 ⁶ / ml, and the medium was replaced 2 h post-transfection. The cells were collected after 24 h, washed with ice-cold PBS supplemented with 0.2 % bovine serum albumin (PBS-BSA), and incubated for 30 minutes on ice with a 1/20 (replicate 1) or 1/40 (replicate 2) dilution of medium containing RBD-sfGFP into PBS-BSA. 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 a BD FACS Aria II at the Roy J. Carver Biotechnology Center. The main cell population was gated by forward/side scattering to remove debris and doublets, and DAPI was added to the sample to exclude dead cells. Of the myc-positive (Alexa 647) population, the top 67% were gated (FIG. IB). Of these, the 15 % of cells with the highest and 20% of cells with the lowest GFP fluorescence were collected (FIG. ID) in tubes coated overnight with fetal bovine serum and containing Expi293 Expression Medium. Total RNA was extracted from the collected cells using a GeneJET RNA purification kit (Thermo Scientific), and cDNA was reverse transcribed with high fidelity Accuscript (Agilent) primed with genespecific oligonucleotides. Diversified regions of ACE2 were PCR amplified as 5 fragments.

[0225] Flanking sequences on the primers added adapters to the ends of the products for annealing to Illumina sequencing primers, unique barcoding, and for binding the flow cell. Amplicons were sequenced on an Illumina NovaS eq 6000 using a 2 x 250 nt paired end protocol. Data were analyzed using Enrich (Fowler et al., Bioinformatics. 27, 3430-3431, 2011), and commands are provided in the GEO deposit. Briefly, the frequencies of ACE2 variants in the transcripts of the sorted populations were compared to their frequencies in the naive plasmid library to calculate a log2 enrichment ratio and then normalized by the same calculation for wild type. Wild type sequences were neither substantially enriched or depleted, and had log2 enrichment ratios of -0.2 to +0.2. [0226] Flow Cytometry Analysis of ACE2-S Binding. Expi293F cells were transfected with pcDNA3-myc-ACE2, pcDNA3-myc-S or pcDNA3-S plasmids (500 ng DNA per ml of culture at 2 x 10 ⁶ / ml) using Expifectamine (ThermoFisher). Cells were analyzed by flow cytometry 24 h post-transfection. To analyze binding of RBD-sfGFP to full length myc-ACE2, cells were washed with ice-cold PBS-BSA, and incubated for 30 minutes on ice with a 1/30 dilution of medium containing RBD-sfGFP and a 1/240 dilution of anti-myc Alexa 647 (clone 9B11, Cell Signaling Technology). Cells were washed twice with PBS- BSA and analyzed on a BD LSR II. To analyze binding of sACE2-sfGFP to full length myc- S, cells were washed with PBS-BSA, and incubated for 30 minutes on ice with a serial dilution of medium containing sACE2-sfGFP and a 1/240 dilution of anti-myc Alexa 647 (clone 9B11, Cell Signaling Technology). Cells were washed twice with PBS-BSA and analyzed on a BD Accuri C6, with the entire Alexa 647-positive population gated for analysis. To measure binding of sACE2-IgGl or sACE2-8h, myc-S or S transfected cells were washed with PBS-BSA and incubated for 30 minutes with the indicated concentrations of purified sACE2 in PBS-BSA. Cells were washed twice, incubated with secondary antibody (1/100 dilution of chicken anti-HIS-FITC polyclonal from Immunology Consultants Laboratory; or 1/250 anti-human IgG-APC clone HP6017 from BioLegend) for 30 minutes on ice, washed twice again, and fluorescence of the total population after gating by FSC-SSC to exclude debris was measured on a BD Accuri C6. Data were processed with FCS Express (De Novo Software) or BD Accuri C6 Software.

[0227] Purification of IgGl-Fc fused proteins. Cleared expression medium was incubated with KANEKA KanCapA 3G Affinity sorbent (Pall; equilibrated in PBS) for 90 minutes at 4 °C. The resin was collected on a chromatography column, washed with 12 column volumes (CV) PBS, and protein eluted with 5 CV 60 mM Acetate pH 3.7. The eluate was immediately neutralized with 1 CV of 1 M Tris pH 9.0, and concentrated with a 100 kD MWCO centrifugal device (Sartorius). Protein was separated on a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences) with PBS as the running buffer. Peak fractions were pooled, concentrated to ~10 mg/ml with excellent solubility, and stored at -80 °C after snap freezing in liquid nitrogen. Protein concentrations were determined by absorbance at 280 nm using calculated extinction coefficients for monomeric, mature polypeptide sequences. [0228] Purification of 8his-tagged proteins. HisPur Ni-NTA resin (Thermo Scientific) equilibrated in PBS was incubated with cleared expression medium for 90 minutes at 4 °C. The resin was collected on a chromatography column, washed with 12 column volumes (CV) PBS, and protein eluted with a step elution of PBS supplemented with 20 mM, 50 mM and 250 mM imidazole pH 8 (6 CV of each fraction). The 50 mM and 250 mM imidazole fractions were concentrated with a 30 kD MWCO centrifugal device (MilliporeSigma). Protein was separated on a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences) with PBS as the running buffer. Peak fractions were pooled, concentrated to ~5 mg/ml with excellent solubility, and stored at -80 °C after snap freezing in liquid nitrogen.

[0229] Other proteins. Untagged sACE22.v2.4 expressed in ExpiCHO-S cells (ThermoFisher) was manufactured and provided by Orthogonal Biologies, Inc.

[0230] Analytical size exclusion chromatography (SEC). Proteins (200 pl at 2 pM) were separated on a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences) equilibrated in PBS. MW standards were from Bio-Rad.

[0231] Biolayer Interferometry. Hydrated anti-human IgG Fc biosensors (Molecular Devices) were dipped in expression medium containing RBD-IgGl for 60 s. Biosensors with captured RBD were washed in assay buffer, dipped in the indicated concentrations of sACE2- 8h protein, and returned to assay buffer to measure dissociation. Data were collected on a BLItz instrument and analyzed with a 1 : 1 binding model using BLItz Pro Data Analysis Software (Molecular Devices). The assay buffer was 10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20, 0.5% non-fat dry milk (Bio-Rad).

[0232] Reagent and data availability. Plasmids are deposited with Addgene under IDs 141183-5, 145145-78, 149268-71, 149663-8 and 154098-106. Raw and processed deep sequencing data are deposited in NCBI’s Gene Expression Omnibus (GEO) with series accession no. GSE147194.

[0233] ACE2 catalytic activity assay. Activity was measured using the Fluorometric ACE2 Activity Assay Kit (BioVision) with protein diluted in assay buffer to 22, 7.4 and 2.5 nM final concentration. Specific activity is reported as pmol MCA produced per min (mU) per pmol of enzyme. Fluorescence was read on an Analyst HT (Molecular Devices). [0234] ELISA. Anti-RBD IgG titers were measured in human serum samples by indirect ELISA as described in Amant et al. (Nat. Med. 5, 562, 2020). Wells of a 96-well plate were coated with 2 pg/ml RBD-8h protein at 4°C overnight. After washing, the wells were blocked with PBS containing 3% non-fat milk at room temperature for 1 hour. Next, various dilutions of heat-inactivated serum (56 °C, 1 hour) were added to blocked wells. After 2 hours at room temperature, wells were washed, followed by incubation with goat antihuman IgG-HRP (ThermoFisher) for 1 hour at room temperature. Any unbound HRP- conjugated antibody was removed by washing, and TMB substrate for HRP was added. The colorimetric reaction was developed for 10 minutes, following which 2N sulfuric acid was added to stop the reaction. Absorbance of the product was measured at 450 nm. For competition assays, dilutions of serum (equivalent to their titers: 1:5000 for Pl, 1:2000 for P2 and 1:1000 for P3) were pre-mixed with various concentrations of sACE2. Serum-sACE2 mixtures were added to blocked plates and the protocol continued as described above.

