Composition for preventing or treating coronavirus infection

Abstract

The present application relates to compositions for preventing or treating infectious diseases. In some embodiments, the present application provides chimeric proteins comprising a target-binding moiety comprising an extracellular binding domain (EBD) or a fragment thereof of angiotensin-converting enzyme 2 (ACE2) protein that specifically binds to S protein, and a positively charged mucoadhesive peptide fragment. Compositions comprising the chimeric proteins described herein are useful for preventing or treating viral infections in individuals, such as coronavirus infections.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Nos. 63 / 331,216, filed April 14, 2022, and 63 / 418,570, filed October 23, 2022, both of which are incorporated by reference in their entirety.

[0002] Electronic Sequence Listing Reference

[0002] The contents of the electronic sequence listing (256442000340SEQLIST.xml; size: 163,869 bytes; and creation date: April 12, 2023) are incorporated herein by reference in their entirety.

[0003] Technical Field

[0003] The present invention relates to compositions and methods for preventing or treating coronavirus infections. [Background technology]

[0004] Respiratory microbial infections, including viral and bacterial infections, are a leading cause of illness and death in adults and children worldwide. There is a significant unmet need for new treatments and prevention strategies that more effectively target these infections. The rapid spread and high morbidity and mortality of coronavirus infections, including SARS-CoV-2 infection, the virus that causes COVID-19, have severely impacted human health and the economy from 2020 to 2022. The number of SARS-CoV-2 infections and hospitalizations has skyrocketed due to increased social activity and mobility, which has led to an increase in viral infections. SARS-CoV-2 infection is primarily transmitted from person to person through infectious droplets released when an infected person speaks, sneezes, or coughs. Infectious droplets can land in the mouths or noses of nearby individuals or be inhaled into the lungs, making the upper respiratory tract mucosal surfaces the initial and primary site of viral infection. Furthermore, airborne transmission of viruses can occur via aerosol particles, which remain in the air for longer periods of time and can travel farther from their source than droplets. While face masks are used as a first line of defense, they are a passive barrier to infection and are imperfectly effective. Treatments and prevention efforts are necessary to contain coronavirus pandemics such as SARS-CoV-2.

[0005] Therapeutic drug development for COVID-19 treatment includes both small molecule and large molecule (e.g., inhibitory polypeptide) drug candidates. Currently, many inhibitory polypeptides targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein have been developed. Angiotensin-converting enzyme 2 (ACE2) mediates viral entry into cells by binding to the S protein via the S1 subunit of the RBD. Therefore, ACE2 inhibitor peptides are promising therapeutic agents for preventing coronavirus infection.

[0006] Current pharmaceutical prevention strategies for COVID-19 focus on the development of SARS-CoV-2 vaccines, but such vaccine approaches may be burdened by viral genetic mutations and the potential for antibody-dependent enhancement (ADE, Ricke, Front Immunol. 2021;12:640093). Therefore, safe and flexible methods for preventing coronavirus infection are urgently needed. Summary of the Invention

[0007]

[0007] The present application provides compositions and methods for preventing or treating an infection caused by a coronavirus or its variants. Accordingly, one aspect of the present application provides a chimeric protein comprising: (a) a target-binding moiety comprising the extracellular binding domain (EBD) or a fragment thereof of angiotensin-converting enzyme 2 (ACE2) protein, which specifically binds to the spike (S) protein; and (b) a mucoadhesive peptide fragment comprising at least about five positively charged amino acid residues, which promotes adhesion of the chimeric protein to a mucosa.

[0008] In some embodiments, the chimeric protein comprises a single polypeptide chain.

[0009] In some embodiments, the chimeric protein comprises two or more polypeptide chains. In some embodiments, the chimeric protein comprises two or more mucoadhesive peptide fragments. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises at least about five positively charged amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises at least about six positively charged amino acid residues.

[0010] In some embodiments, the positively charged amino acid residue is selected from the group consisting of lysine, arginine, histidine, ornithine, and combinations thereof. In some embodiments, the positively charged amino acid residue comprises lysine. In some embodiments, the mucoadhesive peptide fragment comprises about 5, about 6, about 12, or about 30 lysines. In some embodiments, the positively charged amino acid residue comprises arginine. In some embodiments, the mucoadhesive peptide fragment comprises about 5, about 6, about 12, or about 30 arginines. In some embodiments, the positively charged amino acid residue comprises histidine. In some embodiments, the mucoadhesive peptide fragment comprises about 5, about 6, about 12, or about 30 histidines. In some embodiments, the positively charged amino acid residue comprises ornithine. In some embodiments, the mucoadhesive peptide fragment comprises about 5, about 6, about 12, or about 30 ornithines.

[0011] In some embodiments, the mucoadhesive peptide fragment comprises at least five consecutive positively charged amino acids. In some embodiments, the positively charged amino acid residues are interspersed with one or more non-positively charged amino acid residues. In some embodiments, the non-positively charged amino acid residues are non-polar amino acids or polar non-charged amino acids. In some embodiments, the non-positively charged amino acid residues are selected from the group consisting of isoleucine, valine, alanine, tryptophan, leucine, glycine, methionine, proline, phenylalanine, threonine, cysteine, tyrosine, glutamine, serine, and asparagine, and combinations thereof. In some embodiments, at least 50% of the amino acid residues in the mucoadhesive peptide fragment are positively charged amino acid residues.

[0012] In some embodiments, the mucoadhesive peptide fragment is less than about 15 kD. In some embodiments, the mucoadhesive peptide fragment has an isoelectric point (pI) higher than the pH of the mucosa. In some embodiments, the half-life of the chimeric protein on the mucosa is at least 12 hours. In some embodiments, the mucoadhesive peptide fragment does not promote penetration of the chimeric protein into mucosal cells. In some embodiments, the mucoadhesive peptide fragment does not disrupt folding of the chimeric protein in host cells expressing the chimeric protein. In some embodiments, the mucoadhesive peptide fragment does not prevent secretion of the chimeric protein from host cells expressing the chimeric protein. In some embodiments, the mucoadhesive peptide fragment does not interfere with binding between the target binding moiety and the S protein.

[0013]

[0013] In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134.

[0014] In some embodiments, the mucoadhesive peptide fragment is fused to the target binding moiety via a bond.

[0015] In some embodiments, the mucoadhesive peptide fragment is fused to the target binding moiety via a peptide linker. In some embodiments, the peptide linker comprises one or more oligomerization and / or multimerization domains. In some embodiments, the peptide linker comprises the constant region of the heavy chain of a full-length antibody or fragment thereof. In some embodiments, the peptide linker comprises the constant region of the light chain of a full-length antibody or fragment thereof. In some embodiments, the linker comprises a C H1, C H2, C H3, C H4 and / or C H5 domain. Ldomain, or fragments thereof. In some embodiments, the peptide linker comprises a CH2 domain or fragment thereof. In some embodiments, the peptide linker further comprises an antibody hinge domain or fragment thereof. In some embodiments, the linker comprises an Fc region or fragment thereof. In some embodiments, the linker comprises a detectable enzyme tag. In some embodiments, the enzyme tag is alkaline phosphatase. In some embodiments, the enzyme tag is glutathione-S-transferase. In some embodiments, the peptide linker comprises a basic helix-loop-helix leucine zipper (bZIP) domain. In some embodiments, the peptide linker comprises a bZIP isoleucine zipper domain. In some embodiments, the peptide linker comprises a bZIP-leucine / isoleucine zipper domain. In some embodiments, the peptide linker comprises a collagen-like peptide. In some embodiments, the peptide linker comprises a p53 tetramerization domain. In some embodiments, the peptide linker comprises a streptavidin (SA) protein. In some embodiments, the peptide linker comprises an SA protein and a dextran backbone. In some embodiments, the peptide linker comprises an SA protein and one or more maleimide polymers (DMGS). In some embodiments, the peptide linker comprises a bacteriophage T7 fibritin protein or a portion thereof. In some embodiments, the peptide linker comprises a cartilage oligomeric matrix protein (COMP) protein.

[0016] In some embodiments, the mucoadhesive peptide fragment is fused to the C-terminus of the target-binding moiety.

[0017] In some embodiments, the target binding moiety comprises the EBD of the human ACE2 (hACE2) protein, or a fragment or variant thereof. In some embodiments, the target binding moiety comprises amino acids 30-41 of the full-length hACE2 protein, or a variant thereof comprising at least about 90% sequence identity to amino acids 30-41 of the full-length hACE2 protein. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 102, or a variant thereof comprising at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 102. In some embodiments, the target binding moiety comprises amino acids 24-42 of the full-length hACE2 protein, or a variant thereof comprising at least about 90% sequence identity to amino acids 24-42 of the full-length hACE2 protein. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the target binding moiety comprises the amino acid sequence of any one of SEQ ID NOs: 1-7, 9-14, and 135, or a variant thereof comprising at least about 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-7, 9-14, and 135. In some embodiments, the chimeric protein comprises the amino acid sequence of any one of SEQ ID NOs: 84-88 and 136-140, or a variant thereof comprising at least about 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 84-88 and 136-140.

[0018] In some embodiments, the target binding moiety comprises the EBD of an animal ACE2 protein, or a fragment or variant thereof. In some embodiments, the animal ACE2 protein is a mouse, guinea pig, horse, ferret, monkey, chimpanzee, pig, dog, cat, cow, rabbit, mink, or chicken ACE2 protein, or a fragment or variant thereof. In some embodiments, the target binding moiety comprises amino acids 30-41 of a full-length animal ACE2 protein, or a variant thereof comprising at least about 90% sequence identity to amino acids 30-41 of a full-length animal ACE2 protein, wherein the full-length animal ACE2 protein is not a chicken ACE2 protein or a dog ACE2 protein. In some embodiments, the target binding moiety comprises amino acids 29-40 of a full-length animal ACE2 protein, or a variant thereof comprising at least about 90% sequence identity to amino acids 29-40 of a full-length animal ACE2 protein, wherein the full-length animal ACE2 protein is not a chicken ACE2 protein or a dog ACE2 protein. In some embodiments, the target binding moiety comprises the amino acid sequence of any one of SEQ ID NOs: 110-122, or a variant thereof comprising at least about 90% sequence identity to any one of SEQ ID NOs: 110-122. In some embodiments, the target binding moiety comprises the amino acid sequence of any one of SEQ ID NOs: 15-27, or a variant thereof comprising at least about 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 15-27. In some embodiments, the chimeric protein comprises the amino acid sequence of any one of SEQ ID NOs: 89-93, or a variant thereof comprising at least about 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 89-93.

[0019] In some embodiments, the mucosa is selected from the group consisting of nasal mucosa, laryngeal mucosa, tracheal mucosa, bronchial mucosa, pulmonary mucosa, ocular mucosa, and combinations thereof.

[0020]

[0020] In some aspects, provided herein is a pharmaceutical composition comprising the chimeric protein of any of the present embodiments and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a plurality of chimeric proteins, at least two of which are different from one another. In some embodiments, the pharmaceutically acceptable carrier comprises about 0.05% to about 0.2% (w / w) methionine. In some embodiments, the pharmaceutically acceptable carrier has a pH of about 4.5 to about 7.5. In some embodiments, the pharmaceutically acceptable carrier comprises about 20 mM to about 50 mM citrate. In some embodiments, the pharmaceutically acceptable carrier comprises about 100 mM to about 150 mM NaCl. In some embodiments, the pharmaceutically acceptable carrier comprises about 0.01% to about 0.1% (w / w) polysorbate 80. In some embodiments, the pharmaceutically acceptable carrier comprises about 1% to about 10% (w / w) glycerin. In some embodiments, the pharmaceutically acceptable carrier comprises about 0.05% to about 0.2% (w / w) potassium sorbate. In some embodiments, the pharmaceutically acceptable carrier (i) comprises about 0.05% to about 0.2% (w / w) methionine, (ii) has a pH of about 4.5 to about 7.5, (iii) comprises about 20 mM to about 50 mM citrate, (iv) comprises about 100 mM to about 150 mM NaCl, (v) comprises about 0.01% to about 0.1% (w / w) polysorbate 80, (vi) comprises about 1% to about 10% (w / w) glycerin, and / or (vii) comprises about 0.05% to about 0.2% (w / w) potassium sorbate. In some embodiments, the pharmaceutical compositions are formulated for intranasal, intraocular, and / or intrabronchial administration.

[0021]

[0021] In some aspects, provided herein is an isolated nucleic acid or set of isolated nucleic acids encoding the chimeric protein of any of the foregoing embodiments.

[0022] In another aspect, provided herein is a vector or set of vectors comprising a nucleic acid or set of nucleic acids of the foregoing embodiments.

[0023]

[0023] In a further aspect, provided herein is a host cell comprising a chimeric protein of any of the preceding embodiments, a nucleic acid or set of nucleic acids of the preceding embodiments, or a vector or set of vectors of the preceding embodiments.

[0024]

[0024] In a further aspect, provided herein is a method for preparing a chimeric protein, the method comprising: (a) culturing a host cell of the aforementioned embodiment under conditions effective to express the chimeric protein; and (b) obtaining the expressed chimeric protein from the host cell.

[0025]

[0025] In another aspect, provided herein are methods for preventing or treating an infection caused by a virus in an individual, the method comprising administering to the individual an effective amount of a chimeric protein of any of the preceding embodiments or a pharmaceutical composition of any of the preceding embodiments. In some embodiments, the chimeric protein or pharmaceutical composition is administered to the individual before the individual is exposed to the virus. In some embodiments, the chimeric protein or pharmaceutical composition is administered to the individual within about 72 hours after the individual is exposed to the virus. In some embodiments, the chimeric protein or pharmaceutical composition is administered topically to a mucous membrane. In some embodiments, the chimeric protein or pharmaceutical composition is administered via a nasal spray, inhaler, nebulizer, or eye drops. In some embodiments, the chimeric protein or pharmaceutical composition is administered once daily.

[0026] In some aspects, provided herein are in vitro methods for killing or neutralizing a virus, comprising contacting the virus with a chimeric protein of any of the foregoing embodiments in the presence of at least one component of the complement system. In some embodiments, the chimeric protein comprises a peptide linker comprising an Fc region or a fragment thereof. In some embodiments, the peptide linker comprises a C H1, C H2, C H3, C H4, and / or C H5 domain. L domain, or fragments thereof. In some embodiments, the peptide linker comprises a CH2 domain or fragment thereof. In some embodiments, the peptide linker further comprises an antibody hinge domain or fragment thereof. In some embodiments, at least one component of the complement system is C1, C4, or the membrane attack complex (MAC). In some embodiments, at least one component of the complement system is C1. In some embodiments, at least one component of the complement system is C4. In some embodiments, C4 is involved in viral neutralization. In some embodiments, at least one component of the complement system is MAC. In some embodiments, MAC is involved in viral killing.

[0027]

[0027] In another aspect, provided herein are methods of killing or neutralizing a virus in an individual, comprising administering to the individual an effective amount of a chimeric protein of any of the preceding embodiments or a pharmaceutical composition of any of the preceding embodiments. In some embodiments, the chimeric protein or pharmaceutical composition comprising the chimeric protein and a pharmaceutically acceptable carrier comprises a peptide linker comprising an Fc region or a fragment thereof. In some embodiments, the peptide linker comprises a C H1, C H2, C H3, C H4, and / or C H5 domain. L In some embodiments, the peptide linker comprises a CH2 domain or a fragment thereof. In some embodiments, the peptide linker further comprises an antibody hinge domain or a fragment thereof.

[0028] In another aspect, provided herein are methods of activating the complement pathway in an individual, comprising administering to the individual an effective amount of any of the preceding embodiments or the pharmaceutical composition of the preceding embodiments. In some embodiments, the chimeric protein, or pharmaceutical composition comprising the chimeric protein and a pharmaceutically acceptable carrier, comprises a peptide linker comprising an Fc region or a fragment thereof. In some embodiments, the peptide linker comprises a C H1, C H2, C H3, C H4, and / or C H5 domain. L In some embodiments, the peptide linker comprises a CH2 domain or a fragment thereof. In some embodiments, the peptide linker further comprises an antibody hinge domain or a fragment thereof.

[0029] In some embodiments, at least one virus is killed on the mucosa. In some embodiments, at least one virus is neutralized on the mucosa.

[0030]

[0030] In another aspect, provided herein is a method of preventing, treating, or reducing an infection caused by a virus in an individual, comprising administering to the individual an effective amount of any of the preceding embodiments or the pharmaceutical composition of the preceding embodiments, wherein at least one virus is killed or neutralized on the mucosa. In some embodiments, the chimeric protein or pharmaceutical composition comprising the chimeric protein and a pharmaceutically acceptable carrier comprises a peptide linker comprising an Fc region or a fragment thereof. In some embodiments, the peptide linker comprises a C H1, C H2, C H3, C H4, and / or C H5 domain. L In some embodiments, the peptide linker comprises a CH2 domain or a fragment thereof. In some embodiments, the peptide linker further comprises an antibody hinge domain or a fragment thereof.

[0031] In some embodiments, the chimeric protein activates the complement pathway in the individual. In some embodiments, killing or neutralization is achieved through activation of the complement pathway.

[0032] In some embodiments, the virus is a coronavirus. In some embodiments, the virus is selected from the group consisting of SARS-CoV, SARS-CoV-2, and HCoV-NL63. In some embodiments, the S protein comprises the amino acid sequence of any one of SEQ ID NOs: 96-100, 103, 123-127, and 141-143, or a variant thereof comprising at least about 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 96-100, 103, 123-127, and 141-143.

[0033] Also provided are kits and articles of manufacture (eg, nasal sprays) comprising any one of the above-described compositions and instructions for any one of the above-described methods.

[0034]

[0034] The drawings illustrate certain embodiments of the features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended claims in any way. [Brief explanation of the drawings]

[0035] [Figure 1]

[0035] Various human ACE2 protein constructs and key features are shown. [Figure 2]

[0036]

[0023] Figure 1 shows a CLUSTALOmega multiple sequence alignment of several human ACE2 (hACE2) and animal ACE2 sequences. Asterisks indicate conserved amino acid residues, and the spike protein S1 subunit binding site, which serves as the basis for the hACE2 fragment ACE12, is underlined. SEQ ID NOs corresponding to the ACE2 proteins in the alignment, in order of appearance, are as follows: SEQ ID NO:27 (chicken), SEQ ID NO:16 (guinea pig), SEQ ID NO:15 (mouse), SEQ ID NO:20 (pig), SEQ ID NO:23 (bovine), SEQ ID NO:24 (rabbit), SEQ ID NO:18 (monkey), SEQ ID NO:1 (human), SEQ ID NO:19 (chimpanzee), SEQ ID NO:17 (horse), SEQ ID NO:25 (ferret), SEQ ID NO:26 (mink), SEQ ID NO:21 (dog), and SEQ ID NO:22 (cat). [Figure 3A]

[0037] This figure shows that an exemplary ACE2-Fc1-12K chimeric protein binds to the spike protein S1 subunit protein. SARS-CoV-2 wild-type, alpha, beta, delta, and omicron variant BA.1 and BA.2 spike protein S1 subunit proteins were transiently expressed in HEK293F cells. The cells were first incubated with ACE2-Fc1-12K, followed by a PE-Fc secondary antibody, and then sorted using fluorescence-activated cell sorting (FACS). [Figure 3B]

[0038] The exemplary ACE2-Fc1-12K chimeric protein (20 μg / mL) binds well to spike protein S1 subunit proteins (10 μg / mL) from the alpha, delta, and omicron variants of SARS-CoV-2, and is unaffected by the presence of polycationic moieties. Bio-Layer Interferometry with a streptavidin sensor was used to measure the interaction. All steps were aligned with step baseline 2 (sensor position). The negative control was a HIS-tagged hACE2 protein (lacking the C-terminal polycationic peptide). [Figure 3C]

[0039] This shows that an exemplary ACE614-Fc1-12K chimeric protein blocks SARS-CoV-2 pseudovirus infection of ACE2-expressing HEK293F cells ("HEK293F-ACE2 cells"). ACE614-Fc1-12K chimeric protein was incubated with a pseudotyped lentivirus containing an EF1-α-driven luciferase and GFP reporter gene separated by a P2A self-cleaving peptide and then added to HEK293F-ACE2 cells. Infection was determined by detecting luciferase levels or by detecting GFP in infected cells. [Figure 3D]

[0040] We show that the ACE614-Fc1-12K chimeric protein can block pseudoviruses prepared from the recently circulating (2021 and 2022) Omicron BA.4 variants of SARS-CoV-2 from infecting ACE2-expressing HEK293F cells (using a method similar to that shown in Figure 3C). [Figure 3E]

[0041] We show that the ACE614-Fc1-12K chimeric protein can block pseudovirus prepared from the recently circulating (2022–2023) Omicron XBB.1.5 variant of SARS-CoV-2 from infecting ACE2-expressing HEK293F cells (using a method similar to that shown in Figure 3C). [Figure 4]

[0042] We demonstrate that the ACE2-Fc1-12K chimeric protein activates the complement pathway. The ACE2-Fc1-12K chimeric protein was mixed with complement immunoassay reagents using the Creative Biolabs CH50 Functional Test Kit, which includes the spike protein S1 subunit from SARS-CoV-2 (Delta) and a human complement control (Quidel), and then incubated with red blood cells. Complement fixation pathway activity (classical complement pathway activity) was determined by detecting the degree of hemolysis. [Figure 5]

[0043] We demonstrate that ACE2-Fc1-12K chimeric protein prevents SARS-CoV-2 pseudovirus infection of ACE2-expressing HEK293F cells by activating the complement pathway, thereby killing the SARS-CoV-2 pseudovirus and reducing the amount of SARS-CoV-2 pseudovirus available for infection. ACE2-Fc1-12K chimeric protein was mixed with pseudotyped lentivirus containing an EF1-α-driven luciferase reporter gene, then added to HEK293F-ACE2 cells and incubated with human complement IgG / IgM. Infection was determined by detecting luciferase levels. [Figure 6]

[0044] We show that the presence of polylysine peptide significantly increases the attraction of the ACE2-Fc1-12K chimeric protein to mucin proteins compared with ACE2-Fc1 lacking the C-terminal polylysine peptide modification. To compare the binding of ACE2-Fc1-12K to ACE2Fc1 lacking the cationic modification (12 lysines), an ELISA assay using mucin-coated plates was used. Binding was detected using horseradish peroxidase (HRP)-conjugated goat anti-human IgG, and staining was detected at OD450. [Figure 7A]

[0045] Bioluminescence imaging (BLI) of hACE2 transgenic mice challenged with SARS-CoV-2 delta mutant pseudovirus is shown. Mice were first intranasally challenged with ACE2 mucoadhesive protein (right panel) or vehicle alone (buffer, left panel), and then (10 hours later) intranasally challenged with delta pseudovirus particles. The bottom panel shows graphs of luciferase luminescence (bioluminescence) on the day before challenge and 3, 5, and 7 days after challenge. These data demonstrate the ability of the ACE2-Fc1-12K chimeric protein to protect mice from SARS-CoV-2 delta mutant pseudovirus infection in an in vivo animal model. [Figure 7B]

[0046] Bioluminescence images of hACE2 transgenic mice administered SARS-CoV-2 omicron BA.2 mutant pseudovirus using the same methods described herein for the data shown in Figure 7A, demonstrating the ability of ACE2-Fc1-12K chimeric protein to protect mice from SARS-CoV-2 omicron BA.2 mutant pseudovirus infection. [Figure 7C]

[0047] Bioluminescence images demonstrate that the mucoadhesive modification of the ACE2-Fc1-12K chimeric protein plays a role in protecting hACE2 transgenic mice from pseudovirus infection. Seven days after inoculation with a SARS-CoV-2 mutant delta pseudotyped lentivirus, luciferase release from mice pretreated with ACE2-Fc1-12K chimeric protein bearing the polylysine mucoadhesive modification (center panel) was compared with mice pretreated with ACE2-Fc1 lacking the polylysine mucoadhesive modification (right panel). Pretreatment with vehicle alone is shown in the left panel. [Figure 8]

[0048] The binding affinity of the ACE2-Fc1-12K mucoadhesive chimeric protein to the SARS-CoV-2 spike protein (S1 subunit) at various concentrations (90.91 nM ("1" in Figure 8), 45.45 nM ("2" in Figure 8), 22.73 nM ("3" in Figure 8), 11.36 nM ("4" in Figure 8), 5.682 nM ("5" in Figure 8), and 2.841 nM ("6" in Figure 8)) was measured by surface plasmon resonance (top panel). All tested SARS-CoV-2 variants showed high affinity for the S1 subunit, as demonstrated by K values ​​in the nanomolar range (bottom panel). [Figure 9]

[0049] We show that the addition of various mucoadhesive peptides increases the attraction of ACE2-Fc1 to mucin proteins. To compare the mucin-binding ability of the ACE2-Fc1 chimeric protein with that of ACE2-Fc1 without the mucoadhesive peptide, we performed an ELISA assay using mucin-coated plates. Binding was detected using horseradish peroxidase (HRP)-conjugated goat anti-human IgG, and staining was measured at OD450. [Figure 10]

[0050] This shows that the exemplary ACE2-Fc1-12K chimeric protein and various other mucoadhesive chimeric proteins bind to the spike protein S1 subunit protein. The spike protein S1 subunit protein from SARS-CoV-2 omicron variant BA.5 was transiently expressed in HEK293F cells. The cells were first incubated with the ACE2-Fc1 chimeric protein, followed by a PE-Fc secondary antibody, and then sorted using fluorescence-activated cell sorting (FACS). [Figure 11]

[0051] We demonstrate that the exemplary ACE614-Fc1-12K chimeric protein and various other mucoadhesive chimeric proteins block SARS-CoV-2 pseudovirus infection of ACE2-expressing HEK293F cells. ACE740-Fc1 chimeric protein was incubated with a pseudotyped lentivirus containing an EF1-α-driven luciferase and GFP reporter gene separated by a P2A self-cleaving peptide and then added to HEK293F-ACE2 cells. Infection was determined by detecting luciferase levels or by detecting GFP in infected cells. DETAILED DESCRIPTION OF THE INVENTION

[0036]

[0052] The present application provides compositions and methods for preventing or treating infectious diseases caused by viruses that infect an individual via the mucosa (e.g., coronaviruses) by targeting the virus using chimeric proteins ("ACE2 chimeric proteins") comprising an angiotensin-converting enzyme 2 (ACE2) fragment (e.g., the extracellular binding domain (EBD) of the ACE2 protein or a fragment thereof) with a positively charged mucoadhesive peptide fragment, optionally via a peptide linker.

