Binding proteins useful against ACE2-targeted viruses

Abstract

Described herein are binding proteins useful for ACE2-targeting viruses (e.g., SARS-CoV and SARS-CoV-2) and methods for using them. These binding proteins can include the extracellular portion of angiotensin-converting enzyme 2 (ACE2), excluding the collectrin domain, and a flexible polypeptide flexible linker connecting the ACE2 portion to the fragment crystallizable (Fc) domain. These binding proteins dimerize, and the flexible linker can be selected to be of sufficient length to allow simultaneous interaction with multiple spike (S) proteins on ACE2-targeting viruses.

Description

[Technical Field]

[0001] Cross-reference of related applications This patent application claims priority to U.S. Provisional Patent Application No. 63 / 004,823, titled "BINDING PROTEINS USEFUL AGAINST SARS-LIKE CORONAVIRUSES" (filed April 3, 2020), which is incorporated herein by reference in its entirety.

[0002] Built-in based on reference All publications and patent applications referenced herein are incorporated in whole as much as each individual publication and patent application is specifically and individually indicated for reference.

[0003] field This specification describes binding proteins that bind to severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2). These binding proteins may be flexible ACE2 decoys, which may be used in pharmaceutical compositions and in methods thereof to treat subjects suffering from SARS-CoV and / or SARS-CoV-2 infection. [Background technology]

[0004] background The SARS-CoV-2 pandemic had unprecedented and devastating effects on global societies and economies, marking the third known zoonotic transmission of a highly pathogenic coronavirus to a human population. While previous coronavirus outbreaks, SARS-CoV and MERS-CoV, highlighted the need for clinically available therapeutic or preventive interventions, no proven treatments are currently available. Developing effective intervention strategies relies on knowledge of the molecular and cellular mechanisms of coronavirus infection, highlighting the importance of studying virus-host interactions at the molecular level to identify targets for antiviral interventions and to uncover key viral and host determinants that determine the progression of severe illness.

[0005] Mucosal barriers play a crucial potential protective role as barriers preventing exogenous substances from entering the body. Mucosal defense can be further enhanced by local immunity, which elicits a robust immune response in the mucosa of the intestines, urogenital tract, and respiratory system (i.e., surfaces in contact with the external environment). While the mucosal immune system can provide protection against pathogens, it can maintain resistance to harmless symbiotic microorganisms and benign environmental substances. Mucosa is the primary point of contact between the host and its environment, where a vast amount of secondary lymphoid tissue is found. Mucosa-associated lymphoid tissue, or MALT, provides an important component of the mucosal immune response. The mucosal immune system provides three main functions: first-line defense of the body against antigens and infections, suppression of systemic immune responses to symbiotic bacteria and food antigens (primarily food proteins in gastrointestinal lymphoid tissue, so-called oral tolerance), and regulation of appropriate immune responses to commonly encountered pathogens.

[0006] Unfortunately, mucosal immune responses are insufficient and often difficult to elicit the necessary immune applications over a sufficient period of time. See, for example, U.S. Patent Application Publication No. 2015 / 0284451. Some antibodies have been shown to immobilize them in mucus (often a process called a mucus trap) by interacting with mucin and adhesively cross-linking pathogens covered by individual antibodies to mucin, but it would be beneficial to provide antibodies or antibody constructs with an even more improved ability to more effectively prevent exogenous substances (including viruses) from penetrating the mucosa and reaching target cells. Specifically, it would be beneficial to provide binding proteins such as antibodies that can assist in aggregating and / or restraining exogenous objects together in a way that limits their effective penetration through the mucosa.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0008] Summary of the Disclosure Methods and compositions are described herein for enhancing the aggregation, restraint, and / or mucus trapping of one or more ACE2-targeted viruses (such as SARS-CoV and SARS-CoV-2, etc.) and reducing the proportion of ACE2-targeted viruses that can penetrate through mucus. Specifically, genetically engineered binding proteins useful against ACE2-targeted viruses are described herein. These binding proteins may be multivalent for ACE2-targeted viruses and may include two coronavirus binding regions each flexibly linked to an Fc domain by a flexible polypeptide linker. The linker may be long and flexible enough so that both coronavirus binding regions can bind simultaneously to a target (such as a spike protein).

[0009] For example, angiotensin-converting enzyme 2 (ACE2)-immunoglobulin (IgG) hybrid binding proteins (also referred to herein as flexible-linked ACE2 decoys) that dimerize and have picomolar affinity for ACE2-targeted viruses (including, in particular, SARS-CoV-2) are described herein. These proteins may be genetically engineered for "mucus trapping" and may be used to treat or prevent SARS-CoV (e.g., SARS-CoV-2) infection, for example, for topical immunotherapy against targeted viruses including SARS-CoV-2ACE2. These molecules may generally be formed by linking two or more extracellular components of ACE2 (e.g., components of soluble angiotensin-converting enzyme 2) to the Fc portion using a mobile linker (e.g., (GGGGS)n, (EAAAK)n, etc.).

[0010] In some examples described herein, the extracellular portion of ACE2 may correspond to the wild-type extracellular fragment of ACE2; however, an extracellular fragment of ACE2 modified by one or more alterations (mutations) may be used as the extracellular ACE2 fragment (including mutations designed to improve binding to viruses (e.g., SARS-CoV-2) or to eliminate the original catalytic activity of the ACE2 enzyme). The extracellular fragment of ACE2 may exclude the collectrin domain (corresponding to amino acids 615-740 of wild-type human ACE2). Furthermore, any suitable Fc domain may be used, including antibody Fc derived from different IgG isotypes (e.g., IgG3, IgG4) and Fc genetically engineered to have different effector functions (e.g., LALA-PG that suppresses Fcg-R binding, or YTE or LS mutations that improve FcRn binding). Any suitable linker region may be used. For example, the linker region is (GGGGS) for one or both of the linker regions (each of two or more coronavirus binding / decoy domains is linked to the Fc domain). nIt may also be the case that n for each movable linker is 1 to 26, and in detail, where n is 2 to 25, 3 to 24, 4 to 22, 5 to 20, 6 to 20, 3 to 10, 4 to 15, etc., or (EAAK) n (where n is 0-26, in particular n is 2-25, 3-24, 4-22, 5-20, 6-20, 3-10, 4-15, etc.). The length of the mobile linker may be selected such that the average spacing between 2 (or more) coronavirus binding / decoy domains is greater than approximately 14 nm in total (e.g., each linker is approximately 5 nm or more).

[0011] Soluble angiotensin-converting enzyme 2 (ACE2) can act as a decoy molecule capable of neutralizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by blocking the viral spike (S) protein from binding to ACE2 on host cells. Based on the structures of ACE2 and the S protein, ACE2-Fc conjugates were genetically engineered as described herein, and in some examples, included an extracellular segment of ACE2 that does not contain the C-terminal collectrin domain, and ligated to a human Ig domain (e.g., IgG1-Fc) via an extended, mobile linker that allows for improved bivalent binding of the molecule to the S protein on the virus.