[0235] Not Human Subjects Research (NHSR) determination. De-identified serum samples from recovered COVID- 19 patients were provided by the University of Chicago (patients P2 and P3) and by commercial vendor (patient Pl; Ray Biotech). The Office for the Protection of Research Subjects at the University of Illinois determined that the use of the samples in the ELISA study did not meet the criteria for Human Subjects Research as defined in 45CFR46(d)(l) or 21CFR56.102(c)(e) and did not require IRB approval.

[0236] Virus microneutralization assay. Vero E6 cells were cultured and their infection by authentic SARS-CoV-2 were assayed as described in Wee et al. (Science, eabc7424, 2020). Briefly, soluble ACE2 proteins were serially diluted in culture medium and incubated with SARS-CoV-2 (virus isolate 2019-nCoV/USA-WAl-A12/2020; GenBank Acc. No. MT020880.1) for 1 h. The mixture was added to VeroE6 cells at a MOI of 0.2 and incubated for 24hrs. Cells were fixed and immunostained with anti-SARS-CoV-2 nucleocapsid antibody (Sino Biological) and an Alexa Fluor 488-conjugated goat anti-rabbit secondary. Plates were imaged on an Operetta (PerkinElmer) to determine the number of infected cells and compared to virus only control wells to calculate the percent of relative infection. EXAMPLE 4:

[0237] Zoonotic coronaviruses have crossed over from animal reservoirs multiple times in the past two decades, and it is almost certain that wild animals will continue to be a source of devastating outbreaks. Unlike ubiquitous human coronaviruses responsible for common respiratory illnesses, these zoonotic coronaviruses with pandemic potential cause serious and complex diseases, in part due to their tissue tropisms driven by receptor usage. Severe Acute Respiratory Syndrome Coronaviruses 1 (SARS-CoV-1) and 2 (SARS-CoV-2) engage angiotensin-converting enzyme 2 (ACE2) for cell attachment and entry (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 etal., Cell, 2020), doi: 10.1016/j.cell.2020.02.052; Li etal., Nature. 426, 450-454, 2003; Letko et al., Nat Microbiol. 11, 1860, 2020). ACE2 is a protease responsible for regulating blood volume and pressure that is expressed on the surface of cells in 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). The ongoing spread of SARS-CoV-2 and the disease it causes, COVID- 19, has had a crippling toll on global healthcare systems and economies, and effective treatments and vaccines are urgently needed.

[0238] As SARS-CoV-2 becomes endemic in the human population, it has the potential to mutate and undergo genetic drift. To what extent this will occur as increasing numbers of people are infected and mount counter immune responses is unknown, but already a variant in the viral spike protein S (D614G) has rapidly emerged from multiple independent events and effects S protein stability and dynamics (Zhang et al., bioRxiv, 2020.06.12.148726, 2020; Korber et al., Cell. 182, 812-827. el9, 2020). Another S variant (D839Y) became prevalent in Portugal, possibly due to a founder effect (Borges et al., medRxiv, 2020.08.10.20171884, 2020). Coronaviruses have moderate to high mutation rates (measured at 10 ⁴ substitutions per year per site in HCoV-NL63 (Pyre et al., J. Mol. Biol. 364, 964-973, 2006), an alphacoronavirus that also binds ACE2, albeit via a smaller interface that is only partially shared with the RBDs of SARS-associated betacoronaviruses (Wu et al., Proc. Natl. Acad. Sci. U.S. A. 106, 19970-19974, 2009)), and large changes in coronavirus genomes have frequently occurred in nature from recombination events, especially in bats where coinfection levels can be high (Su et al., Trends Microbiol. 24, 490-502, 2016; Boni et al., Nat Microbiol, 1408-1417, 2020). Recombination of MERS-CoVs has also been documented in camels (Sabir et al., Science. 351, 81-84, 2016). This will all have profound implications for the current pandemic’s trajectory, the potential for future coronavirus pandemics, and whether drug resistance in SARS-CoV-2 becomes prevalent.

[0239] The viral spike is a vulnerable target for neutralizing monoclonal antibodies that are progressing to the clinic, yet in tissue culture escape mutations in the spike rapidly emerge to all antibodies tested (Baum et al., Science, eabd0831, 2020). Deep mutagenesis of the isolated receptor-binding domain (RBD) by yeast surface display has easily identified mutations in S that retain high expression and ACE2 affinity, yet are no longer bound by monoclonal antibodies and confer resistance (Greaney et al., bioRxiv, 2020.09.10.292078, 2020). This has motivated the development of cocktails of non-competing monoclonal antibodies (Baum et al., Science, eabd0831, 2020; Tortorici et al., Science, eabe3354, 2020), inspired by lessons learned from the treatment of HIV-1 and Ebola, to limit the possibilities for the virus to escape. This does not yet address future coronavirus spill overs from wild animals that may be antigenically distinct. Indeed, large screening efforts were required to find antibodies from recovered SARS-CoV-1 patients that cross-react with SARS-CoV-2 (Pinto et al., Nature, 2020), doi: 10.1038/s41586-020-2349-y), indicating antibodies have confined capacity for interacting with variable epitopes on the spike surface, and are unlikely to be broad and pan-specific for all SARS-related viruses.

[0240] An alternative protein-based antiviral to monoclonal antibodies is to use soluble ACE2 (sACE2) as a decoy to compete for receptor-binding sites on the viral spike (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; Monteil et al., Cell, 2020, doi: 10.1016/j. cell.2020.04.004; Chan et al., Science. 4, eabc0870, 2020). In principle, the virus has limited potential to escape sACE2-mediated neutralization without simultaneously decreasing affinity for the native ACE2 receptor, rendering the virus less virulent. Multiple groups have now engineered sACE2 to create high affinity decoys for SARS-CoV-2 that rival matured monoclonal antibodies and potently neutralize infection (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 study disclosed herein, deep mutagenesis was used to identify a large number of mutations in ACE2 that increase affinity for S (Chan et al., Science. 4, eabc0870, 2020). These mutations were dispersed across the interface and also at distal sites where they are predicted to enhance folding of the virus -recognized conformation. A combination of three mutations, called sACE22.v2.4, increases affinity 35-fold and binds SARS-CoV-2 S (KD 600 pM) with affinity comparable to the best monoclonal antibodies (Chan et al., Science. 4, eabc0870, 2020). Even tighter apparent affinities are reached through avid binding to trimeric spike expressed on a membrane. Despite engineering being focused exclusively on SARS-CoV-2 affinity, sACE22.v2.4 potently neutralized authentic SARS- CoV-1 and -2 infection in tissue culture, suggesting it's close resemblance to the wild type receptor confers broad activity against ACE2 -utilizing betacoronaviruses generally. Soluble ACE22.V2.4 is dimeric and monodisperse without aggregation, catalytically active, highly soluble, stable after storage at 37 °C for days, and well expressed at levels greater than the wild type protein. Due to its favorable combination of high activity and desirable properties for manufacture, sACE22.v2.4 is a genuine drug candidate for preclinical development.

[0241] Engineered, high affinity decoy receptors, while very similar to natural ACE2, nonetheless have mutations present at or near the interaction surface. There is therefore an opportunity for viral spike variants to discriminate between an engineered decoy and wild type receptors, providing a route towards resistance. It is disclosed herein that the engineered decoy sACE22.v2.4 binds broadly and tightly to the RBDs of diverse SARS-associated betacoronaviruses that use ACE2 for entry. Mutations were not found within the RBD, which directly contacts ACE2 and is where possible escape mutations will most likely reside, that redirect specificity towards the wild type receptor, although many mutations that favor binding of the engineered decoy were found in a competition binding assay. The results demonstrate that resistance to an engineered decoy receptor will be rare, and sACE22.v2.4 targets common attributes for affinity to S in SARS-associated viruses.