[0037]

[0053] Accordingly, one aspect of the present application provides chimeric proteins comprising: (a) a target-binding moiety comprising the extracellular binding domain (EBD) of angiotensin-converting enzyme 2 (ACE2) protein, or a fragment thereof, that specifically binds to the spike (S) protein; and (b) a mucoadhesive peptide fragment comprising at least about five positively charged amino acid residues (e.g., lysine or histidine), which promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the S protein is derived from a coronavirus (e.g., SARS-CoV-2). In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker (e.g., an immunoglobulin Fc region or fragment thereof).

[0038]

[0054] For example, the compositions described herein may comprise a chimeric protein or a cocktail of different chimeric proteins that contain an ACE2 fragment that targets the SARS-CoV-2 spike (S) protein and are modified with a positively charged peptide that prevents the SARS-CoV-2 virus from reaching its primary target cell population in the respiratory tract (e.g., nasal) mucosa for infection of humans or animals. The compositions may be administered via the nasal cavity using a respiratory spray.

[0039]

[0055] The inventors of the present application have developed a chimeric protein comprising an ACE2 fragment fused, optionally via a peptide linker, to a positively charged mucoadhesive peptide fragment that recognizes the S1 subunit of the S protein of coronaviruses such as SARS-CoV-2. The chimeric protein exhibits significantly improved affinity for mucin molecules compared to the unmodified ACE2 fragment, resulting in improved stability in respiratory mucosa. In cell-based assays, this chimeric protein demonstrated improved efficacy in blocking SARS-CoV-2 infection compared to the unmodified ACE2 fragment. Furthermore, the chimeric protein activates innate immune function and can kill the SARS-CoV-2 virus through activation of the complement pathway. Administration of the exemplary chimeric protein into the nares of mice prevents infection in mice challenged with a high-titer SARS-CoV-2 pseudovirus at least 10 hours after the initial treatment. Protection against SARS-CoV-2 is effective in both the nose and lungs 7 days after virus exposure. Exemplary chimeric proteins are highly stable and maintain SARS-CoV-2 neutralizing activity in nasal spray formulations. Nasal sprays of chimeric proteins can be developed as affordable and effective preventative products to protect people from infection due to airborne (e.g., via the nasal passages) SARS-CoV-2 virus exposure. Chimeric proteins can act as universal binders, blockers, and / or capturers for viruses, such as coronaviruses, whereby the chimeric proteins can bind to the S protein of any coronavirus variant, regardless of its mutation, as long as the virus normally enters host cells via the molecule from which the target-binding moiety in the chimeric protein is derived. For example, the ACE2 chimeric proteins disclosed herein are universal binders, blockers, and / or capturers for all coronaviruses that enter host cells via the ACE2 receptor molecule, including SARS-CoV-2, SARS-CoV, and HCoV-NL63.

[0040]

[0056] Compared to other methods of inhibiting microbial infections that involve the systemic administration of molecules (e.g., inhibitory polypeptides) that lack positively charged mucoadhesive peptide fragments, the methods described herein require the administration of much less protein, resulting in significant cost savings that are important in any pandemic situation. The compositions can also be self-administered, significantly reducing the burden on overwhelmed healthcare systems.

[0041]

[0057] Accordingly, one aspect of the present application provides chimeric proteins comprising: (a) a target-binding moiety comprising the extracellular binding domain (EBD) of angiotensin-converting enzyme 2 (ACE2) protein or a fragment thereof that specifically binds to the spike (S) protein; and (b) a mucoadhesive peptide fragment comprising at least about five positively charged amino acid residues (e.g., lysine or histidine), which promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the S protein is derived from a coronavirus (e.g., SARS-CoV-2). In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker (e.g., an immunoglobulin Fc region or fragment thereof).

[0042]

[0058] The chimeric proteins provided herein are useful for treating or preventing infection by coronavirus in an individual, and for killing or neutralizing coronavirus in an individual, through activation of the complement pathway.

[0043]

[0059] I. Definition

[0060] The term "target binding moiety" is used herein to refer to a molecule or fragment thereof that is capable of specifically binding to a target. A target binding moiety may have one or more target binding sites.

[0044]

[0061] As used herein, a "mucoadhesive peptide fragment" refers to a peptide that has one or more positive charges and is capable of interacting with a mucosa, for example, via electrostatic interactions.

[0045]

[0062] As used herein, "receptor" refers to a receptor on a host cell that facilitates or mediates microbial entry into the host cell. The receptor can be a membrane-bound receptor or a soluble receptor.

[0046]

[0063] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing one or more symptoms attributable to a disease, attenuating the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease, preventing or slowing the onset or recurrence of the disease, slowing or slowing the progression of the disease, ameliorating the disease state, providing remission (whether partial or total) of the disease, reducing the dose of one or more other drugs required to treat the disease, slowing the progression of the disease, improving quality of life, and / or prolonging survival. "Treatment" also includes reducing the pathological consequences of a disease. The methods of the present application contemplate any one or more of these therapeutic aspects.

[0047]

[0064] "Preventing," as used herein, includes providing protection against the occurrence or recurrence of a disease in a subject who may have a predisposition to the disease but has not yet been diagnosed with the disease.

[0048]

[0065] An "effective amount" of a drug refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. An effective amount herein can vary depending on factors such as the patient's condition, age, sex, and weight, as well as the ability of the chimeric protein (e.g., target-binding moiety) to elicit a desired response in an individual. An effective amount is also an amount in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, its complications, and intermediate pathological phenotypes exhibited during disease development. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms attributable to the disease, improving the quality of life of a disease sufferer, reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug, e.g., by targeting, delaying disease progression, and / or prolonging survival. For purposes of this application, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, directly or indirectly. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" is considered in the context of administering one or more therapeutic agents, and a single agent is considered to be given in an effective amount if the desired result can or will be achieved in combination with one or more other agents. An effective amount can be ascertained by measuring the relevant physiological effect and can be adjusted in conjunction with dosing regimens, diagnostic analysis of the subject's condition, and the like.

[0049]

[0066] The terms "individual," "subject," and "patient" are used interchangeably herein to refer to a mammal, including a human. In some embodiments, the individual is a human. In some embodiments, the individual is suffering from a respiratory infection. In some embodiments, the individual is in need of treatment.

[0050]

[0067] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or unnatural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this application, "polypeptide" refers to proteins containing modifications (generally conservative in nature) to the native sequence, such as deletions, additions, and substitutions, so long as the protein maintains a desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts producing the protein or errors during PCR amplification.

[0051]

[0068] As used herein, the terms "specifically binds," "specifically recognizes," or "is specific for" refer to a measurable, reproducible interaction, such as binding, between a target and a target-binding moiety that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, a target-binding moiety that specifically recognizes a target (which may be an epitope) is one that binds to this target with higher affinity, avidity, more readily, and / or for a longer duration than to other targets. In some embodiments, a target-binding moiety that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen (e.g., a SARS-CoV-2S protein) with a binding affinity that is at least about 10-fold greater than the binding affinity for other targets (e.g., a MERS-CoVS protein, or a non-respiratory pathogen protein).

[0052]

[0069] The "CH1 domain" (also called the "C1" domain of "H1") of a human IgG Fc region typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0053]

[0070] The "hinge region" is generally defined as extending from Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain S—S bonds in the same positions.

[0054]

[0071] The "CH2 domain" (also called the "C2" domain of "H2") of human IgG Fc region typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in intact native IgG molecules. It has been speculated that the carbohydrates may displace domain-domain pairing and help stabilize the CH2 domains. Burton, Molec Immunol. 22:161-206 (1985).

[0055]

[0072] The "CH3 domain" (also called the "C2" or "H3" domain) comprises the stretch of residues C-terminal to the CH2 domain of the Fc region (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically to amino acid residue 446 or 447 for IgG).

[0056]

[0073] The "CH4 domain" found in IgE and IgM molecules is located C-terminal to the CH3 domain and includes residues 466-572 in human IgM and residues 323-427 in hIgE. As used herein, the term "substantially similar" or "substantially the same" refers to a sufficiently high similarity between two or more values ​​that one skilled in the art would consider the difference between the two or more values ​​to have little or no biological and / or statistical significance in the biological properties measured by the values. In some embodiments, the difference between two or more substantially similar values ​​is no more than one of 5%, 10%, 15%, 20%, 25%, or 50%. As used herein, the term "substantially similar" or "substantially the same" refers to a sufficiently high similarity between two or more values ​​that one skilled in the art would consider the difference between the two or more values ​​to have little or no biological and / or statistical significance in the biological properties measured by the values. In some embodiments, two or more substantially similar values ​​differ by no more than any one of 5%, 10%, 15%, 20%, 25%, or 50%.

[0057]

[0074] A "variant" of a polypeptide refers to a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide, and up to 100% identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the polypeptide. In some embodiments, a variant has at least about 80% sequence identity. In some embodiments, a variant has at least about 90% sequence identity. In some embodiments, a variant has at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0058]

[0075] As used herein, "percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the specific peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be performed using, for example, BLAST, BLAST-2, ALIGN, or MEGALIGN. TM This can be achieved in a variety of ways within the skill of those in the art using publicly available computer software, such as (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0059]

[0076] The term "isolated nucleic acid" as used herein is intended to mean a nucleic acid of genomic, cDNA, or synthetic origin, or some combination thereof, and by its origin, an "isolated nucleic acid" means that (1) the "isolated nucleic acid" is not associated with all or part of a polynucleotide with which it is found in nature, (2) it is operably linked to a polynucleotide with which it is not associated in nature, or (3) it is not found in nature as part of a larger sequence.

[0060]

[0077] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may include introns, to the extent that the nucleotide sequence encoding the protein, in some versions, contains introns.

[0061]

[0078] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0062]

[0079] The term "vector" is used to describe a polynucleotide that can be manipulated to contain one or more cloned polynucleotides that can be propagated in a host cell. A vector can contain one or more elements of an origin of replication, one or more regulatory sequences (e.g., promoters and / or enhancers) that control the expression of a polypeptide of interest, and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0063]

[0080] A "host cell" refers to a cell that is or has been the recipient of a vector or isolated polynucleotide. A host cell can be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, as well as their derivatives, such as 293-6E and DG44 cells, respectively.

[0064]

[0081] As used herein, a "mutant" virus refers to a virus isolate whose genomic sequence differs from that of a reference virus, and the genomic sequence difference confers a new phenotypic characteristic, such as increased fitness, relative to the reference virus. When a viral species, such as SARS-CoV-2, is referred to in this application, it is understood that the species encompasses the variant as well as the reference virus that was originally isolated and identified. In some embodiments, the variant viruses described herein are "variants of interest," i.e., variants with specific genetic markers associated with changes to receptor binding, reduced neutralization by antibodies generated in response to previous infection or vaccination, reduced therapeutic efficacy, potential diagnostic impact, and / or predicted increased transmissibility and / or disease severity. In some embodiments, the variant viruses described herein are "variants of concern," i.e., variants with evidence of increased transmissibility, more severe disease (e.g., increased hospitalization and / or death), significantly reduced neutralization by antibodies generated during previous infection or vaccination, reduced therapeutic or vaccine efficacy, and / or failed diagnostic detection. In some embodiments, the mutant viruses described herein are "high consequence variants," i.e., high consequence variants have clear evidence that the effectiveness of preventative or medical countermeasures (MCMs) is significantly reduced compared to previously circulating variants.

[0065]

[0082] As used herein, "pharmaceutically acceptable" or "pharmaceutically compatible" means a material that is biologically or otherwise undesirable; e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing significant undesirable biological effects or interacting adversely with any of the other components of the composition in which it is included. Preferably, a pharmaceutically acceptable carrier or excipient will have met required standards in toxicological and manufacturing testing and / or will be listed in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0066]

[0083] As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substrate, composition, or vehicle used in the process of drug delivery, which may have one or more components that may include, but are not limited to, one or more additives, one or more binders, one or more diluents, one or more solvents, one or more fillers, and / or one or more stabilizers.

[0067]

[0084] Embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0068]

[0085] Reference herein to "about" a value or parameter includes (and accounts for) variations on that value or parameter itself. For example, reference to "about X" includes reference to "X."

[0069]

[0086] As used herein, a reference to a value or parameter being "not" generally means and describes "other than" the value or parameter.

[0070]

[0087] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0071] II. Chimeric Proteins

[0088] The present application provides chimeric proteins (e.g., fusion proteins, i.e., ACE2 chimeric proteins) comprising: (a) a target-binding moiety comprising the extracellular binding domain (EBD) of angiotensin-converting enzyme 2 (ACE2) protein or a fragment thereof, which specifically binds to a spike (S) protein (e.g., a coronavirus S protein, an S1 subunit of an S protein, or an S1 subunit of a coronavirus S protein); and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues, which promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the positively charged amino acid residues are selected from the group consisting of lysine, arginine, histidine, ornithine, and combinations thereof. In some embodiments, the positively charged amino acid residue is lysine. In some embodiments, the mucoadhesive peptide fragment is a polylysine peptide having at least about 5 (e.g., about 5 to about 30, e.g., about 12) lysines (e.g., comprising at least about 5 (e.g., about 5 to about 30, e.g., about 12) consecutive lysines). In some embodiments, the positively charged amino acid residue is histidine. In some embodiments, the mucoadhesive peptide fragment is a polyhistidine peptide having at least about 5 (e.g., about 5 to about 30, e.g., about 12) histidines (e.g., comprising at least about 5 (e.g., about 5 to about 30, e.g., about 12) consecutive histidines). In some embodiments, the positively charged amino acid residue is arginine. In some embodiments, the mucoadhesive peptide fragment is a polyarginine peptide having at least about 5 (e.g., about 5 to about 30, e.g., about 12) arginines (e.g., comprising at least about 5 (e.g., about 5 to about 30, e.g., about 12) consecutive arginines). In some embodiments, the positively charged amino acid residue is ornithine. In some embodiments, the mucoadhesive peptide fragment is a polyornithine peptide having at least about 5 (e.g., about 5 to about 30, e.g., about 12) consecutive ornithines.In some embodiments, the positively charged amino acid residues are contiguous with one another. In some embodiments, the positively charged amino acid residues are interspersed with non-positively charged amino acid residues. In some embodiments, the mucoadhesive peptide fragment is covalently fused to the target-binding moiety. In some embodiments, the mucoadhesive peptide fragment is non-covalently associated with the target-binding moiety, e.g., via an oligomerization and / or multimerization domain. In some embodiments, the S protein is derived from a virus, e.g., a coronavirus. In some embodiments, the virus causes a respiratory infection, e.g., a coronavirus infection. In some embodiments, the mucosa is selected from the group consisting of nasal mucosa, laryngeal mucosa, tracheal mucosa, bronchial mucosa, pulmonary mucosa, ocular mucosa, and combinations thereof. In some embodiments, the target-binding moiety is fused directly to the mucoadhesive peptide fragment. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of the peptide linkers listed in Table 9.

[0072]

[0089] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the EBD of human ACE2 (hACE2) protein or a fragment thereof that specifically binds to the S protein of a coronavirus; and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the target-binding moiety specifically binds to the S1 subunit of the S protein of a coronavirus. In some embodiments, the S protein is any one of the S proteins listed in Table 5. In some embodiments, the coronavirus is SARS-CoV-2 or a variant thereof. In some embodiments, the target binding moiety comprises the amino acid sequence of any one of the hACE2 proteins or fragments thereof, e.g., as set forth in Table 3, e.g., in some embodiments, the target binding moiety comprises the amino acid sequence of any one of SEQ ID NOs: 1-7, 9-14, and 135, or a variant thereof having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of any one of SEQ ID NOs: 1-7, 9-14, and 135. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 102, or a variant thereof comprising at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 102.In some embodiments, the chimeric protein comprises the amino acid sequence of any one of the hACE2 proteins listed in Table 1; for example, in some embodiments, the chimeric protein comprises the amino acid sequence of any one of SEQ ID NOs: 84-88 and 136-140, or a variant thereof having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of any one of SEQ ID NOs: 84-88 and 136-140. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of the mucoadhesive peptide fragments listed in Table 8. In some embodiments, the target-binding moiety is fused directly to the mucoadhesive peptide fragment. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of the peptide linkers listed in Table 9.

[0073]

[0090] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the EBD of an animal ACE2 protein or a fragment thereof that specifically binds to a coronavirus S protein; and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the target-binding moiety specifically binds to the S1 subunit of a coronavirus S protein. In some embodiments, the S protein is any one of the S proteins listed in Table 5. In some embodiments, the coronavirus is SARS-CoV-2 or a variant thereof. In some embodiments, the target binding moiety comprises the amino acid sequence of any one of the animal ACE2 proteins or fragments thereof, e.g., as set forth in Table 4, e.g., in some embodiments, the target binding moiety comprises the amino acid sequence of any one of SEQ ID NOs: 15-27, or a variant thereof having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of any one of SEQ ID NOs: 15-27. In some embodiments, the chimeric protein comprises the amino acid sequence of any one of the animal ACE2 proteins listed in Table 1; for example, in some embodiments, the chimeric protein comprises the amino acid sequence of any one of SEQ ID NOs: 89-93, or a variant thereof having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of any one of SEQ ID NOs: 89-93. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of the mucoadhesive peptide fragments listed in Table 8. In some embodiments, the target-binding moiety is fused directly to the mucoadhesive peptide fragment. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker.In some embodiments, the peptide linker comprises the amino acid sequence of any one of the peptide linkers listed in Table 9.

[0074]

[0091] In some embodiments, the half-life of the chimeric protein on the mucosa is at least about n hours, where n is selected from 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 48 hours, or more. In some embodiments, the half-life of the chimeric protein on the mucosa is at least 12 hours. In some embodiments, the half-life of the chimeric protein on the mucosa is at least 24 hours.

[0075]

[0092] The half-life of a chimeric protein on mucosal membranes can be determined using in vitro assays known in the art. Given the size and polarity of the chimeric protein, its adhesion to mucosa (e.g., the nasal cavity) is minimal due to its low membrane permeability. However, mucociliary clearance may be involved in the half-life of the chimeric protein. Mucoadhesive peptide fragments can improve the retention time of the chimeric protein on mucosal membranes. For example, using an in vitro model cell system such as mucosal epithelial cells, the amount of chimeric protein remaining on the cell / mucin surface can be measured by FACS or immunofluorescence. Alternatively, mucosal-associated components, such as mucin, can be incubated with the chimeric protein, and the amount of chimeric protein associated with mucin can be measured by ELISA.

[0076]

[0093] In some embodiments, the chimeric protein comprises a full-length hACE2 protein or fragment thereof (e.g., the EBD of an ACE2 protein or fragment thereof, e.g., SEQ ID NO: 1) of between about 12 amino acids (aa) and about 805 aa, e.g., between about 12 aa and about 700 aa, between about 15 aa and about 500 aa, between about 100 aa and about 300 aa, between about 200 aa and about 400 aa, between about 300 aa and about 500 aa, between about 400 aa and about 600 aa, between about 500 aa and about 700 aa, between about 600 aa and about 805 aa, between about 12 aa and about 20 aa, between about 15 aa and about 20 aa, between about 21 aa and about 42 aa, between about 30 aa and about 41 aa, or between about 500 aa and about 805 aa. In some embodiments, the chimeric protein comprises a target binding portion comprising a full-length hACE2 protein or fragment thereof of greater than about 12 aa, e.g., greater than n, where n is selected from 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 30 aa, 40 aa, 50 aa, 100 aa, 150 aa, 200 aa, 250 aa, 300 aa, 350 aa, 400 aa, 450 aa, 500 aa, 550 aa, 600 aa, 650 aa, 700 aa, 750 aa, 800 aa, 850 aa, or more. In some embodiments, the chimeric protein comprises a target binding portion comprising a full-length hACE2 protein or variant thereof of less than about 805 aa, e.g., less than about n, where n is selected from 800 aa, 750 aa, 700 aa, 650 aa, 600 aa, 550 aa, 500 aa, 450 aa, 400 aa, 350 aa, 300 aa, 250 aa, 200 aa, 150 aa, 100 aa, 50 aa, 40 aa, 30 aa, 20 aa, 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, or less. In some embodiments, the chimeric protein comprises a target binding portion comprising any of about 805 aa, about 714 aa, about 596 aa, about 380 aa, about 342 aa, about 331 aa, about 182 aa, about 19 aa, or about 12 aa of a full-length hACE2 protein or a variant thereof.

[0077]

[0094] In some embodiments, the chimeric protein comprises a target-binding portion comprising a full-length animal ACE2 protein or variant thereof between about 12 aa and about 805 aa, e.g., between about 12 aa and about 700 aa, between about 15 aa and about 500 aa, between about 100 aa and about 300 aa, between about 200 aa and about 400 aa, between about 300 aa and about 500 aa, between about 400 aa and about 600 aa, between about 500 aa and about 700 aa, between about 600 aa and about 805 aa, between about 15 aa and about 20 aa, between about 12 aa and about 20 aa, between about 21 aa and about 42 aa, between about 30 aa and about 41 aa, or between about 500 aa and about 805 aa. In some embodiments, the chimeric protein comprises a target binding moiety comprising a full-length animal ACE2 protein or variant thereof of greater than about 12 aa, e.g., greater than n (where n is selected from 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 30 aa, 40 aa, 50 aa, 100 aa, 150 aa, 200 aa, 250 aa, 300 aa, 350 aa, 400 aa, 450 aa, 500 aa, 550 aa, 600 aa, 650 aa, 700 aa, 750 aa, 800 aa, 850 aa, or more) full-length animal ACE2 protein or variant thereof. In some embodiments, the chimeric protein comprises a target-binding portion comprising a full-length animal ACE2 protein or variant thereof of less than about 805 aa, e.g., less than about n, where n is selected from 800 aa, 750 aa, 700 aa, 650 aa, 600 aa, 550 aa, 500 aa, 450 aa, 400 aa, 350 aa, 300 aa, 250 aa, 200 aa, 150 aa, 100 aa, 40 aa, 30 aa, 50 aa, 20 aa, 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, or less. In some embodiments, the chimeric protein comprises a target-binding portion comprising any of about 805 aa, about 714 aa, about 596 aa, about 380 aa, about 342 aa, about 331 aa, about 182 aa, about 19 aa, or about 12 aa of a full-length animal ACE2 protein or a variant thereof.