[0012] This molecular family, referred to herein as divalent and flexibly linked ACE2-Fc decoys (or simply abbreviated as "flexibly linked ACE2 decoys"), exhibits significantly greater binding affinity and neutralizing ability than predicted. Interestingly, the neutralizing ability of these flexibly linked ACE2 decoys is greater than that of full-length ACE2-Fc decoys that either lack a mobile linker region or contain a short linker region. These flexibly linked ACE2 decoys showed picomolar binding affinity (250 pM) and neutralizing ability (IC50: 50 ng / mL). The flexibly linked ACE2 decoys can also effectively trap fluorescent SARS-CoV-2 virus-like particles in fresh human respiratory mucus and can be stably sprayed using commercially available vibrating mesh nebulizers. In hamsters, delayed intranasal administration of flexible ligated ACE2 decoys, two days after infection, resulted in a tenfold reduction in viral load in nasal turbinate tissue by day four. These results strongly support the use of flexible ligated ACE2 decoys for inhalation immunotherapy of COVID-19 and other emerging viruses that use ACE2 as an entry receptor.

[0013] One non-limiting example of a flexible-linked ACE2 decoy is called ACE2-(G4S)6-Fc, which contains two ACE2 extracellular domains (each excluding its C-terminal collectrin domain) flexibly linked to the Fc domain via (GGGGS)6. While this particular example of ACE2-(G4S)6-Fc is described in many of the examples and drawings used herein, it should be understood that other flexible-linked ACE2 decoys have been identified and shown to have similar properties. In general, a flexible-linked ACE2 decoy containing two ACE2 extracellular domains with one or more mutations (see, for example, Table 1; described in more detail below) linked to the Fc domain by a mobile linker longer than approximately 5 nm may function as described herein and share similar affinity and properties with ACE2-(G4S)6-Fc.

[0014] Furthermore, flexible, multivalent (e.g., bispecific) binding proteins genetically engineered for ACE2-targeted viruses that possess only one ACE2 but instead contain one or more coronavirus-binding proteins (e.g., antibody fragments having binding activity against ACE2-targeted viruses) are described herein.

[0015] Any of the binding proteins described herein (e.g., a flexibly linked ACE2 decoy) may be glycosylated (or selected for glycosylation enrichment) which can enhance their mucus-trapping ability. Increasing the G0F content can improve trapping ability by increasing the G0F content to, for example, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0016] For example, isolated binding proteins that bind to ACE2-targeted viruses having the following amino acid sequence are described herein: A-(B) n -C (formula I) During the ceremony A is the extracellular component of angiotensin-converting enzyme 2 (ACE2) or a variant thereof, with the collectrin domain excluded; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; B is a polypeptide mobile linker; C is a fragment crystallization (Fc) domain, The isolated binding protein is dimerized.

[0017] ACE2-targeted viruses include coronaviruses, such as SARS-like coronaviruses (e.g., SARS-CoV and SARS-CoV-2, SARS-CoV-1, and NL63 seasonal coronavirus).

[0018] The binding proteins described herein may include a mobile linker of sufficient length such that the distance between the A domains of the dimer is greater than approximately 14 nm (e.g., greater than approximately 15 nm, greater than approximately 16 nm, greater than approximately 17 nm, greater than approximately 18 nm, greater than approximately 19 nm, greater than approximately 20 nm, etc.). The linker(single or multiple) distance in the dimer may be determined statistically and / or computationally; the distance may refer to the average distance, as understood by those skilled in the art. The length of the mobile linker may vary due to changes in molecular three-dimensional structure in space, but if it is shorter than the minimum length (e.g., 14 nm, 15 nm, 16 nm, etc.), the proportion of proteins that can bivalently bind to the target (e.g., spike protein on ACE2-targeted viruses) may fall below the efficacy threshold.

[0019] For example, the length of a mobile polypeptide linker may be determined based on the number of polypeptide residues. For instance, the number of residues may be 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 20 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, and so on.

[0020] Generally, the binding proteins described herein may include any suitable Fc domain (e.g., the Fc domain described in any of claims 1 or 2), where the Fc domain is a human IgA, IgM, or IgG Fc domain. The Fc domain may also be a human IgG1 Fc domain. The Fc domain may include a YTE mutation, an LS mutation, or an LALA-PG mutation or other modifications to improve function.

[0021] Generally, the extracellular portion of ACE2 may be the extracellular portion of human ACE2 with the collectrin domain excluded. The extracellular sequence may generally correspond to the sequence of the wild-type human ACE2 extracellular domain and is, for example, an amino acid sequence of at least 40% (at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%) in length from residues 18-614. In some cases, the extracellular portion of ACE2 has 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 11. For example, the extracellular portion of ACE2 may have an amino acid sequence with up to 10 amino acid differences within the amino acids of SEQ ID NO: 11. For example, the extracellular portion of ACE2 may have at least one mutation, or in some cases, two or more mutations. The mutation may be at any of the locations specified in Table 1 of Figures 16A-16B.

[0022] The polypeptide mobile linker may have any suitable sequence. For example, the mobile linker may be a sequence such as GGS, GGGS, or GGGGS. The sequence length (n) may be as short as 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., based on the length of the linker region described above. For example, if the mobile linker has the sequence GGGGS, n may be 5 or greater (e.g., 6 or greater, 7 or greater, etc.). In some examples, the binding protein contains the sequences of SEQ ID NO: 2 and SEQ ID NO: 4. In some examples, the binding protein contains the sequences of SEQ ID NO: 11 and SEQ ID NO: 4. In some examples, the binding protein contains a mobile linker such as SEQ ID NO: 2 or SEQ ID NO: 11, (GGGS)n, and SEQ ID NO: 12 or SEQ ID NO: 13, where n is between 5 and 10 (e.g., n=6). Any of these binding proteins may contain a hinge between the mobile linker and the Fc domain.

[0023] In general, any of these binding proteins may contain an oligosaccharide having a G0 glycosylation pattern on its Fc domain. For example, the Fc domain may contain an oligosaccharide having a G0 glycosylation pattern, which includes a branched coagulan structure of Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1, each branch having a terminal N-acetylglucosamine, to enhance the binding protein's ability to trap in mucus.

[0024] Generally, the binding protein may be part of a mixture in which all or some of the binding proteins (e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, etc.) are glycosylated and contain a G0 glycosylation pattern on the Fc domain.

[0025] Accordingly, pharmaceutical compositions comprising any binding protein and pharmaceutically acceptable excipients are described. For example, the excipient, diluent, or carrier may be configured for inhalation. This composition may be configured for one or more of the following: oral administration, parenteral administration, intraperitoneal administration, transmucosal administration, transdermal administration, rectal administration, inhalation administration, and topical administration.

[0026] Furthermore, methods for treating subjects infected with SARS-CoV-2 are described herein, comprising administering a pharmaceutically acceptable amount of a pharmaceutical composition of any of these binding proteins. Administration may include progressive application of the pharmaceutical composition to the patient. In some examples, administration includes application of the pharmaceutical composition to the patient's mucous membrane. Administration may include spraying the pharmaceutical composition.