An engineered decoy receptor broadly binds RBDs from SARS-associated CoVs with tight affinity

[0242] The affinities of the decoy receptor sACE22.v2.4 were determined for purified RBDs from the S proteins of five coronaviruses from Rhinolophus bat species (isolates LYRal 1, Rs4231, Rs7327, Rs4084 and RsSHC014) and two human coronaviruses, SARS- CoV-1 and SARS-CoV-2. These viruses fall within a common clade of betacoronaviruses that use ACE2 as an entry receptor (Letko et al., Nat Microbiol. 11, 1860, 2020). They share close sequence identity within the RBD core while variation is highest within the functional ACE2 binding site (FIGS. 18 and 19), possibly due to a co-evolutionary 'arms race' with polymorphic ACE2 sequences in ecologically diverse bat species (Frank et al., bioRxiv. 5, 562, 2020). Affinity was measured by biolayer interferometry (BLI), with sACE22 (a.a. S19- G732) fused at the C-terminus with the Fc moiety of human IgGl immobilized to the sensor surface and monomeric 8his-tagged RBD used as the soluble analyte. This arrangement excludes avidity effects, which otherwise cause artificially tight (picomolar) apparent affinities whenever dimeric sACE22 in solution is bound to immobilized RBD decorating an interaction surface. Wild type sACE22 bound all the RBDs with affinities ranging from 16 nM for SARS-CoV-2 to 91 nM for LYRal 1, with median affinity 60 nM (Table 7). The measured affinities for the RBDs of SARS-CoV-1 and SARS-CoV-2 are comparable to 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., EMBOJ. 24, 1634-1643, 2005). Engineered sACE22.v2.4 displayed large increases in affinity for all the RBDs, with KDS ranging from 0.4 nM for SARS-CoV-2 to 3.5 nM for isolate Rs4231, with median affinity less than 2 nM (Table 7). The approximate 35-fold affinity increase of the engineered decoy applies universally to coronaviruses in the test panel and the molecular basis for affinity enhancement must therefore be grounded in common attributes of RBD/ACE2 recognition.

[0243] TABLE 7. BLI kinetics for immobilized sACE22-IgGl binding to coronavirus RBDs

A deep mutational scan of the RBD in the context of full-length S reveals residues in the ACE2 binding site are mutationally tolerant

[0244] To explore potential sequence diversity in S of SARS-CoV-2 that may act as a 'reservoir' for drug resistance, the mutational tolerance of the RBD was evaluated by deep mutagenesis (Fowler and Fields, Nat. Methods. 11, 801-807, 2014). Saturation mutagenesis was focused to the RBD (a.a. C336-L517) of full-length S tagged at the extracellular N- terminus with a c-myc epitope for detection of surface expression. The spike library, encompassing 3,640 single amino acid substitutions, was transfected in human Expi293F cells under conditions where cells typically acquire no more than a single sequence variant (Heredia etal., J. Immunol. 200, jil800343-3839, 2018; Park etal., J Biol Chem. 294, 4759- 10 4774, 2019). The culture was incubated with wild type, 8his-tagged, dimeric sACE22 at a sub-saturating concentration (2.5 nM). Bound sACE22-8h and surface-expressed S were stained with fluorescent antibodies for flow cytometry analysis (FIG. 20A). Compared to cells expressing wild type S, the library was poorly expressed, indicating many mutations are deleterious for folding and expression. A cell population was clearly discernable expressing S variants that bind ACE2 with decreased affinity (FIG. 20B). After gating for c-myc-positive cells expressing S, cells with high and low levels of bound sACE22 were collected by fluorescence-activated cell sorting (FACS), called the ACE2-High and ACE2-Low populations, respectively (FIG. 20C). Both the expression and sACE22 binding signals decreased over minutes to hours during sorting, possibly due to shedding of the SI subunit. Cells were therefore collected and pooled from three separate FACS experiments for a combined 8 hours sort time.

[0245] Transcripts in the sorted cells were Illumina sequenced and compared to the naive plasmid library to determine an enrichment ratio for each amino acid substitution (Fowler et al., Bioinformatics. 27, 3430-3431, 2011). Mutations in S that express and bind ACE2 tightly are selectively enriched in the ACE2-High sort (FIG. 21); mutations that express but have reduced ACE2 binding are selectively enriched in the ACE2-Low sort; and mutations that are poorly expressed are depleted from both sorted populations. Positional conservation scores were calculated by averaging the log2 enrichment ratios for each of the possible amino acids at a residue position. By adding conservation scores for both the ACE2- High and ACE2-Low sorts a score was derived for surface expression, which shows that the hydrophobic RBD core is tightly conserved for folding and trafficking of the viral spike (FIG. 22A). By comparison, residues on the exposed RBD surface are mutationally permissive for S surface expression. This matches the mutational tolerance of proteins generally.

[0246] For tight ACE2 binding (e.g., S variants in the AC E2 -High population), conservation increases for RBD residues at the ACE2 interface, yet mutational tolerance remains high (FIG. 22C). The sequence diversity observed among natural betacoronaviruses, which display high diversity at the ACE2 binding site, is therefore replicated in the deep mutational scan, which predicts the SARS-CoV-2 spike tolerates substantial genetic diversity at the receptor-binding site for function. From this accessible sequence diversity SARS-CoV- 2 might feasibly mutate to acquire resistance to monoclonal antibodies or engineered decoy receptors targeting the ACE2-binding site.

Comparison to a deep mutational scan of the isolated RBD by yeast surface display

[0247] Two deep mutational scans have been reported for the isolated RBD displayed on the surface of yeast (Starr et al., bioRxiv, 2020.06.17.157982, 2020; Linsky et al., bioRxiv, 2020.08.03.231340, 2020). The data described herein, from a selection of full-length S expressed in human cells, is compared to the publicly accessible Starr et al. data set.

Important residues within the RBD for surface expression of full-length spike in human cells are closely correlated with data from yeast surface display of the isolated RBD (FIG. 22B), with the exception of a notable region. The surface of the RBD opposing the ACE2-binding site (e.g., V362, Y365 and C391) is free to mutate for yeast surface display, but its sequence is constrained in the present experiments; this region of the RBD is buried by connecting structural elements to the global fold of an S subunit in the closed-down conformation (this is the dominant conformation for S subunits and is inaccessible to receptor binding) (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.el3, 2020). Targeted mutagenesis was used to individually test alanine substitutions for all the cysteines in the RBD (FIG. 23). All cysteine-to-alanine mutations severely diminished S surface expression in Expi293F cells, including C391 A and C525A on the RBD 'backside' that were neutral in the yeast display scan. These differences demonstrate that there are tighter sequence constraints on the RBD in the context of a full spike expressed at a human cell membrane, yet overall the two data sets closely agree.

[0248] For binding to dimeric sACE22, interface residues were more tightly conserved in the Starr et al. data set (FIG. 22D), possibly a consequence of three differences between the deep mutagenesis experiments. First, the selections for ACE2 binding of S variants at the plasma membrane appears to primarily reflect mutational effects on surface expression, which is almost certainly more stringent in human cells. Yeast permit many poorly folded proteins to leak to the cell surface (Rocklin et al., Science. 357, 168-175, 2017). Second, the yeast selections were conducted at multiple sACE2 concentrations from which apparent KD changes were computed; the Starr et al. data in this regard is very comprehensive. Due to the long sort times required for the human cell libraries where only a small fraction of cells express spike, sorting was performed at a single sACE22 concentration that cannot accurately capture a range of different binding affinities quantitatively. Third, dimeric sACE22 may geometrically complement trimeric S densely packed on a human cell membrane, such that avidity masks the effects of affinity -reducing mutations. Nonetheless, there is overall agreement that ACE2 binding often persists following mutations to the RBD surface, and these data simply suggest mutational tolerance may be even greater than that already observed by Starr et al.