[0078]

[0095] In some embodiments, the chimeric protein comprises a target-binding portion comprising a fragment capable of selectively recognizing the S1 subunit of the S protein and disrupting S1 binding to full-length ACE2. In some embodiments, the chimeric protein comprises a target-binding portion comprising amino acids 24-42 of a full-length ACE2 protein (e.g., a full-length hACE2 protein) or a variant thereof. In some embodiments, the chimeric protein comprises a target-binding portion comprising amino acids 24-42 of a full-length ACE2 protein or a variant thereof between about 12 aa and about 19 aa, e.g., between about 12 aa and about 14 aa, between about 13 aa and about 15 aa, between about 14 aa and about 16 aa, between about 15 aa and about 17 aa, between about 16 aa and about 18 aa, between about 17 aa and about 19 aa, or between about 18 aa and about 19 aa. In some embodiments, the chimeric protein comprises a target binding portion comprising at least about 12 aa, e.g., at least n, of SEQ ID NO:8, where n is selected from 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, and 18 aa. In some embodiments, the chimeric protein comprises a target binding portion comprising more than about 12 aa, e.g., more than n, of SEQ ID NO:8, where n is selected from 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, and 18 aa. In some embodiments, the chimeric protein comprises a target binding portion comprising less than about 20 aa, e.g., less than about n, of SEQ ID NO:8, where n is 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, or less. In some embodiments, the chimeric protein comprises a target binding portion comprising about 19 aa of SEQ ID NO:8. In some embodiments, the chimeric protein comprises a target binding moiety comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 8 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%).

[0079]

[0096] In some embodiments, the chimeric protein comprises a target-binding portion comprising amino acids 30-41 of a full-length ACE2 protein (e.g., a full-length animal hACE2 protein or a full-length animal ACE2 protein) or a variant thereof. In some embodiments, the chimeric protein comprises a target-binding portion comprising the amino acid sequence of any one of SEQ ID NOs: 102, 111-120, and 122, or a variant thereof comprising at least about 90% (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity to any one of SEQ ID NOs: 102, 111-120, and 122. In some embodiments, the full-length ACE2 protein or variant thereof is a hACE2 protein or variant thereof. In some embodiments, the full-length ACE2 protein or variant thereof is not a canine or chicken full-length ACE2 protein or variant thereof.

[0080]

[0097] In some embodiments, the chimeric protein comprises a target-binding portion comprising amino acids 29-40 of a full-length ACE2 protein (e.g., a full-length animal ACE2 protein) or a variant thereof. In some embodiments, the chimeric protein comprises a target-binding portion comprising SEQ ID NO: 110 or 121, or a variant thereof comprising at least about 90% sequence identity to SEQ ID NO: 110 or 121 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the full-length ACE2 protein or variant thereof is a canine or chicken ACE2 protein or variant thereof.

[0081]

[0098] Exemplary chimeric proteins are provided herein that include a target-binding moiety that specifically binds to a coronavirus S protein and a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues. In some embodiments, the chimeric protein includes any of the ACE2 proteins or fragments thereof listed in Tables 3 and 4 (e.g., hACE2 or animal ACE2 proteins or fragments thereof, respectively). In some embodiments, the chimeric protein includes any of the mucoadhesive peptide fragments listed in Table 8. In some embodiments, the target-binding moiety is fused directly to the mucoadhesive peptide fragment. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the chimeric protein includes any of the peptide linkers listed in Table 9.

[0082]

[0099] In some embodiments, the chimeric protein comprises: (a) a target-binding moiety comprising the EBD of ACE2 (e.g., SEQ ID NO: 1) or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30) positively charged amino acid residues that promote adhesion of the chimeric protein to mucosa, wherein the target-binding moiety is fused directly to the mucoadhesive peptide fragment. In some embodiments, the chimeric protein comprises any of the mucoadhesive peptide fragments listed in Table 8.

[0083]

[0100] In some embodiments, the chimeric protein comprises: (a) a target-binding portion comprising the EBD of ACE2 (e.g., SEQ ID NO: 1) or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30) positively charged amino acid residues, which promotes adhesion of the chimeric protein to a mucosa, wherein the target binding portion is fused to the mucoadhesive peptide via a peptide linker. In some embodiments, the peptide linker comprises any of the peptide linkers listed in Table 9. In some embodiments, the chimeric protein comprises: (a) a target-binding portion comprising the EBD of ACE2 (e.g., SEQ ID NO: 1) or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30) positively charged amino acid residues, which promotes adhesion of the chimeric protein to a mucosa, wherein the target binding portion is fused to the mucoadhesive peptide fragment via an immunoglobulin Fc region or a fragment thereof. In some embodiments, the chimeric protein comprises any of the mucoadhesive peptide fragments listed in Table 8.

[0084]

[0101] In some embodiments, the chimeric protein comprises: (a) a target-binding portion comprising the EBD of ACE2 (e.g., SEQ ID NO: 1) or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues that promote adhesion of the chimeric protein to a mucosa, wherein the target binding portion is fused to the mucoadhesive peptide fragment via an immunoglobulin Fc region or a fragment thereof, and the mucoadhesive peptide fragment comprises a polycationic peptide. In some embodiments, the polycationic peptide is a polylysine peptide (e.g., a 6K, 12K, or 30K peptide). In some embodiments, the polycationic peptide is a polyhistidine peptide (e.g., a 5H, 6H, 12H, or 30H peptide).

[0085]

[0102] In some embodiments, the chimeric protein comprises: (a) a target-binding moiety comprising ACE614 (e.g., SEQ ID NO: 2) or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30) positively charged amino acid residues that promote adhesion of the chimeric protein to a mucosa, wherein the target-binding moiety is fused directly to the mucoadhesive peptide fragment. In some embodiments, the chimeric protein comprises any of the mucoadhesive peptide fragments listed in Table 8.

[0086]

[0103] In some embodiments, the chimeric protein comprises: (a) a target binding moiety comprising ACE614 (e.g., SEQ ID NO: 2) or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues that promote adhesion of the chimeric protein to a mucosa, wherein the target binding moiety is fused to the mucoadhesive peptide via a peptide linker. In some embodiments, the peptide linker comprises any of the peptide linkers listed in Table 9. In some embodiments, the chimeric protein comprises: (a) a target binding moiety comprising ACE614 (e.g., SEQ ID NO: 2) or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues that promote adhesion of the chimeric protein to a mucosa, wherein the target binding moiety is fused to the mucoadhesive peptide fragment via an immunoglobulin Fc region or a fragment thereof. In some embodiments, the chimeric protein comprises any of the mucoadhesive peptide fragments listed in Table 8.

[0087]

[0104] In some embodiments, the chimeric protein comprises: (a) a target-binding moiety comprising ACE614 (e.g., SEQ ID NO: 2) or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues that promote adhesion of the chimeric protein to a mucosa, wherein the target binding moiety is fused to the mucoadhesive peptide fragment via an immunoglobulin Fc region or a fragment thereof, and the mucoadhesive peptide fragment comprises a polycationic peptide. In some embodiments, the polycationic peptide is a polylysine peptide (e.g., a 6K, 12K, or 30K peptide). In some embodiments, the polycationic peptide is a polyhistidine peptide (e.g., a 5H, 6H, 12H, or 30H peptide).

[0088]

[0105] In some embodiments, the chimeric protein comprises: (a) a target-binding moiety comprising ACE740 (e.g., SEQ ID NO: 135) or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30) positively charged amino acid residues that promote adhesion of the chimeric protein to a mucosa, wherein the target-binding moiety is fused directly to the mucoadhesive peptide fragment. In some embodiments, the chimeric protein comprises any of the mucoadhesive peptide fragments listed in Table 8.

[0089]

[0106] In some embodiments, the chimeric protein comprises: (a) a target binding moiety comprising ACE740 (e.g., SEQ ID NO: 135) or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues, which promotes adhesion of the chimeric protein to a mucosa, wherein the target binding moiety is fused to the mucoadhesive peptide via a peptide linker. In some embodiments, the peptide linker comprises any of the peptide linkers listed in Table 9. In some embodiments, the chimeric protein comprises: (a) a target binding moiety comprising ACE740 (e.g., SEQ ID NO: 135) or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues, which promotes adhesion of the chimeric protein to a mucosa, wherein the target binding moiety is fused to the mucoadhesive peptide fragment via an immunoglobulin Fc region or a fragment thereof. In some embodiments, the chimeric protein comprises any of the mucoadhesive peptide fragments listed in Table 8.

[0090]

[0107] In some embodiments, the chimeric protein comprises: (a) a target-binding moiety comprising ACE740 (e.g., SEQ ID NO: 135) or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues that promote adhesion of the chimeric protein to a mucosa, wherein the target binding moiety is fused to the mucoadhesive peptide fragment via an immunoglobulin Fc region or a fragment thereof, and the mucoadhesive peptide fragment comprises a polycationic peptide. In some embodiments, the polycationic peptide is a polylysine peptide (e.g., a 6K, 12K, or 30K peptide). In some embodiments, the polycationic peptide is a polyhistidine peptide (e.g., a 5H, 6H, 12H, or 30H peptide).

[0091]

[0108] Table 1 lists the sequences of exemplary chimeric proteins provided herein. Table 1. Exemplary chimeric proteins TIFF2025512512000002.tif250170TIFF2025512512000003.tif247170TIFF2025512512000004.tif252170TIFF2025512512000005.tif23217 0TIFF2025512512000006.tif252170TIFF2025512512000007.tif237170TIFF2025512512000008.tif252170TIFF2025512512000009.tif30170

[0092]

[0109] Additional ACE2 chimeric proteins comprising any of the target-binding moieties or variants thereof that specifically bind to the S protein, mucoadhesive peptide fragments, and / or linkers provided herein are also contemplated. It should be understood that various other chimeric proteins comprising target-binding moieties, including the EBD of a known ACE2 protein or fragment thereof (e.g., hACE2 or an animal ACE2 protein or fragment thereof), such as those described in Tables 3 and 4, or variants known in the art, fused to any of the mucoadhesive peptide fragments, such as those described in Table 8 provided herein, and / or linkers, such as those described in Table 9 provided herein, may be encompassed within the scope of the present invention.

[0093]

[0110] Exemplary human-derived hACE2 chimeric proteins and chimeric protein fragments can be designed using the hACE2 protein sequences and fragments thereof disclosed in Table 3. In some embodiments, the chimeric protein comprises: (a) a target-binding portion comprising the amino acid sequence of any one of SEQ ID NOS: 1-7, 9-14, and 135 (i.e., as set forth in Table 3); and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30) positively charged amino acid residues, which promotes adhesion of the chimeric protein to mucosa. In some embodiments, the chimeric protein comprises a target-binding portion comprising the EBD of a hACE2 protein or a fragment thereof and one or more of the mucoadhesive peptide fragments described herein, for example, any of the chimeric proteins include the hACE2 proteins provided in Table 1 above, such as ACE740-Fc1-5H, ACE740-Fc1-6H, ACE740-Fc1-7X-1, ACE740-Fc1-12X-7, ACE740-Fc1-12X-8, ACE614-Fc1-12K, ACE614-Fc1-12H, ACE200-bIZIP-35X-1, ACE200-CH2-CH2-12X-4, and ACE19-SA-50X-1.

[0094]

[0111] In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of any one of SEQ ID NOs: 84-88 and 136-140. In some embodiments, the chimeric protein comprises the amino acid sequence of any one of SEQ ID NOs: 84-88 and 136-140.

[0095]

[0112] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 135 (i.e., ACE740, as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9. In some embodiments, the peptide linker comprises an Fc region (e.g., an immunoglobulin Fc region) or a fragment thereof. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 63, i.e., "Fc1," as listed in Table 9. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 128, i.e., "5H" in Table 8. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the chimeric protein is ACE740-Fc1-5H as described in Table 1. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 28, i.e., "6H" as described in Table 8. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 137.In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the chimeric protein is ACE740-Fc1-6H as described in Table 1. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 129, i.e., "7X-1" as described in Table 8. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., approximately sequence identity, where n is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the chimeric protein is ACE740-Fc1-7X-1 as described in Table 1. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 130, i.e., "12X-7" as described in Table 8. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 139. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, the chimeric protein is ACE740-Fc1-12X-7 as described in Table 1. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 131, i.e., "12X-8," as described in Table 8. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the chimeric protein is ACE740-Fc1-12X-8 as described in Table 1.

[0096]

[0113] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO:2 (i.e., ACE614, as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs:28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs:63-83, 94, and 109, as listed in Table 9. In some embodiments, the peptide linker comprises an Fc region (e.g., an immunoglobulin Fc region) or a fragment thereof. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:63, i.e., "Fc1," as listed in Table 9. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 32, i.e., "12K" in Table 8. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the chimeric protein is ACE614-Fc1-12K as described in Table 1. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 29, i.e., "12H" as described in Table 8. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 85.In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the chimeric protein is ACE614-Fc1-12H as described in Table 1.

[0097]

[0114] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 6 (i.e., ACE200 as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9. In some embodiments, the peptide linker comprises a basic helix-loop-helix zipper (bZIP) domain. In some embodiments, the peptide linker comprises a leucine / isoleucine zipper (bIZIP) domain. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 72, i.e., "bIZIP," as described in Table 9. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 57, i.e., "35X-1" in Table 8. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, the chimeric protein is ACE200-bIZIP-35X-1, as described in Table 1. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 50, i.e., "12X-4," as described in Table 8. In some embodiments, the peptide linker comprises an Fc region (eg, an immunoglobulin Fc region) or a fragment thereof.In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 66, i.e., "CH2," as described in Table 9. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 87. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 87. In some embodiments, the chimeric protein is ACE200-CH2-CH2-12X-4, as described in Table 1.

[0098]

[0115] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 8 (i.e., ACE19 as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 61, i.e., "50X-1" in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9. In some embodiments, the peptide linker comprises a streptavidin (SA) moiety. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 76, i.e., "SA," as described in Table 9. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the chimeric protein is ACE19-SA-50X-1, as described in Table 1.

[0099]

[0116] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 1 (i.e., hACE2 as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30) positively charged amino acid residues, which promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-84, 94, and 109, as listed in Table 9.

[0100]

[0117] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 3 (i.e., ACE360, as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-84, 94, and 109, as listed in Table 9.

[0101]

[0118] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 4 (i.e., ACEΔ360 as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0102]

[0119] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 5 (i.e., ACE732, as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0103]

[0120] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 7 (i.e., ACEΔ420, as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0104]

[0121] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 102 (i.e., ACE12 as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0105]

[0122] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 9 (i.e., ACE2K26R, as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0106]

[0123] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 10 (i.e., ACE2I468V listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0107]

[0124] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 11 (i.e., ACE2N638S, as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0108]

[0125] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 12 (i.e., ACE2N720D, as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0109]

[0126] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 13 (i.e., ACE2HN-HN as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0110]

[0127] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 14 (i.e., ACE2TY-HA as listed in Table 3) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0111]

[0128] Exemplary animal-derived ACE2 chimeric proteins and chimeric protein fragments can be designed using the animal ACE2 protein sequences disclosed in Table 4 and fragments thereof corresponding to the various partial hACE2 fragments disclosed in Table 3 (e.g., fragments having comparable sequence boundaries and lengths). In some embodiments, the target-binding moiety comprising an animal ACE2 protein described herein is a mouse, guinea pig, horse, ferret, monkey, chimpanzee, pig, dog, cat, cow, rabbit, mink, or chicken ACE2 protein, or a fragment or variant thereof. In some embodiments, the chimeric protein comprises: (a) a target-binding moiety comprising the amino acid sequence of any one of SEQ ID NOS: 15-27 (i.e., as set forth in Table 4); and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30) positively charged amino acid residues, which promotes adhesion of the chimeric protein to mucosa. In some embodiments, the chimeric protein comprises: (a) a target-binding portion comprising the amino acid sequence of any one of SEQ ID NOS: 2-7 and 8-14 (e.g., a fragment having sequence boundaries and length equivalent to an animal ACE protein) (i.e., as set forth in Table 3); and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., from about 5 to about 30) positively charged amino acid residues, which promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the chimeric protein comprises a target-binding portion comprising the EBD of an ACE2 protein or a fragment thereof and one or more of the mucoadhesive peptide fragments described herein, e.g., any of the chimeric proteins may comprise an ACE2 protein provided in Table 1 above, e.g., cACE614-Fc1-12H, cACE200-(GPP), or a fragment thereof. 10 -40X-2, mACE614-COMP-6X-5, gACEΔ360-FC1-12O, and nACEΔ420-T4F-12X.

[0112]

[0129] In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of any one of SEQ ID NOs: 89-93. In some embodiments, the chimeric protein comprises the amino acid sequence of any one of SEQ ID NOs: 89-93.

[0113]

[0130] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 104 (i.e., cACE614) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as set forth in Table 8. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 32, i.e., "12K" in Table 8. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 28, i.e., "12H" in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as set forth in Table 9. In some embodiments, the peptide linker comprises an Fc region (e.g., an immunoglobulin Fc region) or a fragment thereof. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 63, i.e., "Fc1," as described in Table 9. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the chimeric protein is cACE614-Fc1-12H, as described in Table 1.

[0114]

[0131] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 105 (i.e., cACE200) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues, which promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as set forth in Table 8. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 58, i.e., "40X-2" in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as set forth in Table 9. In some embodiments, the peptide linker comprises a repeating glycine-XY repeat unit (GPP). n (n≧1). In some embodiments, the peptide linker comprises a collagen-like protein (GPP) comprising the amino acid sequence of SEQ ID NO: 109, i.e., "(GPP) 10 In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 90. In some embodiments, the chimeric protein comprises a cACE200-(GPP) as described in Table 1. 10 -40X-2.

[0115]

[0132] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 106 (i.e., mACE614) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as set forth in Table 8. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 45, i.e., "6X-5" in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as set forth in Table 9. In some embodiments, the peptide linker comprises cartilage oligomeric matrix protein (COMP) or a fragment thereof. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 77, i.e., "COMP," as described in Table 9. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the chimeric protein is mACE614-COMP-6X-5, as described in Table 1.

[0116]

[0133] In some embodiments, a chimeric protein is provided that includes: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 107 (i.e., gACEΔ360) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as set forth in Table 8. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 38, i.e., "120" in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as set forth in Table 9. In some embodiments, the peptide linker comprises an Fc region (e.g., an immunoglobulin Fc region) or a fragment thereof. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 63, i.e., "Fc1," as described in Table 9. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the chimeric protein is gACEΔ360-Fc1-12O, as described in Table 1.

[0117]

[0134] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 108 (i.e., nACEΔ420) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as set forth in Table 8. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of SEQ ID NO: 47, i.e., "12X-1" in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as set forth in Table 9. In some embodiments, the peptide linker comprises a T4 fibritin domain or a fragment thereof. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:74, i.e., "T4F," as described in Table 9. In some embodiments, the chimeric protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO:93. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO:93. In some embodiments, the chimeric protein is nACEΔ420-T4F-12X-1, as described in Table 1.

[0118]

[0135] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 15 (i.e., mouse ACE2 as listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0119]

[0136] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 16 (i.e., guinea pig ACE2 as listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0120]

[0137] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 17 (i.e., equine ACE2 as listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0121]

[0138] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 18 (i.e., monkey ACE2 as listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0122]

[0139] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 19 (i.e., chimpanzee ACE2 as listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0123]

[0140] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 20 (i.e., porcine ACE2 as listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0124]

[0141] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 21 (i.e., canine ACE2 as listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, which promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0125]

[0142] In some embodiments, a chimeric protein is provided that includes: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 22 (i.e., feline ACE2 listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30) positively charged amino acid residues, which promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0126]

[0143] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 23 (i.e., bovine ACE2 as listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0127]

[0144] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 24 (i.e., rabbit ACE2 as listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0128]

[0145] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 25 (i.e., ferret ACE2 as listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0129]

[0146] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 26 (i.e., mink ACE2 as listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0130]

[0147] In some embodiments, chimeric proteins are provided that include: (a) a target-binding moiety comprising the amino acid sequence of SEQ ID NO: 27 (i.e., chicken ACE2 as listed in Table 4) or a fragment thereof; and (b) a mucoadhesive peptide fragment comprising at least about five (e.g., about five to about 30) positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, as listed in Table 8. In some embodiments, the target-binding moiety is fused to the mucoadhesive peptide fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109, as listed in Table 9.

[0131]

[0148] Without being bound by any theory or hypothesis, coronavirus infections (e.g., SARS-CoV-2 infections) occur primarily via respiratory droplets and potentially airborne transmission. The upper respiratory tract surface is the primary and initial site of coronavirus infection. The nasal epithelium creates a physical glycoprotein barrier against inhaled particles, including allergens and pathogens, preventing their penetration through the epithelial surface of mucosal tissue. One of the major components of the mucosal layer of the nose and respiratory tract is mucin, a group of large glycoproteins that coat the surface of the respiratory epithelium. Mucin, the main non-aqueous component of mucus, has a complex, heterogeneous structure and carries a high negative charge. In some embodiments, the inventors of the present application have engineered peptides containing at least five positively charged amino acids (e.g., lysine, histidine, arginine, ornithine, or a combination thereof), which, upon covalent attachment to a target-binding moiety, impart a positive charge to the conjugate (i.e., the ACE2 chimeric protein). A target-binding moiety with a positively charged C-terminal peptide (e.g., the EBD of the ACE2 protein or a fragment thereof) can form a layer of coronavirus-binding moieties in the nasal / respiratory tract, preventing the virus from binding to virus receptor-expressing epithelial cells. The positively charged target-binding moiety may also bind to the negatively charged phospholipid bilayer of cell membranes. This "sticky" property of the polymeric positively charged amino acid chains increases the half-life of the target-binding moiety in the respiratory mucosal epithelium, prolonging the period of protection. Therefore, a target-binding moiety-mucoadhesive polymer conjugate can prevent coronavirus from entering cells in the respiratory cavity, even if the virus passes through the mucosal barrier and reaches virus receptor-positive epithelial cells. In other embodiments, positively charged mucoadhesive amino acids can be mixed with non-positively charged amino acids without destroying the mucoadhesive properties of the chimeric protein. Furthermore, the target-binding moiety of an ACE2 chimeric protein can be fused to a mucoadhesive peptide fragment via a peptide linker.

[0132]

[0149] Different aspects and embodiments are described in further detail in various sections below.

[0133] A. Target binding moiety

[0150] The chimeric proteins described herein comprise a target binding moiety comprising an inhibitory polypeptide, i.e., the EBD of the ACE2 protein (e.g., SEQ ID NO: 101), or a fragment thereof, that specifically binds to the S protein. In some embodiments, the target binding moiety comprises an inhibitory polypeptide that inhibits binding of the S protein to a receptor on a mucosal cell. In some embodiments, the target binding moiety comprises the native receptor of the S protein or a fragment derived from the native receptor. In some embodiments, the target binding moiety comprises the EBD of a native receptor, e.g., ACE2, or a fragment thereof. In some embodiments, the ACE2 is a hACE2 protein. In some embodiments, the ACE2 is an animal ACE2 protein.

[0134]

[0151] In some embodiments, the target binding moiety comprises a purification tag, for example a His tag such as DYKDDDDKHHHHHH (SEQ ID NO: 95).

[0135]

[0152] In some embodiments, the S protein is from a virus, such as a coronavirus. In some embodiments, the virus causes a respiratory infection.

[0136]

[0153] In humans, coronaviruses cause mild to severe respiratory illnesses, ranging from the common cold to more severe illnesses such as coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS). The target-binding moieties described herein can target any of these viruses, or other future viruses that contain the S protein.

[0137]

[0154] Exemplary viruses, targets, and target-binding moieties are further described below.

[0138] coronavirus

[0155] The target-binding moieties of the present invention (e.g., the EBD of the ACE2 protein or a fragment thereof) specifically bind to the spike protein (also called the spike glycoprotein or S protein) of a virus.

[0032] In some embodiments, the virus is a coronavirus. In some embodiments, the virus is selected from the group consisting of SARS-CoV, SARS-CoV-2, and HCoV-NL63 (including variants thereof). In some embodiments, the virus is SARS-CoV-2. In some embodiments, the virus is a reference coronavirus or a coronavirus having substantially the same genomic sequence (e.g., less than any one of 200, 100, 50, 20, 10, 5, 4, 3, 2, or 1 mutation) and phenotype as a reference coronavirus. In some embodiments, the virus is a mutant coronavirus having one or more mutations in the genomic sequence compared to the reference coronavirus, wherein the one or more mutations contribute to a phenotypic difference, such as increased viral fitness, including, for example, infectivity, pathogenicity, and / or drug resistance.