[0027] For example, a method for treating or inhibiting a viral infection by an ACE2-targeted virus is described herein, which includes administering a binding protein, which is any binding protein (e.g., any of the flexibly linked ACE2 decoys described herein), via an inhalation route. As stated above, the ACE2-targeted virus may be SARS-CoV-2.

[0028] Furthermore, isolated binding proteins that bind to ACE2-targeted viruses, having the following amino acid sequence, are described herein: A-(B) n -C (formula I) During the ceremony A is the extracellular component of angiotensin-converting enzyme 2 (ACE2) that excludes the collectrin domain and has more than 80% amino acid sequence identity with the amino acid sequence of Sequence ID No. 11; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; B is a polypeptide mobile linker; C is a fragment crystallization (Fc) domain, The isolated binding protein is dimerized, Furthermore, n is selected such that the distance between the A domains of the dimer is greater than 14 nm.

[0029] Brief explanation of the drawing A better understanding of the features and advantages of the methods and apparatus described herein can be obtained by referring to the following detailed description illustrating exemplary embodiments and the accompanying drawings as follows. [Brief explanation of the drawing]

[0030] [Figure 1A] Figure 1A shows an example of the 3D molecular structure of an example of a binding protein to a SARS-like coronavirus, in which each monomer contains a dimer of ACE2-Fc with a mobile linker, indicated in this example as (GGGGS)n. [Figure 1B] Figure 1B shows an example of an ACE2-Fc dimer that does not have a movable linker. [Figure 2A]Figures 2A and 2B illustrate the fusion of different ACE2-Fc constructs (dimers) to the S protein trimer, showing differences in "intraspike" binding to the S protein. Figure 2A shows that the shape of the ACE2-Fc in this example does not allow the second Fab to bend around towards either of the remaining two available S proteins on the S protein trimer, thus demonstrating that an ACE2-Fc without a mobile linker can only bind to the same S protein spike univalently. [Figure 2B] Figure 2B shows an example of an ACE2-Fc molecule with a mobile linker (a flexible ACE2 decoy) that enables bivalent binding of one ACE2-Fc molecule to an S protein trimer (for example, when the linker is at least 5.7 nm). [Figure 3] Figure 3 shows an example of an ACE2-Fc dimer lacking a mobile linker, illustrating that it can potentially bind to two different spikes (i.e., "spike-to-spike" binding) but only at a limited frequency. The distance between the binding boundaries of the ACE2 domains is approximately 14.6 nm, which is roughly the same as the distance between spikes on the COVID-19 virus surface when the S-proteins are positioned vertically (approximately 14–15 nm). Due to the lack of rotational flexibility in ACE2Fab, the two S trimer spikes likely require a substantially closer distance than 15 nm for ACE2-Fc without a mobile linker to bind divalently. [Figure 4] Figure 4 illustrates a dimer of ACE2-Fc with a mobile linker (a flexible ACE2 decoy) that more easily achieves bivalent binding to two different S protein trimers. The 5.6 nm linker length for both linkers allows the two ACE2 domains to bind to S proteins separated by 15 nm, even when the two S trimers are aligned upright as they naturally occur on the surface of the virus. [Figure 5]Figure 5 shows an example of a proposed bispecific monoclonal antibody derived from CR3022 IgG (an antibody against the human coronavirus SARS-CoV-2 spike glycoprotein S) and ACE2, which can achieve bivalent binding to only one of the three trimer-like S proteins on each S protein spike of COVID-19 without interfering with each other. The N-terminus and C-terminus of CR3022 are separated by 9.8 nm and can be crosslinked by a (GGGGS)6 linker. [Figure 6A] Figures 6A–6C illustrate computer predictions of the hypothetical structures of dimers of different ACE2 fusion proteins. Figure 6A shows an example of an ACE2-Fc fusion (also referred to herein as ACE2(740)-Fc) consisting of an entire extracellular ACE2 molecule containing the collectrin domain linked to IgG1-Fc. As shown, in this example, the ACE2 domain aggregates even when linked through the Fc domain. [Figure 6B] Figure 6B shows an example of an ACE2-Fc fusion that contains the extracellular domain of ACE2 with the collectrin domain removed, but links to the Fc domain without a mobile linker. This example is called ACE2-Fc. [Figure 6C] Figure 6C shows an example of a flexibly linked ACE2 decoy in which two ACE2 fragments lacking a collectrin domain are linked to human IgG1-Fc via a 30-amino acid glycine-serine mobile linker (e.g., ACE2-(G4S)6-Fc). [Figure 7A] Figure 7A illustrates an example of computer predictions for the binding proteins shown in Figure 6B (ACE2-Fc, without mobile linker) and Figure 6C (ACE2-(G4S)6-Fc). As shown in Figure 7A, the computer prediction for ACE2-Fc indicates that ACE2-Fc binds to the S protein via only one RBD domain. [Figure 7B]In contrast, as shown in Figure 7B, ACE2-(G4S)6-Fc (a flexible ACE2 decoy) is predicted to ligate onto the S protein via two of the three RBD domains located in the "upper" position. [Figure 7C] Figure 7C shows the unmodified PAGEs for ACE2-Fc (lane 2) and ACE2-(G4S)6-Fc (lane 3). [Figure 7D] Figure 7D shows size exclusion chromatography of ACE2-(G4S)6-Fc and ACE2-Fc. The elution times and sizes of both are as predicted. The ACE2-(G4S)6-Fc with a flexible ligated ACE2 decoy is slightly larger. [Figure 8A] Figures 8A-8D illustrate the significantly different binding affinities of the ACE fusion proteins shown in Figure 6, as evaluated by SARS-CoV-2 S-protein ELISA. Figure 8A shows representative concentration-dependent binding curves for ACE2-(G4S)6-Fc (black circles), ACE2-Fc (light gray squares), and full-length ACE2 decoy ACE2(740)-Fc (gray triangles). [Figure 8B] Figure 8B shows ELISA-derived EC50 values ​​for different, independent batches of the ACE2 fusion protein from Figure 6 (with the same labeling applied to Figure 8A). CH represents ACE2-(G4S)6-Fc produced in CHO cells. [Figure 8C] Figure 8C shows representative concentration-dependent binding curves for ACE2-(G4S)6-Fc to S proteins derived from different virus strains, including WT (USA-WA1 / 2020), UK (B.1.1.7), and SA (B.1.351). [Figure 8D] Figure 8D shows EC50 data from the same set of stocks. [Figure 9A] Figures 9A–9C illustrate the pseudotype virus-based neutralization capabilities of three different ACE2 fusion proteins shown in Figures 6A–6C above. Figure 9A shows representative infection curves representing the pseudotype SARS-CoV-2 virus across various ACE2-decoy concentrations. [Figure 9B]Figure 9B shows IC50 data for each of the three categories of ACE2 bivalent fusion proteins. [Figure 9C] Figure 9C shows the IC90 values ​​estimated from the binding curves. Each data point represents a different experiment. There are significant differences between the flexibly ligated ACE2 decoy and other fusion proteins. [Figure 10A] Figures 10A-10B illustrate the effectiveness of a flexibly linked ACE2 decoy in a mucus trap. Figure 10A shows a comparison of percentages of rapidly moving SARS-CoV-2 VLPs, demonstrating that ACE2-(G4S)6-Fc effectively traps SARS-2 VLPs with much higher ability than ACE2-Fc or CR3022 (CR3022 is a control anti-SARS-CoV-2 mAb) in human amniotic fluid (AM). [Figure 10B] Figure 10B shows the binding affinity of sprayed ACE2-(G4S)6-Fc, as evaluated by SARS-CoV-2 S-protein ELISA. ACE2-(G4S)6-Fc collected from the upper chamber (filled circles) and the lower chamber (gray squares) are compared with unsprayed protein (triangles). [Figure 11] Figure 11 illustrates a PCR-based assay for viral load in nasal turbinate tissue from SARS-CoV-2 infected hamsters collected four days after infection. [Figure 12A] Figures 12A-12B illustrate the biophysical characteristics of sprayed ACE2-(G4S)6-Fc. Figure 12A shows an example of unmodified PAGE of sprayed ACE2-(G4S)6-Fc. Samples were collected from the upper chamber (lanes 2, 5, 8), the lower chamber (lanes 3, 6, 9), and the residue ("dead volume") after spraying with the spraying device (lanes 4, 7, 10). The data is shown repeated three times. [Figure 12B] Figure 12B shows size exclusion chromatography of ACE2-(G4S)6-Fc, including samples collected from the sample before spraying, the upper chamber of the sprayer, the lower chamber, or the residue. Data representing three repeats are shown. [Figure 13]Figure 13 shows an example of the yield of ACE2-Fc fusion protein compared to ACE2-(G4S)6-Fc (flexibly linked ACE2 decoy) after protein A affinity chromatography. The protein was purified from a 500 mL culture of Expi293T cells. [Figure 14] Figure 14 shows an example of differential scanning fluorescence measurement of ACE2-(G4S)6-Fc. Data for three independent repeats are shown in the figure. [Figure 15] Figure 15 shows the sequence of the full-length ACE2 (human). [Figure 16A] Figure 16A shows Table 1 illustrating mutations to the full-length ACE2 polypeptide that may be made in any of the flexibly linked ACE2 decoys described herein. [Figure 16B] Figure 16B shows Table 1 illustrating mutations to the full-length ACE2 polypeptide that may occur in any of the flexibly linked ACE2 decoys described herein. [Modes for carrying out the invention]