A screen for S variants that preferentially bind wild type ACE2 over the engineered decoy

[0249] Having shown that the ACE2-binding site of SARS-CoV-2 protein S tolerates many mutations, it was investigated whether mutations might therefore be found that confer resistance to the engineered decoy sACE22.v2.4. Resistance mutations are anticipated to lose affinity for sACE22.v2.4 while maintaining binding to the wild type receptor, and are most likely to reside in the RBD where physical contacts are made. Similar reasoning formed the foundation of a deep mutagenesis-based selection of the isolated RBD by yeast surface display to find escape mutations to monoclonal antibodies, and the results were predictive of escape mutations in pseudovirus growth selections (Greaney et al., bioRxiv, 2020.09.10.292078, 2020).

[0250] To address whether escape mutations from the engineered decoy might be found in the RBD, the S protein library was repurposed for a specificity selection. Cells expressing the library, encoding all possible substitutions in the RBD, were co-incubated with wild type SACE22 fused to the Fc region of IgGl and 8his-tagged sACE22.v2.4 at concentrations where both proteins bind competitively (Chan et al., Science. 4, eabc0870, 2020). It was immediately apparent from flow cytometry of the Expi293F culture expressing the S library that there were cells expressing S variants shifted towards preferential binding to SACE22.V2.4, but no significant population with preferential binding to the wild type receptor (FIGS. 24A-24B). Cells expressing S variants that might preferentially bind sACE22(WT)- IgGl or SACE22.V2.4 were gated and collected by FACS (FIG. 24C), followed by deep sequencing of S transcripts to determine enrichment ratios. There was close agreement between two independent replicate experiments (FIGS. 24D-24G). Most RBD mutations were depleted following sorting, consistent with deleterious effects on S folding and expression.

[0251] Soluble ACE22.v2.4 has three mutations from wild type ACE2: T27Y buried within the RBD interface, and L79T and N330Y at the interface periphery (FIG. 25A). A substantial number of mutations in the RBD of S were selectively enriched for preferential binding to sACE22.v2.4 (FIG. 25B, upper-left quadrant). While sACE22.v2.4-specificity mutations could be found immediately adjacent to the sites of engineered mutations in ACE2 (in particular mutations to S-F486 adjacent to ACE2-L79 and S-T500 adjacent to ACE2- N330), major hot spots for sACE22.v2.4-specificity mutations also mapped to RBD loop 498- 506 contacting the region where the ACE2-al helix packs against a [3-hairpin motif (FIG. 25A). By comparison, there were no hot spots in the RBD for sACE22(WT)-specificity mutations. Indeed, only a small number of mutations were selectively enriched for preferential binding to wild type receptor (FIG. 25B), and the abundance of these putative wild type-specific mutations barely rose above the expected level of noise in the deep mutagenesis data. In this competition assay, S binding to wild type sACE22 is therefore more sensitive to RBD mutations than is S binding to engineered sACE22.v2.4.

[0252] To determine whether the potential wild type ACE2-specific mutations found by deep mutagenesis are real as opposed to false predictions due to data noise, 24 mutants of S selectively enriched in the wild type-specific gate by targeted mutagenesis were tested (blue data points in FIG. 25B). Only minor shifts towards binding wild type sACE22 were observed (FIG. 26). Two S mutants were investigated further in sACE22 titration experiments, N501W and N501 Y, which both retained high receptor binding and displayed small shifts towards wild type sACE22 in the competition experiment. N501 of S is located in the 498-506 loop and its substitution to large aromatic side chains might alter the loop conformation to cause steric strain with nearby ACE2 mutation N330Y in sACE22.v2.4. After titrating the concentrations of 8his-tagged sACE22(WT) and sACE22.v2.4 and measuring bound protein to S-expressing cells by flow cytometry, it was found S-N501W and S-N501Y do show enhanced specificity for wild type sACE22, but the effect is small and sACE22.v2.4 remains the stronger binder (FIG. 25C); these mutations therefore will not confer resistance in the virus to the engineered decoy.

[0253] Dimeric sACE22 binds avidly to S protein on a membrane surface; avid interactions are also observed between sACE22 and spikes on authentic SARS-CoV-2 in infection assays (Chan et al., Science. 4, eabc0870, 2020). BLI kinetics measurements, in which immobilized sACE22-IgGl interacts with monomeric RBD, were used to determine how the observed changes in avid sACE22 binding to S-expressing cells translate to changes in monovalent affinity. Both N501W and N501Y mutants of SARS-CoV-2 RBD displayed increased affinity for wild type ACE2 and engineered ACE2.v2.4, with larger affinity gains in favor of the wild type receptor (Table 7). This aligns with the flow cytometry data indicating a small shift in specificity towards wild type ACE2, but not enough to escape the engineered decoy. By comparison, multiple independent escape mutations are readily found in S of SARS-CoV-2 that diminish the efficacy of monoclonal antibodies by many orders of magnitude (Baum et al., Science, eabd0831, 2020; Greaney et al., bioRxiv, 2020.09.10.292078, 2020).

[0254] Finally, 8 representative mutations to S predicted from the deep mutational scan to increase specificity towards sACE22.v2.4 (FIG. 25B) were cloned and 7 were found to have large shifts towards preferential sACE22.v2.4 binding in the competition assay (FIG. 27). These S mutations were Y449K/Q/S, L455G/R/Y and G504K. The basis for why the mutations increase specificity towards engineered sACE22.v2.4 is ambiguous, since RBD residues Y449, L455, and G504 are not in direct contact with engineered sites of the receptor. BLI kinetics between immobilized sACE22-IgGl and monomeric RBD as the analyte showed reduced affinity of a representative mutant, RBD-Y449K, to both wild type and engineered SACE22 (Table 7). However, affinity changes in the picomolar range for sACE22.v2.4 are hidden during avid binding to full-length S-Y449K at the cell surface, whereas avid binding of wild type sACE22 to S-Y449K (with affinity measured by BLI in the moderate nanomolar range) is substantially reduced. This finding might explain why the competition selection found many mutations that shift specificity towards engineered sACE22.v2.4, as mutations causing small decreases in affinity may have larger effects on avid binding of the weaker- bound wild type receptor.

[0255] Overall, validation by targeted mutagenesis confirms that the selection can successfully find mutations in S with altered specificity. The inability to find mutations in the RBD that impart high specificity for the wild type receptor means such mutations are rare or may not even exist, at least within the receptor-binding domain where direct physical contacts with receptors occur. Mutations elsewhere having long-range conformational effects cannot be excluded. Engineered, soluble decoy receptors therefore live up to their promise as broad therapeutic candidates against which a virus cannot easily escape.