[0139]

[0156] In some embodiments, the virus is SARS-CoV-2. In some embodiments, the virus is a reference SARS-CoV-2 (e.g., WIV4, i.e., hCoV-19 / WIV04 / 2019 or betaCoV / WIV04 / 2019), or a SARS-CoV-2 virus having substantially the same genomic sequence (e.g., less than any one of 200, 100, 50, 20, 10, 5, 4, 3, 2, or 1 mutations) and phenotype as a reference SARS-CoV-2. The genomic sequence of reference SARS-CoV-2 WIV4 can be found in Genbank (NCBI Reference Sequence: NC_045512.2) and is also known as 2019-nCoV. In certain embodiments, the SARS-CoV-2 is a variant, such as a variant of interest, a variant of concern, or a variant of significant consequence. In some embodiments, the SARS-CoV-2 is a variant selected from the group consisting of a B.1.1.7 variant, a B.1.351 variant, a B.1.526 variant, a B1.526.1 variant, a B1.617 variant, a B.1.617.1 variant, a B.1.617.2 variant, a B1.617.3 variant, a P.2 variant, a P.1 (also known as B.1.1.28.1) variant, an A.23.1 variant, a CAL.20C variant, a B.1.427 variant, a B.1.429 variant, a B.1.525 variant, a P.1.351 variant, or a B.1.1.529 variant. In some embodiments, the SARS-CoV-2 variant is a B.1.1.7 variant. In some embodiments, the SARS-CoV-2 variant is the B.1.351 variant. In some embodiments, the SARS-CoV-2 variant is the B.1.617.2 variant. In some embodiments, the SARS-CoV-2 variant is the B.1.1.529 variant. Other variants of SARS-CoV-2 are known in the art. See, e.g., Gomezet al., Vaccines 9(3):243, 2021 and Tanget al., Journal of Infection 82:e27-e28 (2021), which are incorporated herein by reference in their entireties. In some embodiments, the SARS-CoV-2 variant has one or more mutations (e.g., insertions, deletions, and / or substitutions) in the S protein.In some embodiments, one or more mutations in the S protein may affect viral fitness, such as infectivity, pathogenicity, and / or drug resistance (e.g., resistance to neutralizing antibodies and / or resistance to vaccines). For example, a SARS-CoV-2 variant may have an L452R and / or E484K substitution in the S protein. In some embodiments, the one or more mutations in the S protein do not substantially alter viral fitness. In certain embodiments, a SARS-CoV-2 variant does not have a mutation in the S protein.

[0140]

[0157] In some embodiments, the S protein is a coronavirus S protein. In some embodiments, the target binding moiety specifically binds to the S1 subunit of the S protein. In some embodiments, the target binding moiety specifically binds to the S2 subunit of the S protein.

[0141]

[0158] In some embodiments, the target binding moiety is an inhibitory polypeptide that inhibits binding of the S protein to a receptor on a mucosal cell. In some embodiments, the target binding moiety comprises the native receptor for the coronavirus S protein or a fragment derived from the native receptor. In some embodiments, the target binding moiety comprises the EBD of the native coronavirus receptor. In some embodiments, the target binding moiety comprises the EBD of ACE2 or a fragment thereof. In some embodiments, the target binding moiety comprises a truncated version of ACE2.

[0142]

[0159] In nature, the S protein of coronaviruses mediates viral entry into host cells. Table 2 below shows the viral receptors identified for various coronaviruses. See also Raj V Setal. Chapter 15 of Helena Jane Maiere et al. (eds.), Coronaviruses: Methods and Protocols, Methods in Molecular Biology, vol. 1282, Springer Science + Business Media New York 2015; Li F. Annu Rev Virol., 3(1):237-261 (2016); Hulswit 2019 and Zhou et al., Nature 579:270 (2020), which are incorporated herein by reference in their entireties. Table 2. Coronavirus host-virus receptors TIFF2025512512000010.tif46170

[0143]

[0160] Provided herein are chimeric proteins comprising a target-binding moiety comprising the EBD of the ACE2 host viral receptor of a coronavirus, or a fragment thereof. The ACE2 protein or a fragment thereof can be derived from a coronavirus that comprises the ACE2 viral receptor. Chimeric proteins based on these host receptors are contemplated herein (see Table 1). In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, or HCoV-NL63, or a variant thereof.

[0144]

[0161] ACE2 is a cellular receptor for coronavirus infections (e.g., SARS-CoV-2 infections) that mediates binding to the viral S protein present on the surface of the virus particle, allowing the virus to enter susceptible host cells in the respiratory tract. ACE2 is an 805-amino acid metallocarboxyl peptidase that consists of an extracellular catalytic domain (e.g., EBD), a transmembrane region, and a short intracellular domain and is highly conserved among vertebrates. The catalytically active fragment of the ACE2 membrane-bound protein is either released from its membrane tether by the action of ADAM10 / ADAM17 metalloproteinases or cleaved at the plasma membrane by the transmembrane protease TMPRSS2. ADAM17 and TMPRSS2 are expressed in lung cells and play an important role in coronavirus invasion into respiratory tract cells. ACE2 and TMPRSS2 are co-expressed in many tissues throughout the body and can be readily detected in the respiratory system (e.g., expression occurs in type II pneumocytes and enterocytes, alveolar cells, bronchial transient epithelial secretory cells, respiratory epithelial cells, as well as the oral cavity and tongue (Beyerstedtetal. 2021; Heurichetal. 2014)). In cells of the nasal epithelium, ACE2 has been shown to be highly expressed in adults but low in children.

[0145]

[0162] 19 amino acid ACE2 fragment QAKTFL DKFNHEAEDLFY Q ("ACE19", SEQ ID NO: 8 in Table 1) (containing the S1 binding site DKFNHEAEDLFY (SEQ ID NO: 102; the underlined portion of ACE19 above)) was found to selectively recognize the SARS-CoV-2 viral S protein S1 subunit and interfere with S1 binding (Kuznetsovetal. IntJPeptResTher. 28:7, 2022; Mohebbietal. FutureVirol. 10:2217-2235 (2020)).

[0146]

[0163] Provided herein are target-binding moieties comprising the EBD of the ACE2 protein or a fragment thereof. In some embodiments, the ACE2 protein is a hACE2 protein or a fragment thereof. In some embodiments, the ACE2 protein is an animal ACE2 protein or a fragment thereof. hACE2 and animal ACE2 proteins are known in the art. Tables 3-4 list exemplary hACE2 (Table 3) and animal ACE2 (Table 4) proteins and fragments thereof. Table 3. Exemplary hACE2 proteins and fragments and variants thereof TIFF2025512512000011.tif232170TIFF2025512512000012.tif252170TIFF2025512512000013.tif252170TIFF2025512512000014.tif252170TIFF2025512512000015.tif252170TIFF2025512512000016.tif252170TIFF2025512512000017.tif21170Table 4. Exemplary Animal ACE2 Proteins and Fragments and Variants Thereof TIFF2025512512000018.tif252170TIFF2025512512000019.tif252170TIFF2025512512000020.tif252170TIFF2025512512000021.tif252170 TIFF2025512512000022.tif252170TIFF2025512512000023.tif252170TIFF2025512512000024.tif248170TIFF2025512512000025.tif209170

[0147]

[0164] Novel ACE2 proteins or fragments thereof can be established for the S protein of a coronavirus or a mutant thereof (e.g., the S1 subunit of the S protein) using techniques known in the art, and the sequence of such proteins or fragments thereof can be used as the target-binding moiety of the chimeric proteins of the present disclosure. In some embodiments, the coronavirus is a known coronavirus. In some embodiments, the coronavirus is a mutant of a known coronavirus. In some embodiments, the coronavirus is a future coronavirus. In some embodiments, the coronavirus is a mutant of a future coronavirus. In some embodiments, the target-binding moiety comprises a derivative of any one of the hACE2 proteins or animal ACE2 proteins described herein (e.g., a fragment of any one of the hACE2 proteins or animal ACE2 proteins described herein, or a variant of any one of the hACE2 proteins or animal ACE2 proteins described herein).

[0148]

[0165] In some embodiments, the target binding moiety is a full-length hACE2 protein or a fragment or variant thereof (e.g., the EBD of the ACE2 protein or a fragment or variant thereof, e.g., SEQ ID NO: 1) between about 12 amino acids (aa) and about 805 aa, e.g., between about 12 aa and about 700 aa, between about 15 aa and about 500 aa, between about 100 aa and about 300 aa, between about 200 aa and about 400 aa, aa, between about 300 aa and about 500 aa, between about 400 aa and about 600 aa, between about 500 aa and about 700 aa, between about 600 aa and about 805 aa, between about 12 aa and about 20 aa, between about 15 aa and about 20 aa, between about 21 aa and about 42 aa, between about 30 aa and about 41 aa, or between about 500 aa and about 805 aa, or a fragment or variant thereof. In some embodiments, the target binding moiety comprises a full-length hACE2 protein of greater than about 12 aa, or a fragment or variant thereof, such as greater than n (where n is selected from 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 30 aa, 40 aa, 50 aa, 100 aa, 150 aa, 200 aa, 250 aa, 300 aa, 350 aa, 400 aa, 450 aa, 500 aa, 550 aa, 600 aa, 650 aa, 700 aa, 750 aa, 800 aa, 850 aa, or more) full-length hACE2 protein or a fragment or variant thereof. In some embodiments, the target binding moiety comprises a full-length hACE2 protein or fragment or variant thereof of less than about 805 aa, e.g., a full-length hACE2 protein or fragment or variant thereof of less than about n, where n is selected from 800 aa, 750 aa, 700 aa, 650 aa, 600 aa, 550 aa, 500 aa, 450 aa, 400 aa, 350 aa, 300 aa, 250 aa, 200 aa, 150 aa, 100 aa, 50 aa, 40 aa, 30 aa, 20 aa, 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, or less.In some embodiments, the target binding portion comprises any of about 805 aa, about 722 a, about 714 aa, about 596 aa, about 380 aa, about 342 aa, about 331 aa, about 182 aa, about 19 aa, or about 12 aa of a full-length hACE2 protein or a fragment or variant thereof.

[0149]

[0166] In some embodiments, the target binding moiety comprises a full-length animal ACE2 protein of between about 12 aa and about 805 aa, or a fragment or variant thereof, such as between about 12 aa and about 700 aa, between about 15 aa and about 500 aa, between about 100 aa and about 300 aa, between about 200 aa and about 400 aa, between about 300 aa and about 500 aa, between about 400 aa and about 600 aa, between about 500 aa and about 700 aa, between about 600 aa and about 805 aa, between about 15 aa and about 20 aa, between about 12 aa and about 20 aa, between about 21 aa and about 42 aa, between about 30 aa and about 41 aa, or between about 500 aa and about 805 aa, or a fragment or variant thereof. In some embodiments, the target binding moiety comprises a full-length animal ACE2 protein of greater than about 12 aa or a fragment or variant thereof, such as greater than n (where n is selected from 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 30 aa, 40 aa, 50 aa, 100 aa, 150 aa, 200 aa, 250 aa, 300 aa, 350 aa, 400 aa, 450 aa, 500 aa, 550 aa, 600 aa, 650 aa, 700 aa, 750 aa, 800 aa, 850 aa, or more) full-length animal ACE2 protein or a fragment or variant thereof. In some embodiments, the target binding moiety comprises a full-length animal ACE2 protein or fragment or variant thereof of less than about 805 aa, e.g., a full-length animal ACE2 protein or fragment or variant thereof of less than about n, where n is selected from 800 aa, 750 aa, 700 aa, 650 aa, 600 aa, 550 aa, 500 aa, 450 aa, 400 aa, 350 aa, 300 aa, 250 aa, 200 aa, 150 aa, 100 aa, 50 aa, 40 aa, 30 aa, 20 aa, 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, or less. In some embodiments, the target binding portion comprises any of about 805 aa, about 722 a, about 714 aa, about 596 aa, about 380 aa, about 342 aa, about 331 aa, about 182 aa, 19 aa, or about 12 aa of a full-length animal ACE2 protein or a fragment or variant thereof.

[0150]

[0167] In some embodiments, the target-binding moiety comprises a fragment that can selectively recognize the S1 subunit of the S protein and disrupt S1 binding to full-length ACE2. In some embodiments, the target-binding moiety comprises amino acids 24-42 of a full-length ACE2 protein (e.g., a full-length hACE2 protein) or a variant thereof. In some embodiments, the target-binding moiety comprises amino acids 24-42 of a full-length ACE2 protein or a variant thereof between about 12 aa and about 19 aa, e.g., between about 12 aa and about 14 aa, between about 13 aa and about 15 aa, between about 14 aa and about 16 aa, between about 15 aa and about 17 aa, between about 16 aa and about 18 aa, between about 17 aa and about 19 aa, or between about 18 aa and about 19 aa. In some embodiments, the target binding moiety comprises at least about 12 aa, e.g., at least n, of SEQ ID NO:8 (where n is selected from 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, and 18 aa). In some embodiments, the target binding moiety comprises more than about 12 aa, e.g., more than n, of SEQ ID NO:8 (where n is selected from 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, and 18 aa). In some embodiments, the target binding moiety comprises less than about 20 aa, e.g., less than about n, of SEQ ID NO:8 (where n is 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, or less). In some embodiments, the target binding moiety comprises about 19 aa of SEQ ID NO:8. In some embodiments, the chimeric protein comprises a target binding moiety comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 8 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%).

[0151]

[0168] In some embodiments, the target-binding moiety comprises amino acids 30-41 of a full-length ACE2 protein (e.g., a full-length animal hACE2 protein or a full-length animal ACE2 protein) or a variant thereof. In some embodiments, the chimeric protein comprises a target-binding moiety comprising the amino acid sequence of any one of SEQ ID NOs: 102, 111-120, and 122, or a variant thereof comprising at least about 90% (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity to any one of SEQ ID NOs: 102, 111-120, and 122. In some embodiments, the full-length ACE2 protein or variant thereof is a hACE2 protein or variant thereof. In some embodiments, the full-length ACE2 protein or variant thereof is not a canine or chicken full-length ACE2 protein or variant thereof.

[0152]

[0169] In some embodiments, the target-binding portion comprises amino acids 29-40 of a full-length ACE2 protein (e.g., a full-length animal ACE2 protein) or a variant thereof. In some embodiments, the chimeric protein comprises a target-binding portion comprising SEQ ID NO: 110 or 121, or a variant thereof comprising at least about 90% sequence identity to SEQ ID NO: 110 or 121 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the full-length ACE2 protein or variant thereof is a canine or chicken ACE2 protein or variant thereof.

[0153]

[0170] In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to amino acids 24-42 of a full-length hACE2 protein. In some embodiments, the target binding moiety comprises amino acids 24-42 of a full-length hACE2 protein. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO:8. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, the target binding moiety comprises ACE19, as described in Table 3.

[0154]

[0171] In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to amino acids 30-41 of a full-length hACE2 protein. In some embodiments, the target binding moiety comprises amino acids 30-41 of a full-length hACE2 protein. In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 102. In some embodiments, the target binding moiety comprises an ACE12 as set forth in Table 3.

[0155]

[0172] In some embodiments, chimeric proteins, or fragments thereof, are provided that include a target-binding moiety comprising the EBD of hACE2 protein that specifically binds to the S protein. Exemplary hACE2 proteins are listed in Table 3 above. In some embodiments, the target-binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of any one of SEQ ID NOs: 1-7, 9-14, and 135. In some embodiments, the target-binding moiety comprises the amino acid sequence of any one of SEQ ID NOs: 1-7, 9-14, and 135.

[0156]

[0173] In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the target binding moiety comprises a full-length hACE2 protein as set forth in Table 3.

[0157]

[0174] In some embodiments, the target binding moiety comprises amino acids 19-614 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the target binding moiety comprises ACE614, as set forth in Table 3.

[0158]

[0175] In some embodiments, the target binding moiety comprises amino acids 19-360 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the target binding moiety comprises ACE360 as set forth in Table 3.

[0159]

[0176] In some embodiments, the target binding moiety comprises amino acids 30-360 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the target binding moiety comprises ACEΔ360 as set forth in Table 3.

[0160]

[0177] In some embodiments, the target binding moiety comprises amino acids 19-732 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the target binding moiety comprises ACE732 as set forth in Table 3.

[0161]

[0178] In some embodiments, the target binding moiety comprises amino acids 19-740 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 135. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the target binding moiety comprises ACE740 as set forth in Table 3.

[0162]

[0179] In some embodiments, the target binding moiety comprises amino acids 19-200 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the target binding moiety comprises ACE200 as set forth in Table 3.

[0163]

[0180] In some embodiments, the target binding moiety comprises amino acids 42-420 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the target binding moiety comprises ACEΔ420 as set forth in Table 3.

[0164]

[0181] In some embodiments, the target binding moiety comprises a K26R point mutation at amino acid 26 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the target binding moiety comprises ACE2K26R as described in Table 3.

[0165]

[0182] In some embodiments, the target binding moiety comprises an I468V point mutation at amino acid 468 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the target binding moiety comprises ACE2I468V as described in Table 3.

[0166]

[0183] In some embodiments, the hACE2 protein or fragment thereof comprises an hACE2 fusion fragment comprising an N638S point mutation at amino acid 638 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the target binding moiety comprises ACE2N638S as described in Table 3.

[0167]

[0184] In some embodiments, the target binding moiety comprises an N720D point mutation at amino acid 720 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the target binding moiety comprises ACE2N720D as described in Table 3.

[0168]

[0185] In some embodiments, the target binding moiety comprises an hACE2 point mutation at amino acid 378 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the target binding moiety comprises an ACE2HN-HN as set forth in Table 3.

[0169]

[0186] In some embodiments, the target binding moiety comprises an hACE2 fragment comprising a T27Y point mutation at amino acid 27 and an H34A point mutation at amino acid 34 of hACE2 (e.g., SEQ ID NO: 1). In some embodiments, the target binding moiety comprises at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the target binding moiety comprises ACE2TY-HA as set forth in Table 3.

[0170]

[0187] In some embodiments, chimeric proteins are provided that include a target-binding moiety comprising the EBD of an animal ACE2 protein that specifically binds to the S protein, or a fragment thereof. In some embodiments, the target-binding moiety comprising an animal ACE2 protein described herein is a mouse, guinea pig, horse, ferret, monkey, chimpanzee, pig, dog, cat, cow, rabbit, mink, or chicken ACE2 protein, or a fragment or variant thereof. Exemplary animal ACE2 proteins and fragments thereof are listed in Table 4 above.

[0171]

[0188] In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to amino acids 30-41 of a full-length animal ACE2 protein. In some embodiments, the target binding moiety comprises amino acids 30-41 of a full-length animal ACE2 protein. In some embodiments, the full-length animal ACE2 protein is a mouse, guinea pig, horse, ferret, monkey, chimpanzee, pig, cat, cow, rabbit, or mink ACE2 protein. In some embodiments, the full-length animal ACE2 protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to any one of SEQ ID NOs: 15-20 and 22-26. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to amino acids 30-41 of a full-length mouse, guinea pig, horse, ferret, monkey, chimpanzee, pig, cat, bovine, rabbit, or mink ACE2 protein. In some embodiments, the target binding moiety comprises the amino acid sequence of amino acids 30-41 of a full-length mouse, guinea pig, horse, ferret, monkey, chimpanzee, pig, cat, cow, rabbit, or mink ACE2 protein. In some embodiments, the target binding moiety comprises the amino acid sequence of any one of SEQ ID NOs: 15-20 and 22-26. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to any one of SEQ ID NOs: 111-120 and 122. In some embodiments, the target binding moiety comprises the amino acid sequence of any one of SEQ ID NOs: 111-120 and 122.In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:111 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO:111. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:112 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO:112. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:113 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO:113. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:114 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO:114. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:115 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO:115. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:116 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO:116.In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:117 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO:117. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:118 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO:118. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 119 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 120 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 120. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 122 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 122. In some embodiments, the full-length animal ACE2 protein is not a chicken ACE2 protein or a canine ACE2 protein (e.g., SEQ ID NOs: 21 and 27).

[0172]

[0189] In some embodiments, the target-binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to amino acids 29-40 of a full-length animal ACE2 protein. In some embodiments, the target-binding moiety comprises amino acids 29-40 of a full-length animal ACE2 protein. In some embodiments, the full-length animal ACE2 protein is not a dog ACE2 protein or a chicken ACE2 protein. In some embodiments, the full-length animal ACE2 protein comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to SEQ ID NO: 21 or 27. In some embodiments, the full-length animal ACE2 protein comprises the amino acid sequence of SEQ ID NO: 21 or 27. In some embodiments, the target-binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to amino acids 29-40 of a full-length dog or chicken ACE2 protein. In some embodiments, the target-binding moiety comprises the amino acid sequence of amino acids 29-40 of a full-length dog or chicken ACE2 protein. In some embodiments, the target-binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to SEQ ID NO: 110 or 121. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 110 or 121. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 110 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 110.In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 121 (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%). In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 121.

[0173]

[0190] In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of any one of SEQ ID NOs: 15-27. In some embodiments, the target binding moiety comprises an amino acid sequence of any one of SEQ ID NOs: 15-27. In some embodiments, the target binding moiety comprises a mouse ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the target binding moiety comprises an amino acid sequence of SEQ ID NO: 15. In some embodiments, the target binding moiety comprises a guinea pig ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the target binding moiety comprises an equine ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the target binding moiety comprises a monkey ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 18.In some embodiments, the target binding moiety comprises a chimpanzee ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the target binding moiety comprises an amino acid sequence of SEQ ID NO: 19. In some embodiments, the target binding moiety comprises a porcine ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the target binding moiety comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, the target binding moiety comprises a canine ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the target binding moiety comprises a feline ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the target binding moiety comprises a bovine ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO:23.In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the target binding moiety comprises a rabbit ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the target binding moiety comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the target binding moiety comprises a ferret ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the target binding moiety comprises a mink ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the target binding moiety comprises an amino acid sequence of SEQ ID NO: 26. In some embodiments, the target binding moiety comprises a chicken ACE2 fusion fragment. In some embodiments, the target binding moiety comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the target binding moiety comprises the amino acid sequence of SEQ ID NO: 27.

[0174]

[0191] Coronaviruses are a group of related viruses that cause disease in mammals and birds. In humans, coronaviruses cause respiratory infections that can range from mild to fatal. Mild illnesses include the common cold (e.g., with symptoms such as fever and sore throat), while fatal illnesses can cause SARS and COVID-19. Coronaviruses can cause pneumonia (either direct viral pneumonia or secondary bacterial pneumonia) and bronchitis (either direct viral bronchitis or secondary bacterial bronchitis).

[0175]

[0192] Coronaviruses are large, polymorphous, spherical particles with bulbous surface projections. The average diameter of a virus particle is approximately 120 nm (0.12 μm). The envelope diameter is approximately 80 nm (0.08 μm), and the spike length is approximately 20 nm (0.02 μm). The viral envelope consists of a lipid bilayer to which membrane (M), envelope (E), and spike (S) structural proteins are anchored. A subset of coronaviruses (particularly members of the betacoronavirus subgroup A) also contain a short, spike-like surface protein called hemagglutinin esterase (HE). Inside the envelope is a nucleocapsid, formed from multiple copies of the nucleocapsid (N) protein, bound to a positive-sense, single-stranded RNA genome in a continuous, beaded structure. The lipid bilayer envelope, membrane proteins, and nucleocapsid protect the virus when it is outside the host cell.

[0176]

[0193] Infection begins when the viral S glycoprotein binds to a complementary host cell receptor. After binding, host cell proteases cleave and activate the spike protein attached to the receptor. Depending on the available host cell proteases, cleavage and activation allow the virus to enter the host cell by endocytosis or by direct fusion of the viral envelope with the host membrane. Upon entry into the host cell, the virus particle is uncoated, and its genome enters the cytoplasm. The coronavirus RNA genome contains a 5' methylated cap and a 3' polyadenylated tail, which allow the RNA to bind to host cell ribosomes for translation. The host ribosome translates the first overlapping open reading frame of the viral genome, forming a long polyprotein. The polyprotein contains its own protease, which cleaves the polyprotein into multiple nonstructural proteins.

[0177]

[0194] Coronaviruses can be classified into five genera: alpha, beta, gamma, delta, and omicron CoVs (Woo et al., 2009). Human CoVs identified to date that cause human disease include the alpha CoVs hCoV-NL63 and hCoV-229E, the beta CoVs HCoV-OC43, HKU1, severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-19; formerly known as 2019-nCoV) (Luet al., 2015; Wevers and van der Hoek, 2009; Zhu et al., 2020). HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63 continuously circulate in the human population and generally cause mild cold-like symptoms in adults and children worldwide.

[0178]

[0195] SARS-CoV is a zoonotic pathogen of animal origin. Detailed studies have shown that SARS-CoV is transmitted from palm civets to humans (Azhare et al., 2014; Gee et al., 2013; Guanet et al., 2003). Bats and birds, being warm-blooded flying vertebrates, are ideal natural hosts for the coronavirus gene pool (bats are hosts for alphacoronaviruses and betacoronaviruses, while birds are hosts for gammacoronaviruses and deltacoronaviruses). Due to the large number of host species of bats and birds and their global habitats, coronaviruses have evolved and spread widely.