[0031] Detailed explanation Generally, methods and compositions (e.g., genetically engineered binding proteins) for binding to one or more ACE2-targeted viruses (e.g., SARS-CoV and SARS-CoV-2) are described herein. These binding proteins may be used for the treatment, prevention, and / or mitigation of infection by SARS-like coronaviruses. In some examples, these binding proteins may be used to enhance the aggregation, restraint, and / or mucus trapping of ACE2-targeted viruses, including reducing the proportion of ACE2-targeted viruses that can penetrate through mucus.

[0032] For example, angiotensin-converting enzyme 2 (ACE2)-immunoglobulin (IgG) hybrid binding proteins (also referred herein as flexible ACE2 decoys) that dimerize and have picomolar affinity for SARS-like coronaviruses (specifically, for SARS-CoV-2) are described herein. These proteins may be genetically engineered for “mucus trapping,” which involves enhancing mucus trapping by specifically selecting binding proteins that are glycosylated in the Fc domain of the binding protein. These binding proteins may be used to treat or prevent SARS-CoV (e.g., SARS-CoV-2) infection, for example, for topical immunotherapy against ACE2-targeted viruses, including SARS-CoV-2. These molecules may generally be fusions of the extracellular portion(s) of ACE2 (e.g., a portion of soluble angiotensin-converting enzyme 2 with the collectrin domain removed) to the Fc portion using a mobile linker (e.g., (GGGGS)n, (EAAAK)n, etc., but not limited to these).

[0033] Furthermore, binding proteins described herein may include two (or, in some examples, more) coronavirus-binding domains that are polyvalent to ACE2-targeted viruses and are flexibly linked to the Fc domain by a mobile polypeptide linker. The linker may be long and flexible enough to allow both coronavirus-binding domains to bind to a target (e.g., a spike protein) simultaneously.

[0034] The binding proteins described herein may enhance aggregation, facilitate the restraint of replication, and / or improve the mucus trapping of ACE2-targeted viruses (e.g., SARS-CoV-2), as described herein. These binding proteins may prevent SARS-like CoVs from penetrating mucus by improving aggregation ability, facilitating the restraint of target replication, and / or enabling mucus trapping, thereby preventing, limiting, and / or treating infection.

[0035] definition Unless otherwise stated, technical terms will be used according to their conventional usage. Definitions of general terms in molecular biology can be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

[0036] Where used herein, the singular forms “a,” “an,” and “the” mean both singular and plural unless the context clearly indicates otherwise. For example, the term “antigen” includes singular or plural antigens and may be considered equivalent to the phrase “at least one antigen.” Where used herein, the term “comprises” means “includes.” Unless otherwise indicated, it should be further understood that any and all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and presented for illustrative purposes only. Many methods and materials similar or equivalent to those described herein may be used, and particularly preferred methods and materials are described herein. In case of conflict, this specification, including the definitions of terms, shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0037] As used herein, the terms “administer” or “to administer” mean, as used herein, introducing the composition into a subject via a selected route. Administration may be local or systemic. For example, if the selected route is intravenous, the composition (e.g., a composition comprising the antibodies disclosed) is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, intrarectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes.

[0038] Unless the context indicates otherwise, it is specifically intended that the various features described herein may be used in any combination. Furthermore, it is also intended that in some embodiments of the present invention, any feature or combination of features shown herein may be excluded or omitted. Where the specification states, for illustrative purposes, that a complex includes components A, B, and C, it is specifically intended that A, B, or C, or any combination thereof, may be omitted and rejected individually or in any combination.

[0039] When the term “about” is used herein to refer to measurable values ​​such as the amount, dose, time, or temperature of the compound or agent of the present invention, it means to include a variable of plus or minus 10%, plus or minus 5%, plus or minus 1%, plus or minus 0.5%, or still plus or minus 0.1% of the specified amount.

[0040] Unless otherwise indicated, all numbers used in this specification and in the claims to express properties such as the amount of components and reaction conditions are understood in all instances to be modified by the term “approximately.” Therefore, unless otherwise stated, numerical parameters expressed in this specification and in the claims are approximate and may vary depending on the desired properties that may be obtained from the subject matter of this disclosure.