[0256] The allure of soluble decoy receptors is that the virus cannot easily mutate to escape neutralization. Mutations that reduce affinity of the soluble decoy will likely also decrease affinity for the wild type receptor on host cells, thereby coming at the cost of diminished infectivity and virulence. However, this hypothesis has not been rigorously tested, and since engineered decoy receptors differ from their wild type counterparts, even if by just a small number of mutations, it is possible a virus may evolve to discriminate between the two. Here, it is demonstrated that an engineered decoy receptor for SARS-CoV-2 broadly binds with low nanomolar KD the spikes of SARS-associated betacoronaviruses that use ACE2 for entry, despite high sequence diversity within the ACE2-binding site. Mutations in S that confer high specificity for wild type ACE2 were not found in a comprehensive screen of all substitutions within the RBD. The engineered decoy receptor is therefore broad against zoonotic ACE2-utilizing coronaviruses that may spill over from animal reservoirs in the future and against variants of SARS-CoV-2 that may arise as the current COVID- 19 pandemic rages on. It is unlikely that decoy receptors will need to be combined in cocktail formulations, as is required for monoclonal antibodies or designed miniprotein binders to prevent the rapid emergence of resistance (Baum et al., Science, eabd0831, 2020; Cao et al., Science, eabd9909, 2020). [0257] Soluble decoy receptors have proven effective in the clinic, especially for modulating immune responses. Etanercept (trade name Enbrel®; soluble TNF receptor), aflibercept (Eylea®; a soluble chimera of VEGF receptors 1 and 2) and abatacept (Orencia®; soluble CTLA-4) are just three examples of soluble receptors that have profoundly impacted the treatment of human disease (Usmani et al., PLoS ONE. 12, e0181748, 2017), yet no soluble receptors for a viral pathogen are approved drugs. There are two main reasons for this. First, the affinity of entry receptors for viral glycoproteins is often moderate to low, which reduces neutralization potency compared to affinity-matured monoclonal antibodies. For SARS-CoV-2, this problem has been solved by engineering ACE2 to have picomolar affinity for viral S (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). Second, virus entry receptors have endogenous functions for normal physiology and their soluble counterparts may impact this normal physiology to exert unacceptable toxicity. For example, the entry receptor for human cytomegalovirus is a growth factor receptor, and growth factor interactions had to be knocked out to make a virus-specific decoy suitable for in vivo administration (Park et al., PLoS Pathog. 16, el008647, 2020). However, ACE2 in this regard is different and its endogenous activity - the catalytic conversion of vasoconstrictive and inflammatory peptides of the renin-angiotensin and kinin systems - may be of direct benefit for addressing COVID- 19 symptoms. During infection, ACE2 activity is downregulated and the renin-angiotensin system becomes imbalanced, possibly driving aspects of acute-respiratory distress syndrome (ARDS) that cause patients to require mechanical ventilation (Imai et al., Nature. 436, 112-5 116, 2005; Treml et al., Crit. Care Med. 38, 596-601, 2010; Verdecchia et al., Eur J Intern Med. 76, 14-20, 2020). Administration of recombinant, soluble ACE2 rescues lost biochemical activity, with potential protective properties for the pulmonary and cardiovascular systems that include decreased lung elastance, increased blood oxygenation, reduced hypertension and diminished fluid accumulation in the lungs due to proteolytic conversion of angiotensin and bradykinin peptides (Imai et al., Nature. 436, 112-116, 2005; Treml et al., Crit. Care Med. 38, 596-601, 2010; Wang et al., Pulm Pharmacol Ther. 58, 101833, 2019; Chung etal., 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 ACE22 has been developed as a drug for ARDS with an acceptable safety profile in humans (Haschke et al., Clin Pharmacokinet . 52, 783-792, 2013; Khan et al., Crit Care. 21, 234, 2017) and is currently under evaluation in a clinical trial by Apeiron. Engineered, high affinity sACE22 decoys, most likely as fusions with immunoglobulin Fc for increased serum stability (Lei et al., Nat Commun. 11, 2070, 2020; Liu et al., Kidney Int. 94, 114-125, 2018; Iwanaga e/ a/., bioRxiv, 2020.06.15.152157, 2020), represent next generation therapeutics with dual mechanisms of action: (i) potent virus neutralization due to high affinity blockade of the viral spike and (ii) proteolytic turnover of peptide hormones for direct relief of COVID-19 symptoms.

EXAMPLE 5:

[0258] This example evaluates pharmacokinetics (PK) of sACE2.v2.4 in mice. The results demonstrate that serum half-life of sACE2.v2.4 following IV administration is increased by fusion to the Fc moiety of human IgGl. The fusion protein is proteolysed to produce long-lived IgGl fragments that persist beyond 7 days, whereas the ACE2 moiety rapidly disappears within hours. By delivering sACE22.v2.4-IgGl directly to the lungs via intratracheal (IT) administration or nebulization, the protein remains at high levels in lung tissue for at least 4 hours with minimal proteolytic degradation. These results demonstrate that direct lung delivery of high affinity sACE2 derivatives is a viable alternative to IV infusion, and offers possible benefits for out-patient clinical care.

[0259] When wild type human sACE22 is administered intraperitoneally in mice, it has a serum half-life of 8.5 hours (Wysocki et al., Hypertension 55, 90-98, 2010), but this is influenced by resorption kinetics into the blood, which is typically delayed by hours for macromolecules (Shoyaib et al., Pharmaceut Res. 37, 12, 2020). When sACE22.v2.4 (0.5 mg/kg) without a fusion partner was injected into the tail veins of male and female mice, the protein was rapidly cleared with a serum half-life estimated to be under 10 minutes, measured by ACE2 ELISA (FIG. 28A) and ACE2 catalytic activity in serum (FIG. 28B). This is much shorter than the serum half-life of wild type sACE22 in humans (2 to 3 hours) (Haschke et al., Clin Pharmacokinet 52, 783-792, 2013). No toxicity was observed when sACE22.v2.4 was IV administered twice daily at 0.5 mg/kg for five days (days 0, 1, 2, 3, and 4). Mice were euthanized on day 7 and blood chemistry, hematology, and tissue pathology showed no differences with mock treated mice.

[0260] To increase serum half-life, a fusion of sACE22 to IgGl Fc was tested. While other groups have explored fusions of sACE22 to IgGl 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 dampen interactions with pro-inflammatory FcyRs, this study used unmodified IgGl (isoallotype nGlml) to recruit effector functions that have been shown in anti-SARS-CoV-2 mAbs to be necessary for optimum protection (Schafer et al., J Exp Med. 218 (2020), doi:10.1084/jem.20201993). Published PK data on IgGl fusions of SACE22 are mixed. While there is unambiguous evidence that a murine sACE22-IgGl fusion persists for days in mice (Liu et al., Kidney Int. 94, 114-125, 2018), results on human sACE22-IgGl fusions are contradictory. Two reports using an ELISA to detect the human IgGl moiety indicated sACE22-IgGl has a serum half-life of days (Iwanaga et al., Biorxiv, in press, doi:10.1101/2020.06.15.152157; Lei etal., Nat Commun. 11, 2070, 2020), but another study using an ELISA to detect the ACE2 moiety reported rapid clearance within hours (Higuchi et al., Biorxiv, in press, doi:10.1101/2020.09.16.299891). Only one published report detected both parts of the fusion protein, using an anti-ACE2 capture antibody with an anti- IgGl detection antibody to measure a long serum half-life of the human fusion protein in mice (Liu et al., Int J Biol Macromol . 165, 1626-1633, 2020). The reasons for the discrepancies are unclear, but possibly indicate cleavage of the fusion protein to produce fragments of differing serum stability.