[0179]

[0196] SARS-CoV-2 is a group 2B betacoronavirus that shares approximately 70% genetic similarity with SARS-CoV. The virus is widely suspected to have originated in bats, as it shares 96% similarity with a bat coronavirus (SARSr-CoV_RaTG13).

[0180]

[0197] Coronaviruses have been recognized as pathogenic agents in veterinary medicine since the 1930s. Coronaviruses primarily infect the upper respiratory and digestive tracts of mammals and birds. They also cause a variety of illnesses, some of which are severe, in livestock and pets, posing a threat to agriculture. Exemplary coronaviruses that infect animals include infectious bronchitis virus (IBV) of chickens, porcine coronavirus (transmissible gastroenteritis coronavirus, TGEV), porcine respiratory coronavirus (PEDV), bovine coronavirus (BCoV), feline enteric coronavirus, feline infectious peritonitis virus (FIPV), ferret enteric coronavirus, ferret systemic coronavirus, canine coronavirus (CCoV), mouse hepatitis virus (MHV), sialadenitis virus (SDAV), and porcine acute diarrhea syndrome coronavirus (SADS-CoV).

[0181]

[0198] The S protein of naturally occurring coronaviruses forms a homotrimer that protrudes from the viral surface. The S protein contains two functional subunits: one responsible for binding to host cell receptors (the S1 subunit) and the other for fusing the viral membrane with the cellular membrane (the S2 subunit). In many CoVs, the S protein is cleaved at the interface between the S1 and S2 subunits and remains noncovalently bound in the pre-fusion conformation of the CoV. The distal S1 subunit contains one or more RBDs and contributes to stabilizing the pre-fusion state of the membrane-anchored S2 subunit, which contains the fusion machinery. In all CoVs, the S protein is further cleaved by host proteases at the so-called S2' site, located immediately upstream of the fusion peptide. This cleavage is proposed to activate the protein for membrane fusion through extensive, irreversible conformational changes. Consequently, coronavirus entry into susceptible cells is a complex process that requires the coordinated action of receptor binding and proteolytic processing of the S protein to promote virus-cell fusion. See Wallsetal., Cell 180, 281-292 (2020).

[0182]

[0199] For example, the S protein of SARS-CoV can be cleaved by trypsin at two distinct sites: the "classical" S1 / S2 site (R667P1 residue), which is located at the interface between S1 and S2, and the S2' site (R797P1 residue). Protease cleavage of SARS-CoV is thought to occur sequentially, with the S1 / S2 cleavage occurring first and facilitating cleavage at S2'. The second cleavage event at S2' is thought to be important for S fusion activation. S1 / S2 cleavage does not appear to be necessary for syncytium formation and virus-cell fusion. See Millet, Virus Research 202:120-134 (2015).

[0183]

[0200] The spike protein of SARS-CoV-2 can be cleaved by furin at the S1 / S2 site and by transmembrane protease / serine (TMPRSS) protease 2, TMPRSS2, at the S'2 site. See Hoffman et al., Cell 181, 271-280, 2020. The furin cleavage site of SARS-CoV-2 is located between amino acids 685 and 686 of the S protein. Both SARS-CoV-2 and SARS-CoV use ACE2 as a receptor to enter human cells. See Zhou et al., Nature 579:270 (2020).

[0184]

[0201] The S1 of the S protein can be further divided into an N-terminal domain (NTD) and a C-terminal domain (CTD), both of which can function as receptor binders (e.g., SARS-CoV utilizes the CTD of S1 to recognize its receptor (also called the receptor-binding domain [RBD])) (Lietal., 2005; Luetal., 2013).

[0185]

[0202] Exemplary coronavirus S proteins and their variants are listed in Table 5. Table 5. Exemplary coronavirus strains and sequences of the receptor-binding domain of their spike proteins TIFF2025512512000026.tif238170TIFF2025512512000027.tif243170 TIFF2025512512000028.tif14170

[0186]

[0203] Provided herein are ACE2 chimeric proteins comprising a target binding moiety that specifically binds to the S protein or a fragment thereof of a coronavirus, such as those set forth in Table 5, or a variant thereof (the amino acid sequence of any one of SEQ ID NOS: 96-100, 103, 123-127, and 141-143, or a variant thereof comprising at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to any one of SEQ ID NOS: 96-100, 103, 123-127, and 141-143). In some embodiments, the target binding moiety specifically binds to the RBD of the S protein of the coronavirus, or a variant thereof. In some embodiments, the RBD is derived from a SARS-CoV, SARS-CoV-2, or HCoV-NL63 coronavirus, or a variant thereof. In some embodiments, the RBD is a reference SARS-CoV-2 (e.g., WIV4, i.e., hCoV-19 / WIV04 / 2019 or BetaCoV / WIV04 / 2019; e.g., SEQ ID NO: 96), or a SARS-CoV-2 virus having substantially the same genomic sequence (e.g., less than any one of 200, 100, 50, 20, 10, 5, 4, 3, 2, or 1 mutations) and phenotype as a reference SARS-CoV-2. In certain embodiments, the SARS-CoV-2 is a variant, such as a variant of interest, a variant of concern, or a variant of significant consequence. In some embodiments, the SARS-CoV-2 is a variant selected from the group consisting of a B.1.1.7 variant, a B.1.351 variant, a B.1.526 variant, a B1.526.1 variant, a B1.617 variant, a B.1.617.1 variant, a B.1.617.2 variant, a B1.617.3 variant, a P.2 variant, a P.1 (also known as B.1.1.28.1) variant, an A.23.1 variant, a CAL.20C variant, a B.1.427 variant, a B.1.429 variant, a B.1.525 variant, a P.1.351, a BA.5.1.1, a BQ.1 variant, an XBB variant, an XBB.1.5 variant, and an XBB.1.16 variant. In some embodiments, the SARS-CoV-2 variant is the B.1.1.7 variant.In some embodiments, the SARS-CoV-2 variant is the B.1.351 variant. In some embodiments, the SARS-CoV-2 variant is the B.1.617.2 variant. In some embodiments, the RBD is a Delta, Omicron BA.1, or Omicron BA.2 SARS-CoV-2 variant (e.g., SEQ ID NOs: 97-100), as described in Table 5. In some embodiments, the RBD is an animal coronavirus, such as a bat coronavirus (e.g., SEQ ID NO: 100), as described in Table 5. In some embodiments, the RBD is a SARS-CoV coronavirus (e.g., SEQ ID NO: 103), as described in Table 5. In some embodiments, the RBD comprises the amino acid sequence of any one of SEQ ID NOs: 96-100, 103, 123-127, and 141-143, as described in Table 5.

[0187] SARS-CoV-2

[0204] In some embodiments, the target binding moiety specifically binds to the S protein of SARS-CoV-2. In some embodiments, the target binding moiety specifically binds to the S1 subunit of the S protein. Exemplary ACE2 proteins or fragments thereof (e.g., hACE proteins or fragments thereof, and animal ACE2 proteins or fragments thereof) that specifically bind to the S1 subunit of the S protein are disclosed herein in Tables 3 and 4. In some embodiments, the target binding moiety specifically binds to the RBD of the S protein of SARS-CoV-2. Exemplary RBD sequences for the S protein of SARS-CoV-2 are shown in Table 5.

[0188]

[0205] The SARS-CoV-2 S protein contains an S1 region containing a signaling peptide (amino acid residues 1-19), an N-terminal domain (NTD; amino acid residues 20-286), and a C-terminal domain (CTD; amino acid residues 319-541), an S2 region (amino acid residues 686-1213), a transmembrane domain (amino acid residues 1214-1236), and a short cytoplasmic domain (amino acid residues 1237-1273). The CTD, particularly amino acid residues 333-527, plays an important role in binding to ACE2. In particular, amino acid residues A475, K417, G446, Y449, G496, Q498, T500, G502, Y489, F486, and N487 contribute to the binding of the SARS-CoV-2 CTD to hACE2. See Wang et al., 2020, Cell 181, 1-11, which is incorporated herein by reference in its entirety.

[0189]

[0206] In some embodiments, the target binding moiety can recognize two or more isolates or clusters of SARS-CoV-2 (e.g., cluster A, B, or C; or any one of the isolates disclosed in Forster et al. Proc Natl Acad Sci (2020)). In some embodiments, the target binding moiety specifically blocks binding to hACE2 of one or more variants of the S1 protein of SARS-CoV-2, including the SARS-CoV-2 S1 S protein variants in Table 5, such as an S1 protein containing one or more mutations selected from the group consisting of D614G, V367F, N439K, A435S, V483A, K458R, G476S, R408I, V503F, A522V, Y508H, L452R, A520S, I472V, T478I, F490S, and / or P384L.

[0190]

[0207] In some embodiments, the target binding moiety can recognize more than one variant of SARS-CoV-2. In some embodiments, the components are derived from a reference SARS-CoV-2. In some embodiments, the components are derived from a SARS-CoV-2 variant. In some embodiments, compositions are provided that include a plurality of chimeric proteins that can recognize multiple SARS-CoV-2 variants and a reference virus. In some embodiments, each of the plurality of chimeric proteins comprises the same target binding moiety. In some embodiments, at least two of the plurality of chimeric proteins comprise different target binding moieties that recognize different SARS-CoV-2 variants.

[0191]

[0208] Exemplary SARS-CoV-2 variants and their characteristics are shown in Table 6 below. The chimeric proteins and compositions described herein can be used to treat any one of the SARS-CoV-2 variants described herein. The SARS-CoV-2 variants described herein are named by the Phylogenetic Assignment of Named Global Outbreak (PANGO) Lineages software. It is understood that the same variant may be referred to using different naming systems and algorithms in the art. SARS-CoV-2 variant classifications and definitions, as well as a list of known SARS-CoV-2 variants, can be found at worldwideweb.cdc.gov / coronavirus / 2019-ncov / variants / variant-info.html. Table 6. SARS-CoV-2 variants and characteristics. TIFF2025512512000029.tif248170TIFF2025512512000030.tif247170TIFF2025512512000031.tif112170

[0192] SARS-CoV

[0209] In some embodiments, the target binding moiety specifically binds to the S protein of SARS-CoV. In some embodiments, the S protein is derived from a reference SARS-CoV. In some embodiments, the S protein is derived from a SARS-CoV variant. In some embodiments, the target binding moiety specifically binds to the S1 subunit of the S protein of SARS-CoV. The sequence of the SARS-CoV 1 protein is known in the art and includes, for example, UniProtKBID: P59594, UniProtKBID: P0DTC2, NCBIRefSeqID: YP_009724390.1, and GeneBank ID: AAP41037.1. In some embodiments, the SARS-CoV 1 protein comprises the amino acid sequence of SEQ ID NO: 103, as set forth in Table 5.

[0193]

[0210] In some embodiments, the target binding moiety is the EBD of the ACE2 protein or a fragment thereof that specifically binds to the S1 subunit of the S protein of SARS-CoV. In some embodiments, the target binding moiety inhibits binding of the S1 subunit of the SARS-CoV S protein to the cell surface. In some embodiments, the target binding moiety binds within the RBD of the S1 protein of SARS-CoV or to an antigen-binding fragment thereof. In some embodiments, the target binding moiety specifically binds to amino acid residues 319-510 of the S1 protein, where numbering is based on SEQ ID NO: 103. In some embodiments, the target binding moiety is capable of cross-neutralizing multiple SARS-CoV isolates. In some embodiments, the target binding moiety binds to distinct conformational epitopes in the RBD of the S1 protein. In some embodiments, SARS-CoV human isolates (e.g., Tor2, GD03T0013) or Pagumalarvata isolate fusion proteins (e.g., Sz3S1-Fc) can be used as immunogens to induce high titer, cross-neutralizing target-binding moieties.

[0194]

[0211] In some embodiments, the target binding moiety is a derivative of any one of the target binding moieties for the S1 protein of SARS-CoV described herein.

[0195]

[0212] The S protein of SARS-CoV is an attractive target for vaccine design because it mediates receptor binding and viral entry into host cells (i.e., viral infection). The S protein is a type I transmembrane glycoprotein with an S1 domain, which comprises amino acid residues 1–672 of the S protein. The central region of the S1 domain, a fragment located at amino acid residues 318–510, is defined as the receptor-binding domain. The receptor-binding domain of the S1 protein is a major determinant of SARS-CoV neutralization. Antibodies targeting the S1 protein, particularly the receptor-binding domain, are a large class of therapeutic agents useful for the prevention and treatment of SARS-CoV infection.

[0196] B. Variant

[0213] In some embodiments, amino acid sequence variants of the target-binding portion of the chimeric proteins provided herein (e.g., amino acid sequence variants of the EBD of the ACE2 protein or a fragment thereof) are contemplated. For example, amino acid sequence variants of the ACE2 proteins or fragments thereof described herein (e.g., the hACE2 proteins and animal ACE2 proteins or fragments thereof described in Tables 3 and 4, respectively) are contemplated. It may be desirable to improve the binding affinity and / or other biological properties of the target-binding portion. Amino acid sequence variants of the target-binding portion (e.g., the EBD of the ACE2 protein or a fragment thereof) can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the target-binding portion or by peptide synthesis. Such modifications include, for example, deletion, insertion, and / or substitution of residues within the amino acid sequence of the target-binding portion (e.g., the EBD of the ACE protein or a fragment thereof). Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct has the desired properties, such as binding to an S protein (e.g., the S1 subunit of the S protein).

[0197]

[0214] In some embodiments, variants with one or more amino acid substitutions are provided. Amino acid substitutions can be introduced into the target-binding moiety of interest (e.g., the EBD of the ACE2 protein or a fragment thereof) and the products screened for a desired activity, e.g., retention / improvement of target binding or reduction in immunogenicity.

[0198]

[0215] Conservative substitutions are shown in Table B below. Table B. Conservative Amino Acid Substitutions TIFF2025512512000032.tif111170Amino acids can be grouped into different classes according to common side chain properties. a. Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. Acidic: Asp, Glu; d. Basic: His, Lys, Arg; e. Residues that affect chain orientation: Gly, Pro; f. Aromatic: Trp, Tyr, Phe

[0199]

[0216] Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

[0200]

[0217] In some embodiments, substitutions, insertions, or deletions can occur within the target-binding moiety, as long as these changes do not substantially reduce the target-binding moiety's ability to bind to an antigen. For example, conservative changes (e.g., conservative substitutions provided herein) can be made that do not substantially reduce binding affinity. In some embodiments of variant target-binding moieties, the target-binding moiety is unchanged or contains no more than one, two, or three amino acid substitutions.

[0201]

[0218] A useful method for identifying residues or regions of a target-binding moiety that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells Science, 244:1081-1085 (1989). In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antigen-antibody interaction is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the target-binding moiety-S protein complex can be determined to identify contact points between the target-binding moiety and the S protein. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

[0202]

[0219] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes a target binding moiety with an N-terminal methionine residue. Other insertional variants of target binding moieties include fusions to the N- or C-terminus of the target binding moiety to an enzyme (e.g., in the case of ADEPT) or to a polypeptide that extends the serum half-life of the target binding moiety.

[0203] C. Mucoadhesive peptide fragments

[0220] The chimeric proteins described herein comprise one or more (eg, 1, 2, 3, 4, or more) mucoadhesive peptide fragments.

[0204]

[0221] In some embodiments, the target-binding moiety comprises the same number of polypeptide chains as the number of mucoadhesive peptide fragments in the chimeric protein. In some embodiments, the target-binding moiety comprises more polypeptide chains than the number of mucoadhesive peptide fragments in the chimeric protein. In some embodiments, each polypeptide of the target-binding moiety is linked (e.g., fused) to a mucoadhesive peptide fragment. In some embodiments, the target-binding moiety comprises a polypeptide chain that is not linked (e.g., fused) to a mucoadhesive peptide fragment.

[0205]

[0222] In some embodiments, the mucoadhesive peptide fragment is fused to the target-binding moiety at any position that does not interfere with binding of the target-binding moiety to the S protein (e.g., the S1 subunit of the S protein). In some embodiments, the mucoadhesive peptide fragment is fused to a site distal to the target-binding site. In some embodiments, the mucoadhesive peptide fragment is fused to the C-terminus of the target-binding moiety.

[0206]

[0223] In some embodiments, the chimeric protein comprises a single polypeptide chain comprising the target-binding moiety and the mucoadhesive peptide fragment.

[0207]

[0224] In some embodiments, the mucoadhesive peptide fragment comprises from about 10 to about 600 amino acid residues (e.g., positively charged amino acid residues plus non-positively charged amino acid residues). In some embodiments, the mucoadhesive peptide fragment comprises from about 10 to about 20 amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises from about 22 to about 30 amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises from about 32 to about 40 amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises from about 42 to about 50 amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises from about 52 to about 60 amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises from about 16 to about 50 amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises from about 20 to about 44 amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises from about 24 to about 40 amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises from about 28 to about 36 amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises about n amino acid residues, where n is selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 300, 400, 500, 600 or more.In some embodiments, the mucoadhesive peptide fragment comprises any one of about n amino acid residues, where n is 10-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151 ~160, 161~170, 171~180, 181~190, 191~200, 201~210, 211~220, 221~230, 231~240, 241~250, 251~260, 261~270, 271~280, 281~290, 291~300, 301~310, 311~320, 321~330, 331~340, 341 ~350, 351~360, 361~370, 371~380, 381~390, 391~400, 401~410, 411~420, 421~430, 431~440, 441~450, 451~460, 461~470, 471~480, 481~490, 491~500, 501~510, 511~520, 521~530, 531 Selected from 540, 541-550, 551-560, 561-570, 571-580, 581-590, 591-600, 10-30, 10-40, 10-50, 16-30, 16-40, 16-50, 20-40, 20-50, 24-40, 24-50, 30-50, 30-60, 10-60, 12-60, and 10-100. In some embodiments, the mucoadhesive peptide fragment comprises at least about n amino acid residues, where n is selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 300, 400, 500, 600 or more.In some embodiments, the mucoadhesive peptide fragment comprises no more than about n amino acid residues, where n is selected from 600, 500, 400, 300, 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, and 10.

[0208]

[0225] In some embodiments, the mucoadhesive peptide fragment comprises about 5 to about 300 positively charged amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises about 5 to about 10 positively charged amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises about 11 to about 15 positively charged amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises about 16 to about 20 positively charged amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises about 21 to about 25 positively charged amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises about 26 to about 30 positively charged amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises about 8 to about 25 positively charged amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises about 10 to about 22 positively charged amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises about 12 to about 20 positively charged amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises about 14 to about 18 positively charged amino acid residues. In some embodiments, the mucoadhesive peptide fragment comprises any one of about n positively charged amino acid residues, where n is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 or more.In some embodiments, the mucoadhesive peptide fragment comprises about n positively charged amino acid residues, where n is about 5-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, 96-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151 to 160, 161 to 170, 171 to 180, 181 to 190, 191 to 200, 201 to 210, 211 to 220, 221 to 230, 231 to 240, 241 to 250, 251 to 260, 261 to 270, 271 to 280, 281 to 290, 291 to 300, 5 to 15, 5 to 20, 5 to 25, 8 to 15, 8 to 20, 8 to 25, 10 to 20, 10 to 25, 12 to 20, 12 to 25, 15 to 25, 15 to 30, 5 to 30, 6 to 30, and 5 to 50. In some embodiments, the mucoadhesive peptide fragment comprises at least about n positively charged amino acid residues, where n is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more. In some embodiments, the mucoadhesive peptide fragment comprises no more than about n positively charged amino acid residues, where n is selected from 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 9, 8, 7, 6, and 5.

[0209]

[0226] In some embodiments, the mucoadhesive peptide fragment comprises at least about 5 positively charged amino acid residues (e.g., lysine, arginine, histidine, ornithine, and combinations thereof). In some embodiments, the mucoadhesive peptide fragment comprises at least about 5 positively charged amino acid residues (e.g., lysine, arginine, histidine, ornithine, and combinations thereof). In some embodiments, the mucoadhesive peptide fragment comprises from about 5 to about 50 positively charged amino acid residues (e.g., lysine, arginine, histidine, ornithine, and combinations thereof). In some embodiments, the mucoadhesive peptide fragment comprises from about 5 to about 30 positively charged amino acid residues (e.g., lysine, arginine, histidine, ornithine, and combinations thereof). In some embodiments, the mucoadhesive peptide fragment comprises about 5, 6, 12, 18, 24, or 30 positively charged amino acid residues (e.g., lysine, arginine, histidine, ornithine, and combinations thereof).

[0210]

[0227] In some embodiments, the chimeric protein comprises two or more mucoadhesive peptide fragments. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises about 5 to about 300 positively charged amino acid residues. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises about 5 to about 10 positively charged amino acid residues. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises about 11 to about 15 positively charged amino acid residues. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises about 16 to about 20 positively charged amino acid residues. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises about 21 to about 25 positively charged amino acid residues. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises about 26 to about 30 positively charged amino acid residues. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises about 8 to about 25 positively charged amino acid residues. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises about 10 to about 22 positively charged amino acid residues. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises about 12 to about 20 positively charged amino acid residues. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises about 14 to about 18 positively charged amino acid residues. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises about n positively charged amino acid residues, where n is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more.In some embodiments, each of the two or more mucoadhesive peptide fragments comprises about n positively charged amino acid residues, where n is 5-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, 96-100, 101-110, 111-120, 121-130, 131-140, 141-150 , 151 to 160, 161 to 170, 171 to 180, 181 to 190, 191 to 200, 201 to 210, 211 to 220, 221 to 230, 231 to 240, 241 to 250, 251 to 260, 261 to 270, 271 to 280, 281 to 290, 291 to 300, 5 to 15, 5 to 20, 5 to 25, 8 to 15, 8 to 20, 8 to 25, 10 to 20, 10 to 25, 12 to 20, 12 to 25, 15 to 25, 15 to 30, 5 to 30, 6 to 30, and 5 to 50. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises at least about n positively charged amino acid residues, where n is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more. In some embodiments, each of the two or more mucoadhesive peptide fragments comprises no more than about n positively charged amino acid residues, where n is selected from 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 9, 8, 7, 6, and 5.

[0211]

[0228] In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments in the chimeric protein is from about 5 to about 600. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments in the chimeric protein is from about 5 to about 20. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments in the chimeric protein is from about 11 to about 30. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments in the chimeric protein is from about 16 to about 40. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments in the chimeric protein is from about 21 to about 50. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments in the chimeric protein is from about 26 to about 60. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments in the chimeric protein is from about 8 to about 50. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments in the chimeric protein is from about 10 to about 44. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments in the chimeric protein is from about 12 to about 40. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments in the chimeric protein is from about 14 to about 36. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments in the chimeric protein is about n positively charged amino acid residues, where n is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 550, 600 or more.In some embodiments, the total number of positively charged amino acid residues in one or more mucoadhesive peptide fragments in the chimeric protein is about n positively charged amino acid residues, where n is 5-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81 ~85, 86~90, 91~95, 96~100, 101~110, 111~120, 121~130, 131~140, 141~150, 151~160, 161~170, 171-180, 181~190, 191~200, 201~210, 211~220, 221~230, 231~240, 241~250, 251~160, 261~270, 271~280, 281~ 290, 291-300, 301-310, 311-320, 321-330, 331-340, 341-350, 351-360, 361-370, 371-380, 381-390, 391-400, 401-410, 411-420, 421-430, 431-440, 441-450, 451-460, 461-470, 471-480, 481-490, 491-500 , 501-510, 511-520, 521-530, 531-540, 541-550, 551-560, 561-570, 571-580, 581-590, 591-600, 5-15, 5-20, 5-25, 8-15, 8-20, 8-25, 10-20, 10-25, 12-20, 12-25, 15-25, 15-30, 5-30, 6-30, and 5-50.In some embodiments, the total number of positively charged amino acid residues in one or more mucoadhesive peptide fragments in the chimeric protein is at least about n positively charged amino acid residues, where n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 79 40, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, or more. In some embodiments, the total number of positively charged amino acid residues in one or more mucoadhesive peptide fragments in the chimeric protein is about n or less positively charged amino acid residues, where n is 600, 590, 580, 570, 560, 550, 540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 9, 8, 7, 6, and 5.