[0041] Where used herein, a range may be expressed as “about” one particular value and / or “about” another particular value. There are many values ​​disclosed herein, and it is understood that each value disclosed herein is also disclosed as “about” a particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0042] The transitional phrase “essentially consisting of” means that the claims are interpreted to include any specific materials or processes mentioned herein, as well as any that do not substantially affect the essential and novel features (singular or plural) of the claimed invention. Any methods and compositions described herein may partially or completely exclude other components (for example, they may consist of or essentially consist of). Generally, any apparatus and methods described herein are comprehensive, but all or a subgroup of components and / or processes may be exclusive, and may be expressed as consisting of or instead essentially consisting of various components, processes, parts of components or parts of processes.

[0043] As used herein, the term “amino acid substitution” means replacing one amino acid in a polypeptide with another amino acid or omitting an amino acid (i.e., deletion). In some examples, an amino acid in a polypeptide may be substituted, for example, with an amino acid from a homologous polypeptide, and an amino acid in a recombinant SARS-CoV or SARS-CoV-2 polypeptide may be substituted with a corresponding amino acid from a different SARS-CoV or SARS-CoV-2 strain.

[0044] As used herein, the term “antibody” means a binding protein that specifically binds to and recognizes an antigen, such as SARS-CoV or SARS-CoV-2 S protein or its antigenic fragment. The term “antibody” is used herein in a broad sense and encompasses a wide range of antibody structures (including, but not limited to, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, recombinant antibodies and their antigen-binding fragments) as long as they exhibit the desired antigen-binding activity.

[0045] As used herein, the term “monoclonal antibody” means an antibody obtained from a substantially homogeneous population of antibodies (i.e., the individual antibodies constituting the population are identical except for naturally occurring mutations that may be present in trace amounts). Monoclonal antibodies are highly specific and directed to a single antigenic epitope. The modifier “monoclonal” indicates the characteristic of antibodies obtained from a substantially homogeneous population of antibodies and is not construed as requiring the production of a population of antibodies by any particular method. In some examples, monoclonal antibodies are produced by a single clone of B lymphocytes or by cells or their progeny cells into which nucleic acids encoding the antibody light and heavy chain variable regions of a single antibody (or its antigen-binding fragment) have been transfected. In some examples, monoclonal antibodies are isolated from the subject. Monoclonal antibodies may have conserved amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are publicly known; see, for example, Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed., Cold Spring Harbor Publications, New York (2013).

[0046] Typically, immunoglobulins have heavy (H) and light (L) chains linked to each other by disulfide bonds. Immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. Two types of light chains exist: lambda and kappa. There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0047] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). In some examples, the heavy and light chain variable regions are combined to specifically bind an antigen. In further examples, only the heavy chain variable region is required. For example, naturally occurring camel antibodies consisting only of heavy chains are functional and stable even without a light chain (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). H The references to "V" or "VH" refer to the variable region of the antibody heavy chain (including antigen-binding fragments, e.g., Fv, scFv, dsFv, or Fab). L The terms "VL" or "VL" refer to the variable domain of the antibody light chain (including those of Fv, scFv, dsFv, or Fab).

[0048] The light and heavy chain variable regions include a “framework” region interrupted by three hypervariable regions, also known as the “complementarity-determining region” or “CDR” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, USD Department of Health and Human Services, 1991). The sequences of various light or heavy chain framework regions are relatively conserved across species. The antibody framework region, i.e., the framework region of the constituent light and heavy chains combined, contributes to the positioning and arrangement of the CDR in three-dimensional space.

[0049] CDRs are primarily responsible for binding antigens to epitopes. The amino acid sequence boundaries of a given CDR can be easily determined using one of many well-known schemes described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). The CDRs on each chain are usually called CDR1, CDR2, and CDR3 (from N-terminus to C-terminus) and are usually identified by the chain on which a particular CDR is located. Therefore, V H CDR3 is a CDR3 derived from the variable domain of the heavy chain of the antibody in which it was found, V LCDR1 is the CDR1 derived from the variable domain of the light chain of the antibody in which it was found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0050] As used herein, the phrase "antigen-binding fragment" refers to a portion of a full-length antibody that has the ability to specifically recognize an antigen of the same species as well as various combinations of such portions. Non-limiting examples of antigen-binding fragments include Fv, Fab, Fab’, Fab’-SH, F(ab)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and bispecific and multispecific antibodies formed from antibody fragments. Antibody fragments include those generated by any modification of an antibody, whether the whole antibody or de novo synthesized antibodies using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2.sup.nd Ed., Springer Press, 2010), that have the ability to specifically recognize an antigen of the same species as well as various combinations of such portions.

[0051] A single-chain antibody (scFv) is a genetically engineered molecule comprising one or more antibody V H and V L domains linked by a polypeptide linker suitable as a genetically fused single-chain molecule (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intra-molecular orientation of the V H -domain and V L -domain in an scFv is generally not critical for scFvs. Thus, scFvs having both possible arrangements (V H -domain-linker-domain-V L -domain; VL -Domain-LinkerDomain-V H -Domain) may be used.

[0052] In dsFv, the heavy chain, light chain, and variable chain have been mutated to stabilize the chain bond by introducing disulfide bonds. H and V L Diabodies, which are bivalent, bispecific antibodies in which a domain is expressed on one polypeptide chain, but the linker is too short to pair the two domains on the same chain, so it is paired with a complementary domain on another chain to create two antigen-binding sites, are also included (see, for example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0053] Antibodies also include genetically modified forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994–1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., WH Freeman & Co., New York, 1997.

[0054] Antibodies that do not exist in nature may be constructed using solid-phase peptide synthesis, generated by recombinant DNA, or obtained by screening a combinatorial library consisting of variable heavy and variable light chains, as described in Huse et al., Science 246:1275-1281 (1989), which is incorporated by reference herein. For example, these and other methods for producing chimeric antibodies, humanized antibodies, CDR-grafted antibodies, single-chain antibodies, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995); each of these is incorporated herein by reference).

[0055] As used herein, the term “humanized” antibody or antigen-binding fragment means one or more CDRs derived from a human framework region and a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment that provides the CDRs is referred to as the “donor,” and the human antibody or antigen-binding fragment that provides the framework is referred to as the “acceptor.” In one example, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. The constant region does not need to be present, but if present, it may be substantially identical to the human immunoglobulin constant region, for example, at least about 85–90%, or for example, about 95% or more. Thus, all parts of the humanized antibody or antigen-binding fragment, except perhaps the CDRs, are substantially identical to the corresponding parts of the natural human antibody sequence.

[0056] As used herein, the term “chimeric antibody” means, as used herein, an antibody that contains sequences derived from two different antibodies, typically from different species. In some examples, a chimeric antibody contains one or more CDRs and / or framework regions derived from one human antibody, and CDRs and / or framework regions derived from another human antibody.