[0261] Using an ELISA for human IgGl, both wild type sACE22-IgGl and sACE22.v2.4-IgGl (SEQ ID NO: 11) showed equivalent serum PK after IV administration (2.0 mg/kg) in male mice, with protein persisting for over 7 days (FIG. 29). It was therefore concluded that the three mutations in the high affinity sACE22.v2.4 variant (T27Y, L79T, and N330Y) did not substantially change PK, consistent with a previous study of another modified sACE2 derivative (Higuchi et al., Biorxiv, in press, doi: 10.1101/2020.09.16.299891). Serum components could not be further characterized due to insufficient material, consequently another PK study was conducted in both male and female mice to more thoroughly track how sACE22.v2.4-IgGl changes in the serum with time. Again, human IgGl protein persisted for days in the serum (FIG. 30A), yet the ACE2 moiety was rapidly cleared within 24 hours based on an ACE2 ELISA (FIG. 30B). Measurement of ACE2 catalytic activity revealed even faster decay (FIG. 30C). Immunoblot for human IgGl confirmed that the fusion protein was being proteolyzed in mouse blood to liberate long-lived IgGl fragments (FIG. 30D). Overall, fusion of sACE22.v2.4 to IgGl Fc provided only a modest increase in serum stability. No toxicity was observed in mice IV administered sACE22.v2.4-IgGl (2.0 mg/kg), followed up 7 days later with blood chemistry, hematology, and tissue pathology analysis. [0262] To improve upon serum PK observed following IV administration, a study was performed to deliver the protein directly to the respiratory tract, which is the primary site of SARS-CoV-2 infection. Following IT delivery (1.0 mg/kg), sACE22.v2.4-IgGl was found to persist at high levels in the lungs for at least 4 hours by ACE2 ELISA, human IgGl ELISA, and anti-human IgGl immunoblot (FIGS. 31A-31C). Levels of sACE22.v2.4-IgGl absorbed into the blood were too low for detection. As observed for IV administration, wild type sACE22-IgGl and sACE22.v2.4-IgGl had equivalent PK in the lungs (within experimental error) following IT delivery. Administration of sACE22.v2.4-IgGl by inhalation was further investigated. In this study, the protein was nebulized for 30 minutes into a chamber holding the mice. While doses in the nebulizer-holding chamber were below that achieved through IT administration, it was nonetheless observed that sACE22.v2.4-IgGl remained high and relatively constant for 4 hours, as measured by ACE2 ELISA, human IgGl ELISA, and immunoblot (FIGS. 31D-31F). Direct delivery to the respiratory tract achieved high levels of protein in the lung tissue with minimal degradation for over 4 hours. The different PK profiles based on route of administration (e.g., protein delivered directly to the lungs persists for hours but does not reach detectable levels in plasma, whereas IV delivered protein achieves high but short lived plasma concentrations) offer clinical opportunities to treat patients in different ways, possibly based on how advanced disease is or whether infection is systemic, or to treat patients using both IV and either IT or inhalation routes of administration.

EXAMPLE 6:

[0263] This example describes experiments performed using SARS-CoV-2 pseudovirus to evaluate whether modified ACE2 polypeptides are capable of blocking virus entry into cells.

[0264] Human A549 lung epithelial cells over-expressing the ACE2 receptor, human A549 lung epithelial cells, and human lung endothelial cells were incubated with a VSV- SARS-CoV-2-luciferase-pseudotype virus and the wild-type sACE22-IgGl or the engineered sACE22.v2.4-IgGl peptides at concentrations of 0, 5 or 25 pg/ml. Each experiment contained a no virus control; all other samples contained the virus at an MOI of 0.01. Cells were harvested and the extent of viral entry was quantified based on expression of the luciferase reporter (FIG. 32). Engineered sACE22.v.2.4-IgGl had superior protection against entry of the SARS-CoV-2 pseudo virus into human lung epithelial cells and human endothelial cells. [0265] In a second study, K18-hACE2 transgenic mice, which express the human ACE2 receptor in epithelial cells, were injected intravenously with either wild-type sACE22-IgGl or sACE22.v2.4-IgGl and intraperitoneally with the VSV-SARS-CoV-2-luciferase-pseudotype virus. The lung and the liver were harvested at 24 hours and the extent of viral entry was quantified by luciferase activity (FIG. 33). Engineered sACE22.v.2.4-IgGl achieved superior protection against SARS-CoV-2 pseudotype virus entry into the lung and liver in human ACE2-expressing mice.

[0266] Taken together, these results demonstrate that the v2.4 derivative of soluble ACE2 more effectively blocks SARS-CoV-2 pseudo virus entry into cells expressing human ACE2, both in tissue culture and in an animal model.

EXAMPLE 7:

[0267] This example describes a study to investigate whether sACE22.v2.4-IgGl exhibits protective and/or therapeutic benefits against SARS-CoV-2-induced lung vascular leakage in a mouse model of COVID- 19. While particular methods are provided, one of skill in the art will recognize that methods that deviate from these specific methods can also be used, including addition or omission of one or more steps.

[0268] The readouts for this study are vascular leakage in the lung and edema formation in the lung. The following animal groups are used for this study:

Group 1 (Control), 4 mice (2 males and 2 females, 2-month old).

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

Group 3 (sACE22.v2.4-IgGl administered IV (10 mg/kg) or IT (2 mg/kg) or by inhalation hours prior to SARS-CoV-2 infection, 5 x 10 ⁴ pfu/mice for 7 days), 4 mice (2 males and 2 females, 2-month old). This group assesses pre-exposure prophylaxis.

Group 4 (sACE22.v2.4-IgGl administered IV (10 mg/kg) or IT (2 mg/kg) or by inhalation after SARS-CoV-2 infection, 5 x 10 ⁴ pfu/mice for 7 days), 4 mice (2 males and 2 females, 2-month old). This group assesses post-infection therapy. [0269] Mice are administered sACE22.v2.4-IgGl polypeptide by one of several methods (e.g., IV, IT, inhalation) and infected with SARS-CoV-2 via the airway to mimic human lung infection.

[0270] It is expected that sACE22.v2.4-IgGl will reduce SARS-CoV-2-induced lung vascular leak and reduce edema formation, which are the primary causes of respiratory failure and death in COVID-19 patients.

EXAMPLE 8:

[0271] This example describes a study to investigate whether sACE22.v2.4-IgGl exhibits a protective and/or therapeutic benefit against SARS-CoV-2-induced lung vascular injury and long term fibrosis in a mouse model of COVID-19. While particular methods are provided, one of skill in the art will recognize that methods that deviate from these specific methods can also be used, including addition or omission of one or more steps.

[0272] The readouts for this study are H&E staining, Masson trichrome and Sirius red staining, MPO assay, and protein lysates to assess signaling shifts and inflammatory pathology. The following animal groups are used for this study:

Group 1 (Control), 4 mice (2 males and 2 females, 2-month old).

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

Group 3 (sACE22.v2.4-IgGl administered IV (10 mg/kg) or IT (2 mg/kg) or by inhalation prior to SARS-CoV-2 infection, 5 x 10 ⁴ pfu/mice for 7 days), 4 mice (2 males and 2 females, 2-month old). This group assesses pre-exposure prophylaxis.

Group 4 (sACE22.v2.4-IgGl administered IV (10 mg/kg) or IT (2 mg/kg) or by inhalation after SARS-CoV-2 infection, 5 x 10 ⁴ pfu/mice for 7 days), 4 mice (2 males and 2 females, 2-month old). This group assesses post-infection therapy.

[0273] It is expected that sACE22.v2.4-IgGl will reduce inflammatory injury and fibrosis in this mouse model of COVID-19. EXAMPLE 9:

[0274] This example describes a study to investigate whether sACE22.v2.4 (with and without fusion to IgGl Fc) blocks the spike proteins of highly transmissible SARS-CoV-2 variants. Mutants of SARS-CoV-2 have emerged that show increased transmission and possibly increased virulence. The virus variants of concern as of March, 2021 are B.1.351 originating from South Africa (Tegally et al., medRxiv, in press, doi: 10.1101/2020.12.21.20248640), P.l from Brazil, and B.1.1.7 from England (Leung et al., Eurosurveillance 26, 2021, doi:10.2807/1560-7917.ES.2020.26.1.2002106; Volz et al., medRxiv, in press, doi: 10.1101/2020.12.30.20249034). All three virus variants share the N501Y mutation in S, which increases monovalent affinity for wild type ACE2 by 20-fold (Example 4 - Table 7). The high affinity v2.4 ACE2 derivative also binds with increased affinity (Example 4 - Table 7). This study tests the apparent monovalent affinity and avid binding of dimeric sACE22-IgGl (wild type and v2.4) with full-length S variants from the P.l, B.l.1.7, and B.1.351 lineages.