[0212]

[0229] In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments per target-binding moiety is from about 5 to about 30. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments per target-binding moiety is from about 8 to about 25. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments per target-binding moiety is from about 10 to about 22. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments per target-binding moiety is from about 12 to about 20. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments per target-binding moiety is from about 14 to about 18. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments per target-binding moiety is about n positively charged amino acid residues, where n is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments per target-binding moiety is about n positively charged amino acid residues, where n is selected from 5-10, 11-15, 16-20, 21-25, 26-30, 5-15, 5-20, 5-25, 8-15, 8-20, 8-25, 10-20, 10-25, 12-20, 12-25, 15-25, 15-30, 5-30, and 6-30. In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments per target-binding moiety is at least about n positively charged amino acid residues, where n is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more.In some embodiments, the total number of positively charged amino acid residues in the one or more mucoadhesive peptide fragments per target-binding moiety is no more than about n positively charged amino acid residues, where n is selected from 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 9, 8, 7, 6, and 5.

[0213]

[0230] The one or more mucoadhesive peptide fragments may comprise any suitable positively charged amino acid residue at the physiological pH of the mucosa, including naturally occurring and synthetic amino acid residues such as lysine, arginine, histidine, ornithine, and combinations thereof. In some embodiments, the mucoadhesive peptide fragment comprises only lysine. In some embodiments, the mucoadhesive peptide fragment comprises only arginine. In some embodiments, the mucoadhesive peptide fragment comprises only histidine. In some embodiments, the mucoadhesive peptide fragment comprises only ornithine.

[0214]

[0231] In some embodiments, the mucoadhesive peptide fragments comprise both lysine and arginine. In some embodiments, the mucoadhesive peptide fragments comprise equal numbers of lysines and arginines. In some embodiments, the mucoadhesive peptide fragments comprise unequal numbers of lysines and arginines. In some embodiments, the mucoadhesive peptide fragments comprise both lysine and histidine. In some embodiments, the mucoadhesive peptide fragments comprise equal numbers of lysines and histidines. In some embodiments, the mucoadhesive peptide fragments comprise unequal numbers of lysines and histidines. In some embodiments, the mucoadhesive peptide fragments comprise both lysine and ornithine. In some embodiments, the mucoadhesive peptide fragments comprise equal numbers of lysines and ornithine. In some embodiments, the mucoadhesive peptide fragments comprise unequal numbers of lysines and ornithine. In some embodiments, the mucoadhesive peptide fragments comprise both arginine and ornithine. In some embodiments, the mucoadhesive peptide fragments comprise equal numbers of arginines and ornithine. In some embodiments, the mucoadhesive peptide fragments comprise unequal numbers of arginines and ornithines. In some embodiments, the mucoadhesive peptide fragments comprise both arginine and histidine. In some embodiments, the mucoadhesive peptide fragments comprise equal numbers of arginines and histidines. In some embodiments, the mucoadhesive peptide fragments comprise unequal numbers of ornithines and histidines. In some embodiments, the mucoadhesive peptide fragments comprise both ornithine and histidine. In some embodiments, the mucoadhesive peptide fragments comprise equal numbers of ornithines and histidines. In some embodiments, the mucoadhesive peptide fragments comprise unequal numbers of ornithines and histidines. In some embodiments, the mucoadhesive peptide fragments comprise lysine, arginine, and ornithine. In some embodiments, the mucoadhesive peptide fragments comprise equal numbers of lysines, arginines, and ornithine. In some embodiments, the mucoadhesive peptide fragments comprise unequal numbers of lysines, arginines, and ornithine. In some embodiments, the mucoadhesive peptide fragment comprises histidine, arginine, and ornithine.In some embodiments, the mucoadhesive peptide fragments comprise equal numbers of histidines, arginines, and ornithine. In some embodiments, the mucoadhesive peptide fragments comprise unequal numbers of histidines, arginines, and ornithine. In some embodiments, the mucoadhesive peptide fragments comprise lysine, histidine, and ornithine. In some embodiments, the mucoadhesive peptide fragments comprise equal numbers of lysines, histidines, and ornithine. In some embodiments, the mucoadhesive peptide fragments comprise unequal numbers of lysines, histidines, and ornithine. In some embodiments, the mucoadhesive peptide fragments comprise lysine, arginine, and histidine. In some embodiments, the mucoadhesive peptide fragments comprise equal numbers of lysines, arginines, and histidines. In some embodiments, the mucoadhesive peptide fragments comprise unequal numbers of lysines, arginines, and histidines. In some embodiments, the mucoadhesive peptide fragments comprise lysine, arginine, histidine, and ornithine. In some embodiments, the mucoadhesive peptide fragment comprises equal numbers of lysines, arginines, histidines, and ornithines. In some embodiments, the mucoadhesive peptide fragment comprises unequal numbers of lysines, arginines, histidines, and ornithines. In some embodiments, the mucoadhesive peptide fragment comprises one or more non-naturally occurring positively charged amino acid residues at the physiological pH of the mucosa.

[0215]

[0232] In some embodiments, the mucoadhesive peptide fragment has an isoelectric point (pI) higher than the pH of the mucosa. The pH values ​​of various human mucosa are known. For example, human nasal mucosa may have a pH range of about 5.5 to about 6.5, about 5.5 to about 6.6, about 6.44 to about 6.91, about 6.4 to about 7.9, or 6.4 to about 6.5. In some examples, human nasal mucosa may have a pH of about 6.6. In some examples, human nasal mucosa may have a pH range of about 6.1 to about 7.9, or a pH of about 6.71. In further examples, human bronchial mucosa may have a pH range of about 5.7 to about 6.6, or 7 to about 7.5. In some examples, human bronchial mucosa may have a pH of about 6.7, about 7.1, about 6.25, about 6.78, or about 6.58. In some examples, the human mucosa may be pathological. In such an example, a smoker may have a sputum mucosal pH of about 7.25 or about 6.82. In another example, a patient suffering from chronic bronchitis may have a sputum mucosal pH of about 7.59 and / or a sputum purulent pH of about 7.83. In another example, a patient suffering from rhinitis may have a nasal mucosal pH range of about 7.2 to about 8.3. In another example, a patient suffering from the common cold may have a mucosal pH range of about 7.2 to about 8.3.

[0216]

[0233] In some embodiments, various properties (e.g., pI, net charge, and molecular weight) of the polycationic peptides described herein can be calculated at nasal pH (e.g., about 6.5). In some embodiments, the equivalent of a 20-mer polypeptide can be calculated at nasal pH (e.g., about 6.5). 20-mer polypeptides are linear, and the size of each polypeptide will be proportional to its molecular weight. The pI and molecular weight at nasal pH of various exemplary 20-mer polypeptides were calculated and are listed in Table 7. The interaction between positively charged polypeptides and mucosal cells or mucins can occur primarily due to charge. Table 7. Properties of exemplary amino acids and corresponding 20-mer polypeptides at nasal pH TIFF2025512512000033.tif36170

[0217]

[0234] In some embodiments, the mucoadhesive peptide fragments have a pI range of at least about n, where n is selected from 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.5, 12, 12.5, 13 or more. In some embodiments, the mucoadhesive peptide fragments have a pI range of about 8 to about 14. In some embodiments, the mucoadhesive peptide fragments have a range of pI values ​​of about 8.8 to about 10.0. In some embodiments, the mucoadhesive peptide fragments have a pI value range of about 11.3 to about 13.3. In some embodiments, the chimeric protein has a pI value range of at least about n, where n is 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.10, 11.11, 11.12, 11.13, 11.14, 11.15, 11.16, 11.17, 11.18, 11.19, 11.20, 11.21, 11.22, 11.23, 11.24, 11.25, 11.26, 11.27, 11.28, 11.29, 11.29, 11.30, 11.31, 11.32, 11.33, 11.34, 11.35, 11.36, 11.37, 11.38, 11.39, 11.40, 11.41, 11.42, 11.43, 11.44, 11.45, 11.46, 11.47, 11.48, 11.49, 11.50, 11. .8, 10.9, 11, 11.5, 12, 12.5, 13 or more, including 8-8.3, 8.3-8.5, 8.5-8.7, 8.7-8.9, 8.9-9.1, 9.1-9.3, 9.3-9.4, 9.4-10, 8-10, 8-9, 9-10, 8-11, 8.5-9.5, 8.76-9.44, 8.77-9.61, and 8.32-9.33.

[0218]

[0235] In some embodiments, the mucoadhesive peptide fragment is a polylysine peptide having about n consecutive lysines, where n is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the mucoadhesive peptide fragment is a polylysine peptide having about n consecutive lysines, where n is selected from 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 5-15, 5-20, 5-25, 8-15, 8-20, 8-25, 10-20, 10-25, 12-20, 12-25, 15-25, 15-30, 5-30, 6-30, and 5-50.

[0219]

[0236] In some embodiments, the mucoadhesive peptide fragment is a polyhistidine peptide having about n consecutive histidines, where n is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the mucoadhesive peptide fragment is a polyhistidine peptide having about n consecutive histidines, where n is selected from 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 5-15, 5-20, 5-25, 8-15, 8-20, 8-25, 10-20, 10-25, 12-20, 12-25, 15-25, 15-30, 5-30, 6-30, and 5-50.

[0220]

[0237] In some embodiments, the mucoadhesive peptide fragment is a polyarginine peptide having about n consecutive arginines, where n is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the mucoadhesive peptide fragment is a polyarginine peptide having about n consecutive arginines, where n is selected from 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 5-15, 5-20, 5-25, 8-15, 8-20, 8-25, 10-20, 10-25, 12-20, 12-25, 15-25, 15-30, 5-30, 6-30, and 5-50.

[0221]

[0238] In some embodiments, the mucoadhesive peptide fragment is a polyornithine peptide having about n consecutive ornithines, where n is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the mucoadhesive peptide fragment is a polyornithine peptide having about n consecutive ornithines, where n is selected from 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 5-15, 5-20, 5-25, 8-15, 8-20, 8-25, 10-20, 10-25, 12-20, 12-25, 15-25, 15-30, 5-30, 6-30, and 5-50.

[0222]

[0239] In some embodiments, the mucoadhesive peptide fragment comprises a contiguous stretch of positively charged amino acid residues, hi some embodiments, the mucoadhesive peptide fragment comprises about n consecutive positive amino acids, such as arginine, histidine, lysine, or ornithine, or a combination thereof, where n is selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the mucoadhesive peptide fragment comprises approximately contiguous positive amino acids, such as arginine, histidine, lysine, or ornithine, or a combination thereof, where n is selected from 2 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 2 to 15, 2 to 20, 2 to 25, 8 to 15, 8 to 20, 8 to 25, 10 to 20, 10 to 25, 12 to 20, 12 to 25, 15 to 25, 15 to 30, 2 to 30, 6 to 30, and 2 to 50. In some embodiments, all of the positively charged amino acid residues are contiguous with one another.

[0223]

[0240] In some embodiments, the mucoadhesive peptide fragment comprises a contiguous stretch of one or more non-positively charged amino acid residues. In some embodiments, the non-positively charged amino acid residues are non-polar amino acids or polar non-charged amino acids. In some embodiments, the mucoadhesive peptide fragment comprises isoleucine, valine, alanine, tryptophan, leucine, glycine, methionine, proline, phenylalanine, threonine, cysteine, tyrosine, glutamine, serine, asparagine, or a combination thereof. In some embodiments, the mucoadhesive peptide fragment comprises one or more alanine, threonine, cysteine, serine, glutamine, asparagine, or a combination thereof. In some embodiments, the mucoadhesive peptide fragment comprises one or more combinations of isoleucine, valine, alanine, tryptophan, leucine, glycine, methionine, proline, phenylalanine, threonine, cysteine, tyrosine, glutamine, serine, or asparagine. In some embodiments, the mucoadhesive peptide fragment comprises a combination of one or more alanine, threonine, cysteine, serine, glutamine, or asparagine. In some embodiments, the mucoadhesive peptide fragment comprises one or more non-naturally occurring non-positively charged amino acid residues at the physiological pH of the mucosa.

[0224]

[0241] In some embodiments, the positively charged amino acid residues are interspersed with non-positively charged amino acid residues. In some embodiments, the positively charged amino acid residues are present at every other position in the mucoadhesive peptide fragment. In some embodiments, the positively charged amino acid residues are present at every third position in the mucoadhesive peptide fragment. In some embodiments, the positively charged amino acid residues are present at every fourth position in the mucoadhesive peptide fragment. In some embodiments, the positively charged amino acid residues are randomly dispersed in the mucoadhesive peptide fragment. In some embodiments, the positively charged residues are present in one or more clusters within the mucoadhesive peptide fragment.

[0225]

[0242] In some embodiments, at least about n% of the amino acid residues in the mucoadhesive peptide fragment are positively charged amino acid residues, where n% is selected from 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more. In some embodiments, all amino acid residues in the mucoadhesive peptide fragment are positively charged. In some embodiments, no more than n% of the amino acid residues in the mucoadhesive peptide fragment are positively charged amino acid residues, where n% is selected from 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, and 10%. In some embodiments, about n% of the amino acid residues in the mucoadhesive peptide fragment are positively charged amino acid residues, where n% is selected from 10% to 99%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 80%, 10% to 100%, 10% to 30%, 30% to 60%, 60% to 90%, 20% to 50%, and 50% to 100%. In some embodiments, at least 50% of the amino acid residues in the mucoadhesive peptide fragment are positively charged amino acid residues.

[0226]

[0243] In some embodiments, at least n% of the amino acid residues in the mucoadhesive peptide fragment are non-positively charged amino acid residues, where n% is selected from 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% or more. In some embodiments, all amino acid residues in the mucoadhesive peptide fragment are positively charged. In some embodiments, no more than n% of the amino acid residues in the mucoadhesive peptide fragment are non-positively charged amino acid residues, where n% is selected from 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1%. In some embodiments, about n% of the amino acid residues in the mucoadhesive peptide fragment are non-positively charged amino acid residues, where n% is selected from 1%-5%, 5%-10%, 10%-25%, 25%-50%, 1%-10%, 5%-15%, 10%-20%, 15%-25%, 20%-30%, 25%-35%, 30%-40%, 35%-45%, or 40%-50%. In some embodiments, 50% or less of the amino acid residues in the mucoadhesive peptide fragment are non-positively charged amino acid residues.

[0227]

[0244] In some embodiments, the mucoadhesive peptide fragment is about 15 kD or less. In some embodiments, the mucoadhesive peptide fragment is about 0.5 kD to about 50 kD. In some embodiments, the mucoadhesive peptide fragment is about 0.5 kD to about 15 kD. In some embodiments, the mucoadhesive peptide fragment is about 2 kD to about 12 kD. In some embodiments, the mucoadhesive peptide fragment is about 4 kD to about 10 kD. In some embodiments, the mucoadhesive peptide fragment is about 6 kD to about 14 kD. In some embodiments, the mucoadhesive peptide fragment is about n, where n is selected from 0.5 kD, 1 kD, 2 kD, 3 kD, 4 kD, 5 kD, 6 kD, 7 kD, 8 kD, 9 kD, 10 kD, 11 kD, 12 kD, 13 kD, 14 kD, 15 kD, 20 kD, 25 kD, 30 kD, 35 kD, 40 kD, 45 kD, 50 kD, or more. In some embodiments, the mucoadhesive peptide fragment is about n, where n is selected from 0.5-1 kD, 1-2 kD, 2-3 kD, 4-5 kD, 5-6 kD, 6-7 kD, 8-9 kD, 9-10 kD, 10-11 kD, 11-12 kD, 12-13 kD, 13-14 kD, 14-15 kD, 15-20 kD, 20-25 kD, 25-30 kD, 30-35 kD, 35-40 kD, 40-45 kD, 45-50 kD, or more. In some embodiments, the mucoadhesive peptide fragment is at least about n, where n is selected from 0.5 kD, 1 kD, 2 kD, 3 kD, 4 kD, 5 kD, 6 kD, 7 kD, 8 kD, 9 kD, 10 kD, 11 kD, 12 kD, 13 kD, 14 kD, 15 kD, 20 kD, 25 kD, 30 kD, 35 kD, 40 kD, 45 kD, 50 kD, or more. In some embodiments, the mucoadhesive peptide fragment is less than or equal to about n, where n is selected from 50 kD, 45 kD, 40 kD, 35 kD, 30 kD, 25 kD, 20 kD, 15 kD, 14 kD, 13 kD, 12 kD, 11 kD, 10 kD, 9 kD, 8 kD, 7 kD, 6 kD, 5 kD, 4 kD, 3 kD, 2 kD, 1 kD, and 0.5 kD.

[0228]

[0245] In some embodiments, the mucoadhesive peptide fragment does not facilitate penetration of the chimeric protein into mucosal cells. In some embodiments, the mucoadhesive peptide fragment does not comprise a cell-penetrating peptide motif. In some embodiments, the mucoadhesive peptide is not a cell-penetrating peptide.

[0229]

[0246] In some embodiments, the mucoadhesive peptide fragment is not a histidine tag. In some embodiments, the mucoadhesive peptide fragment is not a peptide consisting of or consisting essentially of six histidines.

[0230]

[0247] In some embodiments, the mucoadhesive peptide fragment does not disrupt folding of the chimeric protein in a host cell expressing the chimeric protein, hi some embodiments, at least about n% of the chimeric protein expressed in a mammalian host cell is properly folded, where n% is selected from 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0231]

[0248] In some embodiments, the mucoadhesive peptide fragment does not prevent secretion of the chimeric protein from host cells expressing the chimeric protein. In some embodiments, at least about n% of the chimeric protein expressed in mammalian host cells is secreted, where n% is selected from 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Without being bound by any theory or hypothesis, proteins with positively charged fragments tend to be trapped in the Golgi apparatus of cells expressing the protein. The chimeric proteins described herein are readily expressed and secreted in host cells and do not become trapped in the Golgi apparatus.

[0232]

[0249] In some embodiments, the mucoadhesive peptide fragment does not interfere with specific binding between the target binding moiety and the S protein (e.g., the S1 subunit of the S protein). In some embodiments, the mucoadhesive peptide fragment reduces binding between the target binding moiety and the S protein (e.g., the S1 subunit of the S protein) by about n% or less, where n% is selected from 50%, 40%, 30%, 20%, 10%, or less.

[0233]

[0250] Exemplary mucoadhesive peptide fragments for incorporation into the chimeric proteins of the present disclosure are provided herein and shown in Table 8. Table 8: Exemplary mucoadhesive peptide fragments TIFF2025512512000034.tif248170TIFF2025512512000035.tif74170

[0234]

[0251] It should be understood that additional mucoadhesive peptide fragments containing similar proportions of positively charged and / or non-positively charged amino acid residues are also within the scope of the present invention.

[0235]

[0252] In some embodiments, the mucoadhesive peptide fragment comprises an amino acid sequence having at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134. In some embodiments, the mucoadhesive peptide fragment comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134, or a variant thereof comprising about 1, 2, or 3 amino acid substitutions. In some embodiments, the mucoadhesive peptide fragment portion comprises the amino acid sequence of any one of SEQ ID NOs: 28-62 and 128-134.

[0236] D. Linker

[0253] In some aspects, the target-binding moiety is linked to the mucoadhesive peptide fragment via a linker (such as a peptide linker, also referred to herein as a linking peptide). In some embodiments, the target-binding moiety is not covalently attached to the mucoadhesive peptide fragment. In some embodiments, the mucoadhesive peptide fragment is chemically conjugated (i.e., via a chemical linker) to the target-binding moiety.

[0237]

[0254] In some embodiments, a peptide linker is positioned between the target binding moiety and the mucoadhesive peptide fragment of the chimeric protein. In some embodiments, the peptide linker is fused to the polypeptide chain of the target binding moiety. In some embodiments, the linker is fused to the mucoadhesive peptide fragment.

[0238]

[0255] In some embodiments, the mucoadhesive peptide fragment is fused to the polypeptide chain of the target-binding moiety via a peptide linker. In some embodiments, the linker is from about 1 to about 20 amino acid residues. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the linker has the amino acid sequence GGGGS (SEQ ID NO: 79).

[0239]

[0256] The length, degree of flexibility, and / or other properties of the linker may have some effect on the properties of the chimeric proteins described herein (e.g., bispecific immune cell engagers or engineered receptors), including, but not limited to, the affinity, specificity, or avidity for one or more targets. For example, a longer linker may be selected so that two adjacent binding moieties do not sterically interfere with each other. In some embodiments, the linker (e.g., a peptide linker) includes flexible residues (e.g., glycine and serine) to allow adjacent binding moieties to move freely relative to each other. For example, a glycine-serine doublet may be a suitable peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a cleavable linker.

[0240]

[0257] Other linker considerations include their effect on the physical or pharmacokinetic properties of the resulting chimeric protein (e.g., bispecific immune cell engager or engineered receptor), such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (high or low stability as well as targeted degradation), rigidity, flexibility, immunogenicity, modulation of target binding moiety binding, ability to be incorporated into micelles or liposomes, etc.

[0241]

[0258] Any one or all of the linkers described herein can be achieved by any chemical reaction that bonds two molecules together, so long as the components or fragments retain their respective activities. This bonding can involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complexation. In some embodiments, the bond is covalent. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporation of an external bridging molecule. Many bivalent or polyvalent linking agents are useful for coupling protein molecules, such as Fc fragments. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of coupling agents known in the art, but rather is exemplary of the more common coupling agents (see Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansene et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

[0242]

[0259] Linkers that can be applied in the present application have been described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester)). In some embodiments, non-peptide linkers used herein include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (PierceChem. Co. Cat. No. 21651G); (iii) SPDP (succinimidyl-6-[3-(2-pyridyldithio) (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamido]hexanoate (PierceChem. Co. Catalog No. 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamido]hexanoate (PierceChem. Co. Catalog No. 2165-G); and (v) sulfo-NHS (N-hydroxysulfosuccinimide: PierceChem. Co. Catalog No. 24510) conjugated to EDC.

[0243]

[0260] The linkers described above contain components with different properties, resulting in chimeric proteins with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically hindered disulfide bond, which can lead to chimeric proteins with increased stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in reduced chimeric protein yields. Sulfo-NHS, in particular, can enhance the stability of carbodiimide coupling. Carbodiimide coupling (e.g., EDC) combined with sulfo-NHS forms esters that are more resistant to hydrolysis than carbodiimide coupling alone.

[0244]

[0261] Any one or all of the linkers described herein may be peptide linkers. The peptide linkers may have naturally occurring or non-naturally occurring sequences. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. See, for example, WO 1996 / 34103.

[0245]

[0262] The peptide linker can be of any suitable length, hi some embodiments, the peptide linker is at least about n amino acids (aa) long, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100. In some embodiments, the peptide linker is about n aa in length or less, where n is selected from 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or less. In some embodiments, the peptide linker is 1 aa to about 10 aa, about 1 aa to about 20 aa, about 1 aa to about 30 aa, about 5 aa to about 15 aa, about 10 aa to about 25 aa, about 5 aa to about 30 aa, about 10 aa to about 30 aa, about 30 aa to about 50 aa, about 50 aa to about 100 aa, or about 1 aa to about 100 aa in length.

[0246]

[0263] In some embodiments, the peptide linker is a stable linker that is not cleaved by a protease. In one embodiment, the phosphopeptide linker is cleavable by a protease.

[0247]

[0264] In some embodiments, the peptide linker does not adopt a rigid three-dimensional structure, but rather tends to provide flexibility to the polypeptide. In some embodiments, the linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G) n (n≧1), glycine-serine polymers (e.g., GS (GS) n (n≧0 (SEQ ID NO: 80), (GSGGS) n (n≧1) (SEQ ID NO: 81), (GGGGS) n (n≧1) (SEQ ID NO: 82), and (GGGS) n (n≧1) (SEQ ID NO: 83), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can therefore function as neutral tethers between components. Glycine has access to much more phi-psi space than alanine and is much less restricted than residues with long side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those of ordinary skill in the art will recognize that the design of chimeric proteins can include fully or partially flexible linkers, such that the linker can include a flexible linker portion and one or more portions that confer less flexible structure to provide the desired chimeric protein structure.

[0248]

[0265] Natural linkers adopt various secondary conformations, such as helix, β-strand, coil / bend, and turn, to perform their functions. α-helical linkers function as rigid spacers that effectively separate protein domains, potentially reducing unfavorable interactions. Non-helical linkers with pro-rich sequences may increase the rigidity of the linker and reduce interdomain interference.