[0057] A “fully human antibody” or “human antibody” is an antibody that contains sequences from (i.e., derived from) the human genome and does not contain sequences from another species. In some cases, human antibodies contain CDRs, framework regions, and (if present) Fc regions from (i.e., derived from) the human genome. Human antibodies can be identified and isolated using techniques for producing antibodies based on sequences derived from the human genome, for example, by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1” Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23:1117-1125, 2005; Lonberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0058] An antibody may have one or more binding sites. If more than one binding site is present, the binding sites may be identical or different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0059] As used herein, the term “antigen” means a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, and includes compositions that are injected into or absorbed by an animal. Antigens react with specific humoral or cellular immune products, including those induced by heterologous antigens such as the SARS-CoV or SARS-CoV-2 antigens of this disclosure. Examples of antigens include, but are not limited to, polypeptides, peptides, lipids, polysaccharides, combinations thereof (e.g., glycopeptides), and nucleic acids containing antigenic determinants, for example, those recognized by immune cells.

[0060] The term “binding protein,” as used herein, means at least one protein having the ability to bind to a given target. The target may be one or more analytes, antigens, autoantigens, proteins, polypeptides, etc. In some aspects, the binding protein may include a fusion protein. In other aspects, the binding protein of this disclosure may also include one or more other molecules, such as one or more immunoglobulins or immunoglobulin fragments. In some aspects, the binding protein is an antibody or an antibody-binding fragment thereof.

[0061] The term “fusion protein,” as used herein, means a protein comprising at least one first protein genetically bound to at least one second protein. Fusion proteins are constructed through the binding of two or more genes that originally encode distinct proteins. Thus, a fusion protein may contain a multimer of different or identical binding proteins, expressed as a single linear polypeptide. Such a fusion protein may further contain additional domains not involved in target binding (e.g., multimerization moieties, polypeptide tags, polypeptide linkers, etc.).

[0062] As used herein, the term “conservative” as used with respect to amino acid substitutions means substitutions that do not substantially affect or reduce the function of the protein (such as the protein’s ability to induce an immune response when administered to a subject). For example, in some cases, recombinant SARS-CoV or SARS-CoV-2 S protein or S1 fragment may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions compared to the corresponding native SARS-CoV or SARS-CoV-2 protein sequence and may induce an immune response to the SARS-CoV or SARS-CoV-2 S protein in a subject. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

[0063] Furthermore, a person skilled in the art will recognize that individual substitutions, deletions, or additions that change, add, or delete one amino acid or a low percentage of amino acids (e.g., less than 5%, or less than 1% in some examples) in the encoded sequence are conservative changes if the change results in the substitution of an amino acid having a chemically similar amino acid.

[0064] Tables of conserved amino acid substitutions that provide functionally similar amino acids are well known to those skilled in the art. The following six groups are examples of amino acids that are considered to be conserved substitutions of one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0065] Non-conservative substitutions are those that reduce the activity or function (e.g., the ability to reduce the immune response when administered to a target) of a protein (e.g., the SARS-CoV or SARS-CoV-2 S protein). For example, if an amino acid residue is essential to the function of a protein, even a substitution that would otherwise be conservative can disrupt its activity. Therefore, conservative substitutions do not alter the fundamental function of the protein of interest.

[0066] As used herein, the term “expression” means the transcription or translation of a nucleic acid sequence. For example, a gene is expressed when its DNA is transcribed into RNA or RNA fragments (and in some cases processed into mRNA). A gene may also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or protein fragment. In a detailed example, a heterologous gene is expressed when it is transcribed into RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. In this specification, the term “expression” is used to mean either transcription or translation. Regulation of expression may include control over transcription, translation, RNA transport and processing, the degradation of intermediate molecules such as mRNA, or control through the activation, inactivation, sclerature or degradation of a particular protein molecule after it has been produced.

[0067] As used herein, the term “expression regulatory sequence” means a nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence to which it is operably ligated. An expression regulatory sequence is operably ligated to a nucleic acid sequence in such a way that it controls and regulates the transcription and, where appropriate, the translation of the nucleic acid sequence. Thus, an expression regulatory sequence may include appropriate promoters, enhancers, transcriptional terminators, a start codon (ATG) before a protein-coding gene, a splicing signal for an intron, maintenance of a precise reading frame that causes the gene to be properly translated into mRNA, and a stop codon. The term “regulatory sequence” is intended to include, at a minimum, components whose presence may affect expression, and may also include further components whose presence is beneficial, such as a leader sequence and a fusion partner sequence. An expression regulatory sequence may include a promoter.

[0068] A promoter is the minimum sequence sufficient to direct transcription. It also contains promoter elements sufficient to enable promoter-dependent gene expression regulation, such as cell-type specific, tissue-specific, or induceable by external signals or drugs; such elements may be located within the 5' or 3' region of the gene. Both constitutive and inductive promoters are included (e.g., Bitter et al., Method in Enzymology 153:516-544, 1987). For example, in bacterial cloning, inductive promoters (e.g., bacteriophage lambda pL, plac, ptrp, ptac (ptrp-lac hybrid promoter)) may be used. In one example, in mammalian cell cloning, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (retroviral long-terminal repeats; adenovirus late promoter; vaccinia virus 7.5K promoter, etc.) may be used. Recombinant DNA or promoters produced by synthetic techniques can also be used to provide transcriptions of nucleic acid sequences.

[0069] Polynucleotides can be inserted into expression vectors that contain promoter sequences that facilitate the efficient transcription of the inserted host gene sequence. Expression vectors typically include an origin of replication, a promoter, and specific nucleic acid sequences that enable expression type selection in transformed cells.

[0070] As used herein, the term “expression vector” means a vector comprising a recombinant polynucleotide containing an expression regulatory sequence operably ligated to the nucleotide sequence to be expressed. An expression vector comprises a sufficient cis-operated element for expression; and other elements for expression that may be supplied by a host cell or an in vitro expression system. Expression vectors include all known in the art, e.g., cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0071] As used herein, the term “heterogeneous” means originating from a different gene source. A nucleic acid molecule that is heterogeneous to the cell in which it is expressed originates from a gene source other than that cell. In one specific, non-limiting example, a heterogeneous nucleic acid molecule encoding recombinant SARS-CoV or SARS-CoV-2 polypeptide or a specific antibody is expressed in a cell, for example, a mammalian cell. Methods for introducing heterogeneous nucleic acid molecules into cells or organisms are well known in the art and include, for example, nucleic acid transformation (including electroporation, lipofection, particle gun acceleration, and homologous recombination).

[0072] As used herein, the term “host cell” means a cell on which a vector can grow and on which its DNA can be expressed. This cell may be a prokaryotic or a eukaryotic cell. The term also includes any progeny cells of the host cell in question. It is understood that all progeny cells may not be identical to the parent cell because mutations may occur during replication. However, such progeny cells are included when the term “host cell” is used.

[0073] As used herein, “IgA” refers to a polypeptide belonging to a class of antibodies substantially encoded by the recognized immunoglobulin alpha gene. In humans, this class or isotype includes IgA1 and IgA2. IgA antibodies can exist as monomers, polymers (called pIgA), primarily in a dimerized form, and secreted IgA. The constant chain of wild-type IgA contains an 18-amino acid elongation portion called the tailpiece (tp) at its C-terminus. Polymeric IgA is secreted by plasma cells as a 15-kDa peptide called two IgA monomers linked in a J-chain via a conserved cysteine ​​residue in the tailpiece.