[0275] S proteins are expressed in human Expi293F cells with N-terminal c-myc tags for measuring surface expression with a fluorescent anti-myc antibody and flow cytometry. Cells are incubated with a dilution series of sACE2-8his and sACE2.v2.4-8his (monomer: ACE2 residues 19-615), washed, and bound protein is measured by flow cytometry using anti-his fluorescent antibody staining. Cells are also incubated with a dilution series of sACE22-IgGl and sACE22.v2.4-IgGl (dimer: ACE2 residues 19-732), washed, and bound protein is measured by flow cytometry using an anti-human IgGl fluorescent antibody. Based on the previously described deep mutagenesis (Example 4), it is expected that the results will confirm that highly transmissible virus variants remain susceptible to tight binding by the engineered v2.4 derivative of sACE2.

[0276] 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 disclosure and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim all that comes within the scope and spirit of these claims. EXAMPLE 10:

[0277] This example show the binding affinity of sACE22.v2.4 to S proteins of SARS- CoV-2 variants.

[0278] SARS-CoV-2 has a moderate mutation rate (10' ³ substitutions per site per year (47)) and variants have emerged with increased transmissibility and virulence, most notably the B.1.1.7 (Alpha), B.1.351 (Beta), P.l (Gamma), and B.1.617.2 (Delta) VOCs. The variants have multiple mutations in S, including changes in immunodominant epitopes that cause partial immune escape and aN501Y substitution within the RBD that increases monovalent affinity for ACE2 by 20-fold. sACE2.v2.4 was assessed for efficacy against these new variants.

[0279] Using flow cytometry (a non-equilibrium experiment from which apparent binding strength was measured), binding of human cells expressing full-length trimeric S to wild type sACE2 and engineered sACE2.v2.4 was determined, using both monomeric sACE2 (a.a. 19-615) and dimeric sACE22-IgGl (ACE2 a.a. 19-732) to assess monovalent and avid binding, respectively (FIG. 34). Compared to wild type, monomeric sACE2.v2.4 showed markedly increased binding to all S proteins tested. The enhanced binding was less when avidity was considered, but nonetheless, the engineered derivative remained tightly bound. Indeed, the engineered decoy receptor had increased binding signal to S from the newly circulating variants compared to the original SARS-CoV-2 isolate, consistent with some S variants showing tighter binding to ACE2. The level of sACE2 bound to S-expressing cells diminished at high concentrations, consistent with shedding of ACE2-bound SI. sACE2 binding to S variants from the B.1.429 (Epsilon), B.1.617.2 (Delta) and C.37 (Lambda) lineages (FIG. 35) was further investigated. Again, monomeric sACE2.v2.4 bound considerably tighter than the wild type decoy, with smaller differences for sACE22-IgGl proteins where avidity masks affinity changes. Due to the decoy inducing SI loss, detection at the cell surface of an N-terminal tag on S decreased.

[0280] Monoclonal antibodies in clinical development have shown mixed results for broad affinity against SARS-CoV-2 variants. Using flow cytometry, binding to S proteins from four VOCs was compared between sACE22.v2.4-IgGl and four mAbs that have received EUA and been used in the treatment of COVID-19 patients (FIG. 36): REGN10933 (casirivimab), REGN10987 (imdevimab), VIR-7831 (sotrovimab), and LY-CoV555 (bamlanivimab). In this assay, sACE22.v2.4-IgGl consistently showed high binding to S, better than VIR-7831 and LY-CoV555, but lower than REGN10987. The monoclonal antibodies REGN10933 and LY-CoV555 show markedly different binding strengths for the different S proteins. REGN10933 and sACE22.v2.4-IgGl engage the ACE2 interaction motif on S and both induce SI shedding at high concentrations. Overall, sACE22.v2.4-IgGl broadly binds S proteins of SARS-CoV-2 VOCs and with comparable strength to mAbs that have shown efficacy in the clinic.

[0281] Using flow cytometry, mAbs were tested for whether they could bind in combination with sACE22.v2.4-IgGl to SARS-CoV-2 Spike expressed on the surface of human cells. These studies were limited to testing the combinations at sub-saturating levels due to sACE22.v2.4-IgGl and REGN10933 inducing SI shedding and signal loss at high concentrations. As sACE22.v2.4-IgGl concentration is increased, no loss of mAh binding is observed (FIG. 37); the proteins can bind together to S-expressing cell membranes when their concentrations are low, as may occur in the in vivo therapeutic setting. To explore competition between the mAbs at saturation, biolayer interferometry (BLI) was used, in which the mAbs were immobilized to a sensor surface and bound to his-tagged RBD from the Delta or Gamma VOCs. The sensors were then transferred to a solution of his-tagged SACE22.V2.4 to determine if additional binding to the captured RBD could be detected (Figure 38). VIR-7831 and sACE22.v2.4 bound non-competitively to RBD, whereas SACE22.V2.4 was unable to bind RBD bound to REGN10933 or REGN10987.

EXAMPLE 11

[0282] This example describes an in vitro study to investigate the ability of combinations of (i) sACE2.v2.4-8his and anti-SARS-CoV-2 spike protein monoclonal antibody (e.g, REGN10933, REGN10987, LY-CoV555, or VIR-7831) or (ii) sACE2 ₂ .v2.4- IgGl and anti-SARS-CoV-2 spike protein monoclonal antibody (e.g, REGN10933, REGN10987, LY-CoV555, or VIR-7831), to neutralize of SARS-CoV-2 infectivity.

[0283] Pseudotyped vesicular stomatitis viruses (VSV) are generated by transiently transfecting 293T cells with a plasmid encoding SARS-CoV-2 spike protein by standard transfection methods known in the art. Transfected cells are cultured for 24 hours, and supernatant comprising VS V-SARS-CoV-2 S is collected and kept on ice, or frozen, until use. [0284] Vero cells are plated onto a 96-well black microplate in culture media containing (i) sACE2.v2.4-8his and anti-SARS-CoV-2 spike protein monoclonal antibody (e.g, REGN10933, REGN10987, LY-CoV555, or VIR-7831), (ii) sACE2 ₂ .v2.4-IgGl and anti- SARS-CoV-2 spike protein monoclonal antibody (e.g., REGN10933, REGN10987, LY- CoV555, or VIR-7831), (iii) sACE2.v2.4-8his and isotype control monoclonal antibody, (iv) sACE22.v2.4-IgGl and isotype control monoclonal antibody, (v) sACE2.v2.4-8his alone, (vi) sACE22.v2.4-IgGl, (vii) anti-SARS-CoV-2 spike protein monoclonal antibody alone (e.g, REGN10933, REGN10987, LY-CoV555, or VIR-7831), (viii) isotype control monoclonal antibody alone, or (ix) media alone. Serial dilutions of VSV-SARS-CoV-2 is added to corresponding test wells except media control wells and cells are incubated for 24 hours at 37 °C, 5% CO2. Following incubation, supernatant is removed and replaced with phosphate buffered saline (PBS). Neutralization of VSV-SARS-CoV-2 is measured using a neutralization fluorescence focus assay and read on an imaging cytometer.

EXAMPLE 12:

[0285] This example describes an in vivo study to investigate the efficacy of combinations of (i) sACE2.v2.4-8his and anti-SARS-CoV-2 spike protein monoclonal antibody (e.g, REGN10933, REGN10987, LY-CoV555, or VIR-7831), or (ii) sACE2 ₂ .v2.4- IgGl and anti-SARS-CoV-2 spike protein monoclonal antibody (e.g, REGN10933, REGN10987, LY-CoV555, or VIR-7831), for the treatment of SARS-CoV-2 infection.