[0249]

[0266] For stability purposes, additional linkers may be used in the chimeric proteins of the present application. For example, in some embodiments, the linker stabilizes the chimeric protein. In some embodiments, the linker increases the serum half-life of the chimeric protein in vivo, the avidity of the chimeric protein for S protein in vitro, the number of chimeric proteins in vitro, and / or the effective amount of chimeric protein delivered to the nasal cavity in vivo. In some embodiments, the linker comprises an oligomerization or multimerization domain. In some embodiments, the oligomerization or multimerization domain is derived from a naturally occurring protein. In some embodiments, the oligomerization or multimerization domain is derived from a non-naturally occurring protein. Exemplary linkers (e.g., peptide linkers and domains contained in the linker) are shown in Table 9. Table 9. Exemplary peptide linkers and linker domains TIFF2025512512000036.tif250170TIFF2025512512000037.tif245170TIFF2025512512000038.tif239170

[0250]

[0267] Any of the linkers listed in Table 9 are compatible with the chimeric proteins provided herein. In some embodiments, the mucoadhesive peptide fragment is fused to the polypeptide chain of the target-binding moiety via any of the linkers provided in Table 9, or a variant thereof. In some embodiments, the mucoadhesive peptide fragment is fused to the polypeptide chain of the target-binding moiety via a linker comprising at least about 90% sequence identity (e.g., about n% sequence identity, where n% is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 63-83, 94, and 109. It should be understood that the linkers provided in Table 9 are exemplary and that other linkers having similar properties are similarly compatible with the chimeric proteins provided herein.

[0251]

[0268] In some embodiments, the peptide linker comprises an enzymatic tag, such as a detectable enzymatic tag. In some embodiments, the enzymatic tag functions as a dimer. In some embodiments, the enzymatic tag is alkaline phosphatase. In some embodiments, the enzymatic tag is glutathione-S-transferase (GST).

[0252]

[0269] Additional peptide linkers or useful domains to be included in the linkers described herein can be used to promote stable protein:protein interactions in the chimeric proteins of the invention. In some embodiments, the linker comprises a domain that promotes protein:protein interactions. In some embodiments, the linker comprises one or more heptad repeats. The term "heptad repeat" as used herein refers to a structural motif consisting of a repeating pattern of seven amino acids. In some embodiments, the heptad repeat comprises the repeating pattern: "HPPHCPC," where "H" represents a hydrophobic amino acid residue, "C" represents a typically charged amino acid residue, and "P" represents a polar (hydrophilic) amino acid residue. In some embodiments, the linker comprises a heptad repeat of a basic helix-loop-helix leucine zipper (bZIP) domain. In some embodiments, the linker comprises a heptad repeat of a basic isoleucine bZIP domain. In some embodiments, the heptad repeat forms a protein trimer.

[0253]

[0270] Glycine-XY repeats (e.g., GPP (GPP) n (n≧0) (SEQ ID NO: 73)) have been shown not to interfere with the functionality or safety profile of the chimeric protein. In some embodiments, the linker comprises one or more (GPP) n (n≧1) motif. In some embodiments, the linker comprises a collagen-like protein. In some embodiments, the collagen-like protein forms a protein trimer.

[0254]

[0271] Other higher-order multimerization domains may be incorporated into the linkers provided herein. In some embodiments, the multimerization domains may form self-assembled complexes. In some embodiments, the linker comprises an affinity moiety. In some embodiments, the linker comprises streptavidin (SA) protein. In some embodiments, the streptavidin protein forms a tetramer within a biotin molecule. In some embodiments, the linker comprises a dextran backbone domain. In some embodiments, the linker comprises an SA protein and a dextran backbone domain. In some embodiments, the linker comprises one or more maleimide polymers (DMGS). In some embodiments, the linker comprises one or more maleimide polymers and an SA protein. In some embodiments, the linker comprises a p53 tetramerization domain. In some embodiments, the linker comprises a bacteriophage T7 fibritin protein, or a portion thereof. In some embodiments, the linker comprises the C-terminal 27 amino acids of the bacteriophage T7 fibritin protein. In some embodiments, the C-terminal 27 amino acids of the bacteriophage T7 fibritin protein form a trimeric complex. In some embodiments, the linker comprises one or more coiled-coil structural domains. In some embodiments, the linker comprises cartilage oligomeric matrix protein (COMP) or a portion thereof. In some embodiments, the liner comprises the coiled-coil domain of COMP. In some embodiments, the coiled-coil domain of COMP forms a pentameric complex.

[0255] Antibody and Fc region linker

[0272] In some embodiments, the peptide linker comprises a constant region of a full-length antibody or a fragment thereof. The fragment refers to a fragment of the constant region of a full-length antibody. In some embodiments, the peptide linker comprises the entire constant region of a full-length antibody. In some embodiments, when the full-length antibody is IgG, IgA, or IgD, the peptide linker comprises a CH1, CH2, and CH3 domain. In some embodiments, when the full-length antibody is IgE or IgM, the peptide linker comprises a CH1, CH2, CH3, and CH4 domain. In some embodiments, the peptide linker comprises a fragment of the constant region of a full-length antibody. In some embodiments, the peptide linker comprises the constant region of the light chain of a full-length antibody or a fragment thereof. In some embodiments, the peptide linker comprises a CH1 domain or a fragment thereof. In some embodiments, the peptide linker comprises a CH2 domain or a fragment thereof. In some embodiments, the peptide linker comprises a CH3 domain or a fragment thereof. In some embodiments, the peptide linker comprises a CH4 domain or a fragment thereof. In some embodiments, the peptide linker comprises a CH L In some embodiments, the peptide linker further comprises an antibody hinge domain or a fragment thereof.

[0256]

[0273] The Fc region or a fragment thereof can be used as a peptide linker or a portion thereof. The terms "Fc region," "Fc domain," or "Fc" refer to the C-terminal non-antigen-binding region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region can be present or absent without affecting the structure or stability of the Fc region. Unless otherwise specified herein, the numbering of amino acid residues within an IgG or Fc region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The Fc region may promote dimerization and retain antibody-like properties, including physicochemical properties for expression, purification, and storage, as well as a long serum half-life in vivo. Such properties may be advantageous for the chimeric proteins provided herein. In some embodiments, the peptide linker comprises an Fc region or a fragment thereof. In some embodiments, the Fc region comprises CH2 and CH3 domains. In some embodiments, the Fc region comprises CH2, CH3, and CH4 domains.

[0257]

[0274] In some embodiments, the Fc fragment comprises an immunoglobulin IgG heavy chain constant region, including the hinge region (starting at Cys226), the IgG CH2 domain, and the CH3 domain. As used herein, the term "hinge region" or "hinge sequence" refers to the amino acid sequence located between the linker and the CH2 domain. In some embodiments, the chimeric protein comprises an Fc fragment comprising the hinge region. In some embodiments, the chimeric protein comprises an Fc fragment without the hinge region.

[0258]

[0275] In some embodiments, the peptide linker comprises an Fc fragment selected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. The Fc fragments of IgG', IgA', and IgD comprise CH2 and CH3, while the Fc fragments of IgE and IgM comprise CH2, CH3, and CH4. In some embodiments, the Fc fragment is derived from human IgG. In some embodiments, the Fc fragment comprises the Fc region of human IgG1, IgG2, IgG3, IgG4, or combination or hybrid IgG. In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the Fc fragment comprises the CH2 and CH3 domains of IgG1. In some embodiments, the Fc fragment is an IgG4 Fc fragment. In some embodiments, the Fc fragment comprises the CH2 and CH3 domains of IgG4. IgG4 Fc is known to have lower effector activity than IgG1 Fc and is therefore desirable for some applications. In some embodiments, the Fc fragment is derived from a mouse immunoglobulin.

[0259]

[0276] In some embodiments, the IgG CH2 domain begins at Ala231. In some embodiments, the CH3 domain begins at Gly341. It is understood that the C-terminal Lys residue of human IgG can optionally be deleted. It is also understood that conservative amino acid substitutions in the Fc region are contemplated within the scope of the present invention without affecting the desired structure and / or stability of the Fc.

[0260]

[0277] In some embodiments of the chimeric proteins disclosed herein, particularly those comprising an Fc region or a fragment thereof, such as CH2, the chimeric protein binds to or recruits a component of the complement system known as the C1 complex (C1qC1r2C1s2). Such recruitment can initiate a cleavage cascade involving C2, C3, C4, and C5, which can subsequently trigger microbial clearance. Microbial clearance can be achieved through the so-called "classical complement pathway," which relies on further downstream complement components, such as the membrane attack complex (MAC), thereby killing microbial targets, such as bacterial cells or enveloped viruses. See Mellorsetal., 2020. Microbial clearance can also be achieved through a C1- and C4-dependent antiviral mechanism that is independent of downstream complement components. With the help of C1 recruited by Fc or simply CH2 (a fragment of Fc), C4 directly inactivates the viral capsid and neutralizes the virus. See Bottermannetal., Cell Host & Microbe, 25:617-629 (2019). As used herein, the term "activation of the complement pathway" includes activation of the classical complement pathway and / or the C4-dependent antiviral pathway.

[0261]

[0278] Additionally, peptide linkers comprising any of the Fc variants known in the art or combinations thereof are contemplated. In some embodiments, the Fc fragment comprises a sequence that has been modified or otherwise altered to enhance C1 recruitment, complement-dependent cytotoxicity (CDC), or antibody-dependent cellular cytotoxicity (ADCC) effector function.

[0262]

[0279] In some embodiments, each chain of the Fc fragment is fused to the same entity. In some embodiments, the chimeric protein comprises two identical target-binding moieties described herein (e.g., the same ACE2 protein or fragment thereof), each fused to one chain of the Fc fragment. In some embodiments, the two chains of the Fc fragment are identical. In some embodiments, the chimera comprising the Fc fragment is a homodimer.

[0263]

[0280] In some embodiments, each chain of the Fc fragment is fused to a different entity. In some embodiments, the target binding moiety comprises two different target binding moieties (e.g., two different ACE2 proteins or fragments thereof), each fused to one chain of the Fc fragment. In some embodiments, the two target binding moieties are different, but both specifically recognize the S protein (e.g., the S1 subunit of the S protein). In some embodiments, the target binding is monovalent, i.e., only one target binding moiety is fused to one chain of the Fc fragment, and the second chain of the Fc fragment is not fused to a target binding moiety. In some embodiments, the target binding moiety comprising an Fc fragment is a heterodimer.

[0264]

[0281] Heterodimerization of non-identical polypeptides of target binding moieties can be promoted by methods known in the art, including, but not limited to, heterodimerization via knobs-into-hole technology. The structure and assembly methods of knobs-into-hole technology are described, for example, in U.S. Pat. Nos. 5,821,333 and 7,642,228, U.S. Patent Application Publication No. 2011 / 0287009, and PCT / US2012 / 059810, the entire contents of which are incorporated herein by reference. This technology was developed by substituting large amino acid residues for small amino acid residues in the CH3 domain of one Fc to introduce a "knob" (or protrusion), and substituting one or more large amino acid residues for small amino acid residues in the CH3 domain of the other Fc to introduce a "hole" (or cavity). In some embodiments, one chain of the Fc fragment in the chimeric protein constitutes the knob, and the second chain of the Fc fragment constitutes the hole.

[0265]

[0282] Preferred residues for forming the knob are commonly naturally occurring amino acid residues, preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Most preferred are tryptophan and tyrosine. In one embodiment, the original residues for forming the knob have small side chain volumes, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. Exemplary amino acid substitutions in the CH3 domain to form the knob include, but are not limited to, T366W, T366Y, or F405W substitutions.

[0266]

[0283] Preferred residues for hole formation are commonly naturally occurring amino acid residues, preferably selected from alanine (A), serine (S), threonine (T), and valine (V). In one embodiment, the original residue for hole formation has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. Exemplary amino acid substitutions in the CH3 domain to generate holes include, but are not limited to, T366S, L368A, F405A, Y407A, Y407T, and Y407V substitutions. In a specific embodiment, the knob comprises a T366W substitution and the hole comprises a T366S / L368A / Y407V substitution. It is understood that other modifications to the Fc region known in the art that promote heterodimerization are also contemplated and are encompassed by the instant application.

[0267] III. Methods of Prevention and Treatment, Killing Viruses, Neutralizing Viruses, and Activating the Complement Pathway

[0284] The present application further provides methods for preventing or treating an infection caused by a coronavirus in an individual, the method comprising administering to the individual an effective amount of any one of the chimeric proteins described herein or a cocktail composition of chimeric proteins described herein. In some embodiments, the method comprises administering to the individual a pharmaceutical composition, such as any of the pharmaceutical compositions provided herein, comprising an effective amount of any one of the chimeric proteins described herein or a cocktail composition of chimeric proteins described herein. In some embodiments, the chimeric protein comprises a peptide linker comprising an Fc region or a fragment thereof. In some embodiments, the peptide linker comprises a C1, C2, C3, C4, and / or C5 domain. Ldomain, or fragments thereof. In some embodiments, the peptide linker comprises a CH2 domain or fragment thereof. In some embodiments, the peptide linker further comprises an antibody hinge domain or fragment thereof. In some embodiments, the method is for preventing an infection caused by a coronavirus, such as SARS-CoV-2, including SARS-CoV-2 variants, in an individual. In some embodiments, the method is for treating an infection caused by a coronavirus, such as SARS-CoV-2, including SARS-CoV-2 variants, in an individual. In some embodiments, the method is for activating the complement pathway in an individual. In some embodiments, the method is an in vitro method for killing or neutralizing a coronavirus. In some embodiments, the method is for killing or neutralizing a coronavirus, such as SARS-CoV-2, including SARS-CoV-2 variants. In some embodiments, a method for preventing, treating, or reducing an infection caused by a coronavirus in an individual, wherein at least one virus is killed or neutralized on a mucosal surface. Also provided is the use of the chimeric proteins in the prevention or treatment of infectious diseases, and in the preparation of a medicament for the prevention or treatment of infectious diseases, for activating the complement pathway, or for killing or neutralizing viruses. Methods of veterinary use are also contemplated herein.

[0268]

[0285] In some embodiments, methods are provided for preventing or treating an infection caused by a coronavirus or a variant thereof that is transmitted via the mucosa in an individual, the method comprising administering to the individual an effective amount of a chimeric protein comprising: (a) a target-binding moiety comprising the EBD of the ACE2 protein or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to the mucosa.

[0269]

[0286] In some embodiments, methods are provided for activating the complement pathway in an individual infected with a coronavirus or a variant thereof, comprising administering to the individual an effective amount of a chimeric protein comprising: (a) a target binding moiety comprising the EBD of the ACE2 protein or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five positively charged amino acid residues, the mucoadhesive peptide fragment promoting adhesion of the chimeric protein to a mucosa. In some embodiments, the chimeric protein comprises a peptide linker comprising an Fc region or a fragment thereof. In some embodiments, the peptide linker comprises a C H1, C H2, C H3, C H4, and / or C H5 region. L In some embodiments, the peptide linker comprises a CH2 domain or a fragment thereof. In some embodiments, the peptide linker further comprises an antibody hinge domain or a fragment thereof. In some embodiments, at least one coronavirus is killed or neutralized on a mucosal surface.

[0270]

[0287] In some embodiments, methods are provided for killing or neutralizing coronavirus in an individual, comprising administering to the individual an effective amount of a chimeric protein comprising: (a) a target-binding moiety comprising the EBD of the ACE2 protein or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five positively charged amino acid residues, the mucoadhesive peptide fragment promoting adhesion of the chimeric protein to a mucosa. In some embodiments, the chimeric protein comprises a peptide linker comprising an Fc region or a fragment thereof. In some embodiments, the peptide linker comprises a C H1, C H2, C H3, C H4, and / or C H5 region. L In some embodiments, the peptide linker comprises a CH2 domain or a fragment thereof. In some embodiments, the peptide linker further comprises an antibody hinge domain or a fragment thereof. In some embodiments, at least one coronavirus is killed or neutralized on the mucosa. In some embodiments, killing or neutralization is achieved via activation of the complement pathway.

[0271]

[0288] In some embodiments, an in vitro method for killing or neutralizing coronavirus is provided, comprising contacting a coronavirus, in the presence of at least one component of the complement system, with a chimeric protein comprising: (a) a target-binding portion comprising the EBD of the ACE2 protein or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five positively charged amino acid residues, the mucoadhesive peptide fragment promoting adhesion of the chimeric protein to a mucosa. In some embodiments, at least one component of the complement system is C1, C4, or the membrane attack complex (MAC). In some embodiments, at least one component of the complement system is C1. In some embodiments, at least one component of the complement system is C4. In some embodiments, C4 is involved in virus neutralization. In some embodiments, at least one component of the complement system is MAC. In some embodiments, MAC is involved in virus killing. In some embodiments, the chimeric protein comprises a peptide linker comprising an Fc region or a fragment thereof. In some embodiments, the peptide linker comprises a CH1, CH2, CH3, CH4, and / or C5 domain. L In some embodiments, the peptide linker comprises a CH2 domain or a fragment thereof. In some embodiments, the peptide linker further comprises an antibody hinge domain or a fragment thereof. In some embodiments, at least one coronavirus is killed or neutralized on the mucosa. In some embodiments, killing or neutralization is achieved via activation of the complement pathway.

[0272]

[0289] In some embodiments, methods are provided for preventing, treating, or reducing an infection caused by a coronavirus in an individual, comprising administering to the individual a chimeric protein comprising: (a) a target-binding moiety comprising the EBD of the ACE2 protein or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five positively charged amino acid residues, the mucoadhesive peptide fragment promoting adhesion of the chimeric protein to a mucosa, wherein at least one coronavirus is killed or neutralized on the mucosa. In some embodiments, the chimeric protein comprises a peptide linker comprising an Fc region or a fragment thereof. In some embodiments, the peptide linker comprises a C1, C2, C3, C4, and / or C5 domain. L In some embodiments, the chimeric protein comprises a chimeric protein comprising a CH2 domain or a fragment thereof. In some embodiments, the peptide linker further comprises an antibody hinge domain or a fragment thereof. In some embodiments, the chimeric protein elicits an immune response in an individual. In some embodiments, the chimeric protein activates the complement pathway in an individual. In some embodiments, at least one coronavirus is killed or neutralized on the mucosa via activation of the complement pathway.

[0273]

[0290] In some embodiments, the chimeric protein is administered to an individual before the individual is exposed to a coronavirus or a variant thereof. In some embodiments, the chimeric protein is administered to an individual within about n hours after the individual is exposed to a coronavirus virus or a variant thereof, where n is selected from 72, 48, 36, 24, 12, 6, 4, or less. In some embodiments, administration of the chimeric protein to an individual protects the individual from infection by a coronavirus for about n days, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more. In some embodiments, the chimeric protein is administered topically to a mucosa. In some embodiments, the chimeric protein is administered via a nasal spray, inhaler, nebulizer, or eye drops. In some embodiments, the chimeric protein is administered to both nostrils of an individual. In some embodiments, the chimeric protein is administered every other day, once daily, or twice daily.

[0274]

[0291] In some embodiments, the individual is a mammal (e.g., a human, a non-human primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc. In some embodiments, the individual is under about 60 years of age (e.g., including under about n years of age, where n is selected from 50, 40, 30, 25, 20, 15, and 10). In some embodiments, the individual is about 60 years of age or older (e.g., including over about n years of age, where n is selected from 70, 80, 90, and 100). In some embodiments, the individual has not been exposed to a coronavirus virus or a variant thereof. In some embodiments, the individual is diagnosed with a coronavirus infection, such as a SARS-CoV-2 infection. In some embodiments, the individual is diagnosed with a SARS-CoV-2 variant infection (such as a variant selected from the group consisting of a B.1.1.7 variant, a B.1.351 variant, a B.1.526 variant, a B1.526.1 variant, a B1.617 variant, a B.1.617.1 variant, a B.1.617.2 variant, a B1.617.3 variant, a P.2 variant, a P.1 (B.1.1.28.1) variant, an A.23.1 variant, a CAL.20C variant, a B.1.427 variant, a B.1.429 variant, a B.1.525 variant, a P.1.351 variant, a BA.5.1.1, a BQ.1 variant, an XBB variant, an XBB.1.5 variant, and an XBB.1.16 variant). In some embodiments, the individual is at risk of developing severe symptoms of an infection (e.g., a coronavirus infection). In some embodiments, the individual has an underlying disease, such as cardiovascular disease, diabetes, a chronic respiratory disease, and / or cancer.

[0275]

[0292] In some embodiments, the methods are for preventing or treating infection with one or more coronavirus variants (e.g., SARS-CoV-2 variants). In some embodiments, the methods prevent or treat infection with multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) coronavirus variants. In some embodiments, the methods prevent or treat infection with multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) SARS-CoV-2 variants. In some embodiments, the one or more SARS-CoV-2 variants are selected from the group consisting of a B.1.1.7 variant, a B.1.351 variant, a B.1.526 variant, a B1.526.1 variant, a B1.617 variant, a B.1.617.1 variant, a B.1.617.2 variant, a B1.617.3 variant, a P.2 variant, a P.1 (B.1.1.28.1) variant, an A.23.1 variant, a CAL.20C variant, a B.1.427 variant, a B.1.429 variant, a B.1.525 variant, a P.1.351 variant, a BA.5.1.1, a BQ.1 variant, an XBB variant, an XBB.1.5 variant, and an XBB.1.16 variant.

[0276]

[0293] In some embodiments, methods are provided for treating or preventing infection in an individual with multiple coronavirus variants (e.g., SARS-CoV-2 variants), comprising administering to the individual an effective amount of a pharmaceutical composition (e.g., a cocktail composition) comprising multiple chimeric proteins, each of the multiple chimeric proteins comprising: (a) a target-binding moiety comprising the EBD of the ACE2 protein or a fragment thereof that specifically binds to the S protein; and (b) a mucoadhesive peptide fragment comprising at least about five positively charged amino acid residues, wherein the mucoadhesive peptide fragment promotes adhesion of the chimeric protein to a mucosa.

[0277]

[0294] In some embodiments, each of the multiple chimeric proteins comprises a different target-binding moiety that specifically recognizes a different coronavirus variant (e.g., a SARS-CoV-2 variant). For example, a pharmaceutical composition can include a cocktail of chimeric proteins, each comprising a target-binding fragment derived from a different ACE2 protein or fragment thereof described herein or known in the art. In some embodiments, the mucoadhesive peptide fragment comprises at least five positively charged amino acid residues interspersed with one or more non-positively charged amino acid residues. In some embodiments, the chimeric protein is administered via a nasal spray. In some embodiments, a chimeric protein, e.g., any of the chimeric proteins comprising an ACE2 peptide or fragment thereof of the present application, is administered as a single agent or in combination with a second, third, or fourth agent (including, e.g., an antiviral agent, convalescent plasma, an anti-inflammatory agent, etc.) to treat an infection, kill a virus, neutralize a virus, and / or activate the complement pathway.

[0278]

[0295] The effectiveness of treatment can be assessed, for example, by viral load (e.g., via detection of viral DNA), survival time, quality of life, viral protein expression and / or activity, detection of serological antibodies to the coronavirus or its variants, assessment of respiratory function, and / or computed tomography (CT) imaging.

[0279] IV. Nucleic acids and preparation methods

[0296] Nucleic acid molecules encoding the chimeric proteins described herein (e.g., chimeric proteins comprising ACE2 or a fragment thereof) are contemplated. In some embodiments, the nucleic acid molecule encodes a chimeric protein described herein. In some embodiments, a nucleic acid (e.g., an isolated nucleic acid) encoding any of the chimeric proteins described herein is provided. In some embodiments, a nucleic acid (e.g., an isolated nucleic acid) encodes the complete amino acid sequence or sequences of any of the chimeric proteins described herein. In some embodiments, a set of nucleic acids (e.g., a set of isolated nucleic acids) encoding any of the chimeric proteins described herein is provided. For example, different polypeptides of a chimeric protein, such as any of the chimeric proteins described herein, can be encoded by different nucleic acids (e.g., isolated nucleic acids) within a set of nucleic acids (e.g., a set of isolated nucleic acids). Nucleic acid molecules can be constructed using recombinant DNA techniques routine in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0280]

[0297] Also provided are vectors comprising a polynucleotide encoding a chimeric protein described herein. In some embodiments, the vector comprises a polynucleotide encoding a chimeric protein described herein. In some embodiments, the vector comprises a nucleic acid encoding any of the chimeric proteins described herein. In some embodiments, the vector comprises a nucleic acid encoding the complete amino acid sequence or sequences of any of the chimeric proteins described herein. In some embodiments, a set of vectors is provided comprising different nucleic acids encoding different polypeptides of the chimeric proteins described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc.

[0281]

[0298] In various embodiments, the chimeric proteins described herein can be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast cells), plant cells, insect cells, and mammalian cells. In some embodiments, the chimeric proteins described herein can be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast cells), plant cells, insect cells, and mammalian cells. In some embodiments, a nucleic acid encoding any of the chimeric proteins described herein can be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast cells), plant cells, insect cells, and mammalian cells. In some embodiments, a set of nucleic acids encoding any of the chimeric proteins described herein can be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast cells), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells (including COS7 cells); 293 cells (including 293-6E cells); CHO cells (including CHO-S, DG44.Lec13 CHO cells, and FUT8 CHO cells); PER.C6® cells (Crucell); and NSO cells.