[0074] As used herein, “IgG” means a polypeptide belonging to a class or isotype of antibody substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4.

[0075] As used herein, the term “isolated” means a biological component (such as a protein, e.g., an antigen encoding a nucleic acid as described herein) that is substantially separated from other biological components (such as other naturally occurring components such as other chromosomes and extrachromosomal DNA, RNA, and proteins). “Isolated” proteins, peptides, and nucleic acids include proteins purified by standard purification methods. The term also encompasses proteins or peptides prepared by recombinant expression in host cells, as well as chemically synthesized proteins, peptides, and nucleic acid molecules. Isolates do not necessarily require absolute purity and may include proteins, peptides, or nucleic acid molecules that are at least 50% isolated, e.g., at least 75%, 80%, 90%, 95%, 98%, 99%, or still 99.9% isolated.

[0076] As used herein, “linker” is a bifunctional molecule that can be used to link two molecules into a single contiguous molecule, for example, to link a carrier molecule to a polypeptide. A non-limiting example of a peptide linker is a glycine-serine linker, e.g., (GGGGS). n A linker is given (where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25).

[0077] As used herein, the terms “conjugating,” “joining,” “bonding,” or “linking” refer to the process of making two molecules into a single continuous molecule; for example, linking two polypeptides into a single continuous polypeptide, or covalently bonding a carrier molecule or another molecule to a polypeptide. Linking may be by chemical or recombinant means. “Chemical means” refers to a reaction between a polypeptide moiety and a carrier molecule, for example, in which a covalent bond is formed between the two molecules to form a single molecule.

[0078] As used herein, “Severe Acute Respiratory Viral Syndrome Coronavirus” or “SARS-CoV” refers to a β-coronavirus, a forward-directed single-stranded RNA virus belonging to the Coronavirus subfamily that causes severe respiratory syndrome in humans. SARS-CoV has the same structural proteins as three other known groups of coronaviruses: spike glycoprotein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N). The coronavirus N protein is required for coronavirus RNA synthesis and has RNA chaperone activity that may be involved in template switching.

[0079] The SARS-CoV spike glycoprotein is 1255 amino acids long and has low amino acid similarity (20-27%) compared to other coronaviruses. Its carboxyl terminus (C-terminus) consists of a transmembrane domain and a cytoplasmic tail. The extracellular domain of the SARS-CoV spike glycoprotein is composed of two 7-amino acid repeat regions known as 7-amino acid repeat region 1 (HR1) and 7-amino acid repeat region 2.

[0080] The SARS-CoV spike glycoprotein has two functional domains: S1 and S2. S1 is responsible for binding to its receptor, angiotensin-converting enzyme 2 (ACE2), on the host cell and defines the host range of the virus. S2 is a transmembrane subunit that facilitates viral-cellular membrane fusion. Membrane fusion occurs when a fusion core is formed due to conformational changes in the HR. The protein's HR folds into a coiled-coil structure (called the membrane fusion state), folding the HR domain of the S protein into a hairpin-like formation. This hairpin structure brings the cell membrane and viral membrane together, resulting in final fusion.

[0081] Other known β-coronaviruses include SARS-CoV-2 and MERS-CoV, both of which cause severe and potentially fatal respiratory infections. The genome sequence of SARS-CoV-2 is 96.2% identical to that of bat CoV RaTG13 and 79.5% identical to that of SARS-CoV. Sequences of SARS-CoV-2 from many different samples have been described in various publications, e.g., Lu et al., Lancet, 395:565-574 (February 2020) and https: / / www.ncbi.nlm.nih.gov / genbank / sars-cov-2-seqs / , and these are incorporated herein by reference where necessary.

[0082] As used herein, the term “neutralizing antibody” means an antibody that reduces the infectivity titer of an infectious agent by binding to a specific antigen on that infectious agent. In some cases, this infectious agent is a virus. In some cases, an antibody specific to the SARS-CoV or SARS-CoV-2 S protein neutralizes the infectivity titer of SARS-CoV or SARS-CoV-2. A “broad-spectrum neutralizing antibody” is an antibody that binds to a relevant antigen (e.g., an antigen that shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the antigenic surface of the antigen) and inhibits its function. With respect to an antigen derived from a pathogen, such as a virus, an antibody may bind to or inhibit the function of an antigen derived from one or more classes and / or subclasses of the pathogen. For example, with respect to SARS-CoV or SARS-CoV-2, this antibody may be able to bind to or inhibit the function of the antigen, such as the SARS-CoV or SARS-CoV-2 S protein derived from one or more strains of SARS-CoV or SARS-CoV-2. In one example, a broad-spectrum neutralizing antibody against the SARS-CoV or SARS-CoV-2 S protein is quite different from other antibodies against the SARS-CoV or SARS-CoV-2 S protein in that it neutralizes a high proportion of many types of circulating SARS-CoV or SARS-CoV-2.

[0083] As used herein, the term “nucleic acid” means polymers, related naturally occurring structural variants, and related synthetic analogs thereof, composed of nucleotide units linked via phosphodiester bonds (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and related synthetic analogs thereof). Therefore, the term includes nucleotide polymers in which nucleotides and their linkages include synthetic analogs not present in nature (e.g., phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids (PNAs), etc.). Such polynucleotides may be synthesized, for example, using an automated DNA synthesizer. The term “oligonucleotide” typically refers to short polynucleotides, generally about 50 nucleotides or less. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will be understood that this also includes RNA sequences (i.e., A, U, G, C) in which “U” is replaced by “T”.

[0084] As used herein, the term “nucleotide” means, but is not limited to, a monomer containing a base linked to a sugar, such as a pyrimidine, purine, or their synthetic analogues, or a base linked to an amino acid, such as in peptide nucleic acids (PNAs). A nucleotide is a single monomer in a polynucleotide. A nucleotide sequence means the sequence of bases in a polynucleotide.

[0085] Conventional notation is used herein to describe nucleotide sequences: the left end of a single-stranded nucleotide sequence is the 5' end; the left direction of a double-stranded nucleotide sequence is called the 5' direction; the direction of nucleotide addition from 5' to 3' in a nascent RNA transcript is called the transcription direction; the DNA strand having the same sequence as mRNA is called the "coding strand"; the sequence in the DNA strand transcribed from DNA and having the same sequence as mRNA located at 5' relative to the 5' end of the RNA transcript is called the "upstream sequence"; the sequence in the DNA strand having the same sequence as RNA located at 3' relative to the 3' end of the coding RNA transcript is called the "downstream sequence".

[0086] "cDNA" refers to DNA that is complementary to or identical to mRNA, whether in single-stranded or double-stranded form.