[0286] 6 to 8 week old humanized mice are injected peritoneally with (i) sACE2.v2.4-

8his and anti-SARS-CoV-2 spike protein monoclonal antibody (e.g, REGN10933, REGN10987, LY-CoV555, or VIR-7831), (ii) sACE2 ₂ .v2.4-IgGl and anti-SARS-CoV-2 spike protein monoclonal antibody (e.g, REGN10933, REGN10987, LY-CoV555, or VIR- 7831), (iii) sACE2.v2.4-8his and isotype control monoclonal antibody, (iv) sACE22.v2.4- IgGl and isotype control monoclonal antibody, (v) sACE2.v2.4-8his alone, (vi) sACE22.v2.4-IgGl, (vii) anti-SARS-CoV-2 spike protein monoclonal antibody alone (e.g, REGN10933, REGN10987, LY-CoV555, or VIR-7831), (viii) isotype control monoclonal antibody alone, or (ix) sterile saline control at 1 day pre-infection, or 1 day post-infection. Mice are anaesthetized and intranasally inoculated with PBS or SARS-CoV-2. Mice are weighed prior to infection and every day during infection to assess SARS-CoV-2 induced weight loss. Mice are euthanized at day 2 or day 4 post-infection. Lungs are collected, homogenized, and RNA extracted to assess percent SAR-CoV-2 RNA relative to infected mice treated with sACE2.v2.4-8his alone, sACE22.v2.4-IgGl alone, anti-SARS-CoV-2 spike protein monoclonal antibody alone (e.g., REGN10933, REGN10987, LY-CoV555, or VIR- 7831), or isotype control monoclonal antibody alone. Supernatants from lung homogenates are also used to perform plaque assays on Vero cells to quantitate levels of virus at the time of euthanization. Tissue samples are also collected from infected mice for histological analysis.

EXAMPLE 13:

High affinity binding of sACE2 ₂ .v2.4-IgGl to S proteins of variants of concern (VOCs)

[0287] Flow cytometry was used to determine the binding of human cells expressing full-length trimeric S to wild type sACE2 and sACE2.v2.4 using monomeric sACE2 (residues 19-615, a protease domain-only construct) and dimeric sACE22-IgGl (ACE2 residues 19-732, a region that includes protease and dimerization domains and forms a stable dimer even before fusion to IgGl Fc, itself a disulfide-bonded dimer). As shown in FIGs. 39A-39D, compared to wild type, monomeric sACE2.v2.4 showed markedly increased binding to S proteins from original and 3 VOCs (alpha, beta, and gamma). The enhanced binding was less when avidity was considered, nonetheless the engineered derivative remained tightly bound.

[0288] As shown in FIG. 2, consistent with tighter ACE2 affinity, the sACE2.v2.4 engineered decoy showed markedly increased binding to S from three VOCs (i.e. alpha, beta, and gamma).

[0289] The level of sACE2 bound to S-expressing cells diminished at high concentrations, suggesting shedding of ACE2-bound SI. Further analysis of sACE2 binding to S from B. 1.429 (Epsilon), B. 1.617.2 (Delta), and C.37 (Lambda) lineages demonstrated tighter binding of sACE2.v2.4 and decreased detection at the cell surface of an N-terminal c- myc tag on S, consistent with SI loss (Figure 3). sACE2-induced shedding of SI may irreversibly neutralize virions even if sACE2 proteins dissociate.

[0290] Monoclonal antibodies have shown mixed results for broad affinity against SARS-CoV-2 variants. Using flow cytometry, binding to S proteins from four VOCs (alpha, beta, gamma, and delta) was compared between sACE22.v2.4-IgGl and mAbs in clinical use (FIGs. 1E-1H): REGN10933 (casirivimab), REGN10987 (imdevimab), VIR-7831 (sotrovimab), and LY-CoV555 (bamlanivimab). In this assay, sACE22.v2.4-IgGl consistently showed high binding to S variants whereas REGN10933 and LY-CoV555 showed widely different binding affinities. REGN10933 and sACE22.v2.4-IgGl directly engage the ACE2 interaction motif on S and both induced SI shedding at high concentrations. Furthermore, only VIR-7831 and sACE22.v2.4-IgGl bound to the more distantly related S protein of SARS-CoV-1 (FIG. II), again emphasizing the exceptional breadth of sACE22.v2.4-IgGl against betacoronaviruses that use ACE2 as an entry receptor. [0291] Conformational accessibility of epitopes and avidity influence the binding to trimeric S at the cell surface. Therefore, monovalent affinities for soluble RBD from delta and gamma VOCs were measured using biolayer interferometry (BLI) (FIG. 4; Table 8). VIR-7831 and sACE22.v2.4-IgGl had the tightest monovalent affinities with excellent breadth. Overall, sACE22.v2.4-IgGl broadly bound S proteins of SARS-CoV-2 VOCs with comparable strength to the assessed mAbs, which have shown efficacy in clinical settings.

(1) Dimeric IgG proteins were immobilized to an anti -human IgG sensor. Sensors were transferred to monomeric RBD-8h at 200, 66.7, 22.2 and 7.41 nM.

(2) % ² and R ² were determined from curves fitted to the entire data.

(3) ND, not determined due to no binding.

EXAMPLE 14:

Additional mutations further enhance the affinity of sACE2.v2.4 for S of delta

[0292] Using the mutational landscape of ACE2 for binding to S from an original Wuhan isolate (FIG. 2), 1 to 3 mutations were added to sACE2.v2.4-8h (ACE2 residues 19- 615; a monomer) (Table 9). The proteins were expressed, purified, and tested by flow cytometry for binding to Expi293F cells expressing the full-length spike of delta. For comparison, sACE2-CDY14 carrying 6 mutations was included in the test set. CDY14 has the highest affinity for S of any sACE2 derivative reported in the literature to date. The screen identified two new mutants of ACE2, v2.4.2 (carrying the v2.4 mutations plus Q76V and L91P) and v2.4.10 (carrying the v2.4 mutations plus K31Y, L91P, and R51G) with moderately increased binding to S of delta (FIG. 43 A). Binding of CDY14 was comparable to the ACE2.v2.4.2 and ACE2.v2.4.10 variants.

¹ Measured by flow cytometry using monomeric sACE2-8h constructs.

² ND, not determined due to poor expression and purification of sACE2-8h protein that was monomeric by size exclusion chromatography.

EXAMPLE 15: sACE2 ₂ .v2.4-IgGl tightly binds S from the omicron SARS-CoV-2 variant

[0293] Flow cytometry was used to measure binding of sACE ₂ 2.v2.4-IgGl to full-length S of omicron. As shown in FIG. 43B, sACE22.v2.4-IgGl retains tight binding that is higher than wild type sACE22-IgGl. The monoclonal antibodies REGN10933, REGN10987, and LY-CoV555 have no significant affinity for the omicron spike protein, which aligns with their reported loss of efficacy in neutralization experiments. VIR-7831 binds the omicron spike protein, although over an order of magnitude weaker than sACE22-IgGl. sACE22.v2.4.2-IgGl, which binds delta S with greater affinity than sACE22.v2.4-IgGl, did not show enhanced affinity for omicron spike protein as compared to sACE22.v2.4-IgGl, suggesting that additional engineering of sACE2 for affinity against selected viral variants does not necessarily increase affinity universally and that it may be advantageous to formulate a mixture of engineered decoys together with wild type sACE2 to ensure that there always remains some basal S binding activity as SARS-CoV-2 continues to evolve. INCORPORATION BY REFERENCE

[0003] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[0004] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the disclosure described herein. Various structural elements of the different embodiments and various disclosed method steps may be utilized in various combinations and permutations, and all such variants are to be considered forms of the disclosure. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.