[0282]

[0299] Introduction of one or more nucleic acids into a desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd Cold Spring Harbor Laboratory Press, 2001. The nucleic acid may be transiently or stably transfected into the desired host cell according to any suitable method.

[0283]

[0300] The present invention also provides host cells comprising any of the nucleic acids or vectors described herein. In some embodiments, the present invention provides host cells comprising the chimeric proteins described herein. In some embodiments, the present invention provides host cells comprising the chimeric proteins described herein. In some embodiments, the present invention provides host cells comprising nucleic acids encoding any of the chimeric proteins described herein. In some embodiments, the nucleic acids encode the complete amino acid sequence or sequences of any of the chimeric proteins described herein. In some embodiments, the present invention provides host cells comprising a set of nucleic acids encoding any of the chimeric proteins described herein. In some embodiments, the present invention provides host cells comprising vectors comprising nucleic acids encoding any of the chimeric proteins described herein. In some embodiments, the vectors comprise nucleic acids encoding the complete amino acid sequence or sequences of any of the chimeric proteins described herein. In some embodiments, the present invention provides host cells comprising a set of vectors comprising different nucleic acids encoding different polypeptides of any of the chimeric proteins described herein. For the purpose of isolating genes encoding polypeptides of interest, any host cell capable of overexpressing heterologous DNA can be used. Non-limiting examples of mammalian host cells include, but are not limited to, COS cells, HeLa cells, and CHO cells. Suitable non-mammalian host cells include prokaryotes (such as E. coli or Bacillus subtilis) and yeast (such as S. cerevisiae, S. pombe; or K. lactis).

[0284]

[0301] The chimeric proteins described herein, isolated nucleic acids encoding any of the chimeric proteins described herein, and / or sets of isolated nucleic acids encoding any of the chimeric proteins described herein can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ligands that bind to the target-binding moieties described herein. For example, Protein A, Protein G, Protein A / G, or affinity columns can be used to bind the target-binding moieties and purify the chimeric proteins. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be suitable for purifying some polypeptides, such as inhibitory polypeptides. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be suitable for purifying some polypeptides. Mixed-mode chromatography (e.g., reversed-phase / anion exchange, reversed-phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be suitable for purifying some polypeptides. Many methods for purifying polypeptides are known in the art.

[0285] V. Pharmaceutical Compositions, Kits, and Articles of Manufacture A. Pharmaceutical Compositions

[0302] One aspect of the present application provides a composition (e.g., a pharmaceutical composition) comprising any one of the chimeric proteins described herein. In some embodiments, the pharmaceutical composition is suitable for nasal administration. In some embodiments, the pharmaceutical composition is suitable for respiratory (e.g., upper airway) administration. In some embodiments, the pharmaceutical composition is suitable for administration by inhalation. In some embodiments, the pharmaceutical composition is a nasal spray formulation.

[0286]

[0303] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a citrate buffered saline carrier. In some embodiments, the pharmaceutical composition comprises a stabilizer, viscosity enhancer, surfactant, and / or preservative. In some embodiments, the pharmaceutical composition comprises 25 mM citrate buffer (pH 6.5), 100 mM NaCl, 0.1% methionine, 0.02% polysorbate 80, and 0.1% potassium sorbate. In some embodiments, the pharmaceutical composition comprises 25 mM citrate buffer (pH 6.5), 125 mM NaCl, 5% glycerin, 0.1% methionine, 0.02% polysorbate 80, and 0.1% potassium sorbate.

[0287]

[0304] In some embodiments, the pharmaceutical composition comprises one chimeric protein. In some embodiments, the pharmaceutical composition comprises at least two chimeric proteins, wherein the two chimeric proteins have different target-binding moieties. In some embodiments, the pharmaceutical composition comprises a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) chimeric proteins, wherein the target-binding moieties of the chimeric proteins differ from one another. In some embodiments, the pharmaceutical composition comprises a cocktail of chimeric proteins that target different components of the same coronavirus and / or the same component of different variants (e.g., strains) of coronavirus. In some embodiments, each of the chimeric proteins in the cocktail composition comprises one or more of the same mucoadhesive peptide fragments. In some embodiments, each of the chimeric proteins in the cocktail composition comprises one or more different mucoadhesive peptide fragments.

[0288]

[0305] In some embodiments, the pharmaceutical composition is formulated for topical administration to mucous membranes, such as nasal mucosa, laryngeal mucosa, tracheal mucosa, bronchial mucosa, pulmonary mucosa, ocular mucosa, and combinations thereof. In some embodiments, the pharmaceutical composition is formulated for administration via nasal spray, inhaler, nebulizer, or eye drops.

[0289]

[0306] In some embodiments, a pharmaceutical composition is provided, comprising: (a) a chimeric protein comprising a target-binding moiety including the EBD of an ACE2 protein or a fragment thereof that specifically binds to the S protein; and a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30, e.g., about 12) positively charged amino acid residues (e.g., lysine, histidine, arginine, ornithine, or a combination thereof) that promotes adhesion of the chimeric protein to the mucosa; (b) a stabilizer that maintains the mildly reducing environment of the nose; (c) a buffering agent; and (d) an osmolality adjuster, wherein the pharmaceutical composition has a pH of about 4.5 to about 7.5 (e.g., about 6.0 to about 7.0) and an osmolality of about 230 to about 330 Osm / kg (e.g., about 250 to about 300 Osm / kg). In some embodiments, the S protein is derived from a coronavirus (e.g., ASARS-CoV-2 virus). In some embodiments, the target-binding moiety specifically binds to the S1 subunit of the S protein. In some embodiments, the target binding moiety is any one of the target binding moieties described in Section II. In some embodiments, the chimeric protein is any one of the chimeric proteins described in Section II. In some embodiments, the positively charged amino acid residues are interspersed with one or more non-positively charged amino acid residues. In some embodiments, the pharmaceutical composition further comprises a viscosity enhancing agent. In some embodiments, the pharmaceutical composition further comprises a surfactant. In some embodiments, the pharmaceutical composition further comprises a preservative.

[0290]

[0307] In some embodiments, a pharmaceutical composition for nasal administration is provided, comprising: (a) a chimeric protein comprising a target-binding portion comprising the EBD of an ACE2 protein or a fragment thereof that specifically binds to the S protein; and a mucoadhesive peptide fragment comprising at least about five (e.g., about 5 to about 30, e.g., about 12) positively charged amino acid residues (e.g., lysine, histidine, arginine, ornithine, or a combination thereof) that promotes adhesion of the chimeric protein to a mucosa; (b) methionine; (c) a buffering agent; (d) an osmolality adjusting agent; (e) a viscosity-enhancing agent; (f) a surfactant; and (g) a preservative; the pharmaceutical composition has a pH of about 4.5 to about 7.5 (e.g., about 6.0 to about 7.0) and an osmolality of about 230 to about 330 Osm / kg (e.g., about 250 to about 300 Osm / kg). In some embodiments, the S protein is derived from a coronavirus (e.g., ASARS-CoV-2 virus). In some embodiments, the target binding moiety specifically binds to the S1 subunit of the S protein. In some embodiments, the target binding moiety is any one of the target binding moieties described in Section II. In some embodiments, the chimeric protein is any one of the chimeric proteins described in Section II. In some embodiments, the positively charged amino acid residues are intermixed with one or more non-positively charged amino acid residues.

[0291]

[0308] In some embodiments, a pharmaceutical composition for nasal administration is provided, the pharmaceutical composition comprising: (a) a chimeric protein comprising a target-binding moiety that includes the EBD of an ACE2 protein or a fragment thereof that specifically binds to the S protein; and a mucoadhesive peptide fragment that includes at least about five (e.g., about 5 to about 30, e.g., about 12) positively charged amino acid residues (e.g., lysine, histidine, arginine, ornithine, or a combination thereof) that promotes adhesion of the chimeric protein to a mucosa; (b) methionine; (c) a citrate buffer; and (d) NaCl, the pharmaceutical composition having a pH of about 4.5 to about 7.5 (e.g., about 6.0 to about 7.0) and an osmolality of about 230 to about 330 Osm / kg (e.g., about 250 to about 300 Osm / kg). In some embodiments, the S protein is derived from a coronavirus (e.g., ASARS-CoV-2 virus). In some embodiments, the target-binding moiety specifically binds to the S1 subunit of the S protein. In some embodiments, the target binding moiety is any one of the target binding moieties described in Section II. In some embodiments, the chimeric protein is any one of the chimeric proteins described in Section II. In some embodiments, the positively charged amino acid residues are interspersed with one or more non-positively charged amino acid residues. In some embodiments, the pharmaceutical composition further comprises a viscosity enhancing agent (e.g., glycerin). In some embodiments, the pharmaceutical composition further comprises a surfactant (e.g., polysorbate 80). In some embodiments, the pharmaceutical composition further comprises a preservative (e.g., potassium sorbate).

[0292]

[0309] In some embodiments, a pharmaceutical composition for nasal administration comprises: (a) a chimeric protein comprising a target-binding portion comprising the EBD of an ACE2 protein or a fragment thereof that specifically binds to an S protein, and a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30, e.g., about 12) positively charged amino acid residues (e.g., lysine, histidine, arginine, ornithine, or a combination thereof), which promotes adhesion of the chimeric protein to a mucosa at a concentration of about 0.6 mg / mL to about 1 mg / mL (e.g., about 1 mg / mL to about 3 mg / mL); and (b) about 0.05% to about 0.2% (e.g., about 0.075% to about 0.125%) (w / w) of a chimeric protein comprising a target-binding portion comprising the EBD of an ACE2 protein or a fragment thereof that specifically binds to an S protein, and a mucoadhesive peptide fragment comprising at least about 5 (e.g., about 5 to about 30, e.g., about 12) positively charged amino acid residues (e.g., lysine, histidine, arginine, ornithine, or a combination thereof), which promotes adhesion of the chimeric protein to a mucosa at a concentration of about 0.6 mg / mL to about 1 mg / mL (e.g., about 1 mg / mL to about 3 mg / mL). (c) about 20 mM to about 50 mM (e.g., about 20 mM to about 30 mM) citrate buffer, (d) about 100 mM to about 150 mM (e.g., about 110 mM to about 130 mM) NaCl, (e) about 1% to about 10% (e.g., about 2.5% to about 7.5%) (w / w) glycerin, (f) about 0.01% to about 0.1% (e.g., about 0.01% to 0.05%) (w / w) polysorbate 80, and (g) about 0.05% to about 0.2% (e.g., about 0.075% to about 0.125%) (w / w) potassium sorbate, and having a pH of about 4.5 to about 7.5 (e.g., about 6.0 to about 7.0). In some embodiments, the pharmaceutical composition comprises about 25 mM citrate (pH 6.5), about 125 mM NaCl, about 5% glycerin, about 0.1% methionine, about 0.02% polysorbate 80, and about 0.1% potassium sorbate. In some embodiments, the S protein is derived from a coronavirus (e.g., ASARS-CoV-2 virus). In some embodiments, the target binding moiety specifically binds to the S1 subunit of the S protein. In some embodiments, the target binding moiety is any one of the target binding moieties described in Section II. In some embodiments, the chimeric protein is any one of the chimeric proteins described in Section II. In some embodiments, the positively charged amino acid residues are intermixed with one or more non-positively charged amino acid residues.

[0293]

[0310] In some embodiments, the pharmaceutical compositions described herein are for administration via nasal spray. In some embodiments, the pharmaceutical compositions are for prophylactic use. In some embodiments, the pharmaceutical compositions maintain the stability (including physical and chemical stability) of the target-binding moiety at 37°C for at least about n days, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days, including any values ​​and ranges therebetween. In some embodiments, the pharmaceutical composition promotes adhesion of the target-binding moiety to mucous membranes, such as the nasal mucosa. In some embodiments, the pharmaceutical composition extends the residence time of the target-binding moiety in the nares and other upper respiratory tract regions by at least about n, e.g., compared to the target-binding moiety in PBS, where n is selected from 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more. In some embodiments, the pharmaceutical composition is neutral and gentle on nasal surfaces. In some embodiments, the pharmaceutical composition is a solution of the target binding moiety. In some embodiments, the pharmaceutical composition is an aqueous solution.

[0294]

[0311] Nasal spray pharmaceutical composition parameters and excipients are described, for example, in Kulkami, Candshaw, D., Inhalation, 10-11 (2021); Thorat, Scholars Journal of Applied Medical Sciences (SJAMS), 4(8D):2976-2985 (2016), which are incorporated by reference in their entireties. Excipients commonly used in nasal spray pharmaceutical compositions include, but are not limited to, tonicity or osmolality adjusting agents, buffers, purging agents, preservatives, surfactants, chelating agents, suspending agents, cosolvents, antioxidants, and humectants. Pharmaceutical compositions for various routes of administration, including nasal pharmaceutical compositions, are described, for example, in Cui Y. et al., Drug Development and Industrial Pharmacy, 11:28 (2017), which is incorporated by reference herein. Any excipient that complies with FDA guidelines for nasal spray pharmaceutical compositions and / or pharmaceutical compositions may be used herein.

[0295]

[0312] In some embodiments, the pharmaceutical composition has a pH that is compatible with the nasal environment. The average reference pH for the human nose is about 6.3. The optimal pH of a pharmaceutical composition also depends on factors such as the pI of the target-binding moiety (including positively charged mucoadhesive peptides), protein stability, and the net charge of the target-binding moiety. In some embodiments, the pharmaceutical composition has a pH of about 4.5 to about 7.5, e.g., a pH of about n, where n is selected from 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, and 7.5, including any value or range therebetween. In some embodiments, the pharmaceutical composition has a pH of about n, where n is selected from 4.5-5.0, 5.0-5.5, 5.5-6.0, 6.0-6.5, 4.5-5.5, 5.5-6.5, 5.0-6.5, 4.5-6.0, 6.5-7.0, 7.0-7.5, 6.0-7.5, 5.5-7, 6-7, and 6.5-7.5. In some embodiments, the pharmaceutical composition has a pH of about 6.5.

[0296]

[0313] Physiologically acceptable acids, bases, salts, and combinations thereof can be used to adjust and buffer the pH. Suitable additives for lowering the pH or as an acidic component of a buffer system are strong mineral acids, particularly sulfuric acid and hydrochloric acid. Furthermore, medium-strength inorganic and organic acids and acid salts, such as phosphoric acid, citric acid, tartaric acid, succinic acid, fumaric acid, methionine, acidic hydrogen phosphate with sodium or potassium, lactic acid, glucuronic acid, and the like, can be used. Suitable additives for raising the pH or as a basic component of a buffer system include, in particular, mineral bases, such as sodium hydroxide or other alkali and alkaline earth hydroxides and oxides, including, in particular, magnesium hydroxide and calcium hydroxide, basic ammonium salts such as ammonium hydroxide and ammonium acetate, basic amino acids such as lysine, carbonates such as sodium carbonate or magnesium carbonate, sodium bicarbonate, and citrates such as sodium citrate.

[0297]

[0314] In some embodiments, the pharmaceutical composition comprises a citrate buffer. In some embodiments, the citrate buffer comprises citric acid and sodium citrate. The citrate buffer has a pKa of about 6.4. In some embodiments, the citrate buffer is present at a concentration of about 20 mM to about 50 mM, e.g., about nmM, where n is selected from 20, 25, 30, 35, 40, 45, and 50, including any value or range therebetween. In some embodiments, the citrate buffer is present at a concentration of about nmM, where n is selected from 20-30, 30-40, 40-50, 25-50, 25-35, and 25-40. In some embodiments, the pharmaceutical composition comprises about 25 mM citrate buffer.

[0298]

[0315] In some embodiments, the pharmaceutical composition comprises a phosphate buffer, which has a pKa of about 7.2.

[0299]

[0316] In some embodiments, the pharmaceutical composition has an osmolality that approximates the nasal environment. In some embodiments, the pharmaceutical composition has an osmolality that facilitates adhesion of the target-binding moiety to mucous membranes (e.g., nasal mucosa). In some embodiments, the pharmaceutical composition minimizes penetration of the target-binding moiety into the bloodstream. In some embodiments, the pharmaceutical composition has an osmolality of about 230 Osm / kg to about 330 Osm / kg, e.g., about n Osm / kg, where n is selected from 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, and 330, including any value or range therebetween. In some embodiments, the pharmaceutical composition has an osmolality of about n Osm / kg, where n is selected from 230-250, 250-270, 270-290, 290-310, 310-330, 230-275, 275-300, 300-330, 230-280, 280-330, or 260-320. In some embodiments, the pharmaceutical composition has an osmolality of about 280 Osm / kg. One of skill in the art can readily convert these osmolality values ​​to osmolality.

[0300]

[0317] In some embodiments, the pharmaceutical composition includes a osmotic adjuster. Exemplary osmotic adjusters or isotonicity adjusters include, but are not limited to, sodium chloride, sodium sulfate, sodium phosphate, calcium chloride, calcium sulfate, calcium phosphate, magnesium chloride, magnesium sulfate, or magnesium phosphate. In some embodiments, the osmotic adjuster is sodium chloride. Calcium and magnesium salts can have a positive or supporting effect on the inhalation of the active agent solution, possibly by attenuating local irritation caused by administration. Alternatively, physiologically safe organic compounds can be used as osmotic adjusters. Particularly suitable are water-soluble substances with a relatively low molecular weight, e.g., 300 or less, more preferably 200 or less, and correspondingly high osmotic activity. Examples of such additives are sugars and sugar alcohols, particularly trehalose, mannitol, sorbitol, and isomalt.

[0301]

[0318] In some embodiments, the pharmaceutical composition comprises about 100 mM to about 150 mM NaCl, e.g., about nmM NaCl, where n is selected from 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, and 150, including any value or range therebetween. In some embodiments, the pharmaceutical composition comprises about nmM NaCl, where n is selected from 100-120, 120-140, 100-125, 125-150, 130-150, and 110-130. In some embodiments, the pharmaceutical composition comprises about 125 mM NaCl.

[0302]

[0319] In some embodiments, the pharmaceutical composition comprises one or more stabilizers. In some embodiments, the stabilizers maintain the mildly reducing environment of the nose. In some embodiments, the one or more stabilizers comprise methionine. In some embodiments, the one or more stabilizers comprise glycerin. In some embodiments, the one or more stabilizers comprise trehalose, e.g., 10% trehalose. In some embodiments, the pharmaceutical composition comprises about 0.05% (w / w) to about 0.2% (w / w) methionine, e.g., about n (w / w) methionine, where n is selected from 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, and 0.2%, including any value or range therebetween. In some embodiments, the pharmaceutical composition comprises about n (w / w) methionine, where n is selected from 0.05%-0.1%, 0.75%-1.25%, 0.1%-0.15%, 0.15%-0.2%, 0.1%-0.2%, 0.125-0.175%, 0.8%-1.6%, and 0.5%-0.15%. In some embodiments, the pharmaceutical composition comprises about 0.1% methionine.

[0303]

[0320] In some embodiments, the pharmaceutical composition comprises a viscosity enhancing agent. In some embodiments, the viscosity enhancing agent is selected from the group consisting of glycerin, dextran, and hydroxyethylcellulose. In some embodiments, the viscosity enhancing agent is glycerin. In some embodiments, the pharmaceutical composition comprises about 1% (w / w) to about 10% (w / w) glycerin, e.g., about n (w / w) glycerin, where n is selected from 1%, 2%, 3%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, including any value or range therebetween. In some embodiments, the pharmaceutical composition comprises about n (w / w) glycerin, where n is selected from 1%-4%, 2%-6%, 3%-7%, 5%-8%, 7%-10%, 2.5%-7.5%, 4%-6%, 1%-2.5%, 2.5%-5%, 5%-7.5%, and 7.5%-10%. In some embodiments, the pharmaceutical composition comprises about 5% glycerin.

[0304]

[0321] In some embodiments, the pharmaceutical composition comprises a surfactant. In some embodiments, the surfactant allows the target-binding moiety to cross mucous membranes (e.g., nasal mucosa) and / or allows the target-binding moiety to be absorbed across the mucous membrane. Suitable surfactants include, in particular, those considered safe for oral or nasal inhalation or mucosal administration. Examples of surfactants with particularly good physiological compatibility include tyloxapol, polysorbates (such as polysorbate 20 and polysorbate 80), PEG 400, PEG 3500, polyoxyl 400 stearate, vitamin E-TPGS, and macrogol hydroxystearates, such as macrogol-15-hydroxystearate. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.01% (w / w) to about 0.1% (w / w) polysorbate 80, e.g., about n (w / w) polysorbate 80, where n is selected from 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, and 0.1%, including any value or range therebetween. In some embodiments, the pharmaceutical composition comprises about n (w / w) polysorbate 80, where n is selected from 0.01%-0.02%, 0.02%-0.05%, 0.05%-0.1%, 0.01%-0.05%, 0.02%-0.04%, 0.04%-0.08%, 0.02%-0.08%, and 0.02%-0.1%. In some embodiments, the pharmaceutical composition comprises about 0.02% polysorbate.

[0305]

[0322] In some embodiments, the pharmaceutical composition comprises a preservative. In some embodiments, the preservative maintains the sterility of the pharmaceutical composition. Exemplary preservatives include, but are not limited to, benzyl alcohol, benzalkonium chloride, chlorobutanol, methylparaben, phenylethyl alcohol, propylparaben, and potassium sorbate. In some embodiments, the preservative is potassium sorbate. In some embodiments, the pharmaceutical composition comprises about 0.05% (w / w) to about 0.2% (w / w) potassium polysorbate, for example, about n (w / w) potassium polysorbate, where n is selected from 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, and 0.2% (w / w), including any value or range therebetween. In some embodiments, the pharmaceutical composition comprises about n (w / w) potassium sorbate, where n is selected from 0.05%-0.1%, 0.75%-1.25%, 0.1%-0.15%, 0.15%-0.2%, 0.1%-0.2%, 0.125-0.175%, 0.8%-1.6%, and 0.5%-0.15%. In some embodiments, the pharmaceutical composition comprises about 0.1% potassium sorbate.

[0306]

[0323] In some embodiments, the target binding moiety is present in the pharmaceutical composition at a concentration of about 0.6 mg / mL to about 6 mg / mL, e.g., about nmg / mL, where n is selected from 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6, including any value or range therebetween. In some embodiments, the target binding moiety is present in the pharmaceutical composition at a concentration of about nmg / mL, where n is selected from 0.6-1, 1-2, 2-3, 3-4, 4-5, 5-6, 0.6-2.5, 2.5-5, 2-4, 4-6, 0.6-3, 3-6, and 2-5.

[0307]

[0324] Nasal sprays containing inhibitory polypeptides (e.g., ACE2 protein or fragments thereof) can be used as vaccines, therapeutics, and complement other preventive measures against the spread of coronaviruses (e.g., SARS-CoV-2). Nasal sprays offer many advantages. One advantage of using chimeras in prophylactic nasal sprays is the established manufacturing process and scalability. Another advantage is that the inhibitory polypeptides used in nasal sprays require much smaller amounts (approximately 10,000-fold less) than therapeutics. This significantly reduces costs and allows for wider use. Furthermore, the use of human ACE2 inhibitory polypeptides significantly reduces the risk of immunogenicity, an important consideration for prophylactic nasal spray pharmaceutical compositions that are used repeatedly over long periods of time. The modified inhibitory polypeptides in nasal spray pharmaceutical compositions are stable at room temperature for extended periods, facilitating routine use and storage.

[0308]

[0325] In some embodiments, the pharmaceutical composition (e.g., a nasal spray pharmaceutical composition or an eye drop pharmaceutical composition) is administered at a dose of about 0.1 mg to about 1 mg of the target-binding moiety, e.g., per nostril or per eye. In some embodiments, about 100 μL of the pharmaceutical composition (e.g., a nasal spray pharmaceutical composition or an eye drop pharmaceutical composition) is administered at a time, e.g., to both nostrils of an individual (e.g., 100 μL per nostril or 100 μL per eye).

[0309]

[0326] Suitable pharmaceutical compositions can be obtained in the form of a lyophilized pharmaceutical composition or aqueous solution by mixing one or more chimeric proteins having the desired purity as described herein with any pharmaceutically acceptable carrier, additive, or stabilizer (Remington's Pharmaceutical Sciences 23rd edition, Adejare, A. Ed. (2020)). Acceptable carriers, additives, or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. azoles); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as pol...

Claims

[Claim 1] It is a chimeric protein, (a) A target binding site comprising the extracellular binding domain (EBD) or a fragment thereof of the angiotensin-converting enzyme 2 (ACE2) protein that specifically binds to the spike (S) protein; and (b) A mucosal-adhering peptide fragment comprising at least about five positively charged amino acid residues, which promotes the adhesion of chimeric proteins to mucosa. A chimeric protein containing [this protein].

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