[0087] As used herein, the term “encoding” refers to the intrinsic properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, and the biological properties arising therefrom, which serve as a template for the synthesis of other polymers and macromolecules in a biological process having either a given nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a given amino acid sequence. Thus, a gene codes for a protein when the gene produces a protein in a cell or other biological system, resulting in the transcription and translation of mRNA. Both the coding strand (where the nucleotide sequence is identical to the mRNA sequence and is usually provided by a sequence listing) and the non-coding strand (used as a template for transcription) of a gene or cDNA can be said to code for a protein or other products of that gene or cDNA. Unless otherwise specified, “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and code for the same amino acid sequence. Protein-coding nucleotide sequences and RNAs may contain introns.

[0088] When a polynucleotide whose first sequence specifically hybridizes with a polynucleotide whose second sequence, the first sequence is "antisense" with respect to the second sequence.

[0089] As used herein, the term "operably ligated" means that a first nucleic acid sequence is operably ligated to a second nucleic acid sequence when the first nucleic acid sequence is positioned in a functional relation to the second nucleic acid sequence. For example, a promoter, such as the CMV promoter, is operably ligated to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably ligated DNA sequences are contiguous and, if necessary, link two protein-coding regions within the same reading frame.

[0090] As used herein, the term “pharmaceutically acceptable carriers” means conventional and conventional carriers known in the art, such as those described in Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995. Generally, the properties of the carrier depend on the specific mode of administration in which it is used. For example, parenteral formulations typically include injectable solutions containing a pharmaceutically and physiologically acceptable liquid, such as water, saline, equilibrated salt solution, dextrose aqueous solution, glycerol, etc., as a vehicle. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutical composition may contain trace amounts of non-toxic adjuvants, such as wetting agents or emulsifiers, preservatives, and pH buffering agents (e.g., sodium acetate or sorbitan monolaurate). In detail, a carrier suitable for administration to a subject may be sterile and / or suspended, or may be contained in a unit dosage form containing one or more measured doses of a composition suitable for inducing a desired anti-SARS-CoV or SARS-CoV-2 immune response. This may be accompanied by pharmacotherapy for its use for therapeutic purposes. The unit dosage form may be, for example, contained in a sealed container or syringe for injection to a subject, or may be lyophilized for subsequent dissolution and administration, or may be contained in a solid or sustained-release form.

[0091] As used herein, the term “polypeptide” means any chain of amino acids, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). “Polypeptide” applies to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as amino acid polymers containing one or more amino acid residues that are non-natural amino acids (e.g., artificial chemical mimics of corresponding naturally occurring amino acids). “Residue” means an amino acid or amino acid mimetic incorporated into a polypeptide by an amide bond or mimetic amide bond. Polypeptides have an amino terminus (N-terminus) and a carboxyl terminus (C-terminus). “Polypeptide” is used interchangeably with peptides or proteins, and in this specification, it is used to mean a polymer of amino acid residues.

[0092] Amino acids in peptides, polypeptides, or proteins are generally chemically linked via amide bonds (CONH). Furthermore, amino acids may also be linked by other chemical bonds. For example, bonds for amino acids or amino acid analogs may include CH2NH--, --CH2S--, --CH2--CH2-CH=CH-- (cis and trans), --COCH2--CH(OH)CH2--, and --CHH2SO-- (these and others are referenced in Spatola, in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p.267 (1983); Spatola, AF, Vega Data (March 1983), Vol.1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci pp.463-468, 1980; Hudson, et al., Int J Pept Prot Res 14:177-185, 1979; Spatola et al. Life Sci (This can be found in 38:1243-1249,1986; Harm J. Chem. Soc Perkin Trans. 1307-314,1982; Almquist et al. J. Med. Chem. 23:1392-1398,1980; Jennings-White et al. Tetrahedron Lett 23:2533,1982; Holladay et al. Tetrahedron Lett 24:4401-4404,1983; and Hruby Life Sci 31:189-199,1982).

[0093] As used herein, the terms “sample” or “biological sample” mean a biological specimen obtained from a subject, containing genomic DNA, RNA (including mRNA), proteins, or combinations thereof. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsy, microneedle aspiration, surgical specimens, and autopsy materials.

[0094] As used herein, the term “sequence identity” refers to the similarity between amino acid sequences; if not referred to as sequence identity, it can be expressed as similarity between sequences. Sequence identity is often measured as a percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologues, or variants of polypeptides exhibit a relatively high degree of sequence identity when aligned using standard methods.

[0095] Methods for aligning sequences for comparison are well known in the field. Various programs and alignment algorithms are described below: Smith & Waterman, Adv.Appl.Math.2:482,1981; Needleman & Wunsch, J.Mol.Biol.48:443,1970; Pearson & Lipman, Proc.Natl.Acad.Sci.USA 85:2444,1988; Higgins & Sharp, Gene,73:237-44,1988; Higgins & Sharp, CABIOS 5:151-3,1989; Corpet et al., Nuc.Acids Res.16:10881-90,1988; Huang et al. Computer Appls.in the Biosciences 8,155-65,1992; and Pearson et al. Altschul et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Bio. 215:403-10, 1990. These papers present a detailed discussion of sequence alignment methods and homology calculations.

[0096] Once the sequences are placed side by side, the number of matches is determined by counting the number of positions where identical nucleotide or amino acid residues exist in both sequences. The sequence identity percentage is determined by dividing the number of matches by the length of the sequence shown in the identified sequence or the segmented length (such as 100 consecutive nucleotide or amino acid residues from the sequence shown in the identified sequence), and then multiplying the resulting value by 100. For example, a peptide sequence with 1166 matches when placed side by side with a test sequence having 1554 amino acids is 75.0% identical to the test sequence (1166 / 1554*100=75.0). The sequence identity percentage value is rounded to one decimal place. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value is always an integer.

[0097] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol.Biol.215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions on how to determine sequence identity using this program are available on the NCBI website.

[0098] polypeptide homologs and variants are typically characterized by having at least about 75% sequence identity, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as counted over the full-length alignment with the amino acid sequence of interest. Proteins with greater similarity to the reference sequence will show an increase in the percentage of identity when evaluated by this method (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity). When sequences that are less than a complete sequence are compared for sequence identity, homologs and variants typically have at least 80% sequence identity over a short window of 10–20 amino acids, and may have at least 85% or at least 90% or 95% sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity across such short windows are available on the NCBI website. Those skilled in the art will understand that these sequence identity ranges are for guidance purposes only, and that it is entirely possible to obtain highly meaningful homologs from outside the presented ranges.

[0099] For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence, and the test sequence is compared to it. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, and if necessary, the lower sequence coordinates are specified, along with the sequence algorithm program parameters. Default program parameters are used. The method for aligning sequences for comparison is well known in the field. The optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv.Appl.Math.2:482,1981; by the homology alignment algorithm of Needleman & Wunsch, J.Mol.Biol.48:443,1970; by the search for similarity method of Pearson & Lipman, Proc.Nat'l.Acad.Sci.USA 85:2444,1988; by computer implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.); or by manual alignment algorithms and visual inspection (e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor, NY, 2012) and Ausubel et al. (In Current This can be done (see Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). One example of a useful algorithm is PILEUP. PILEUP uses a simplified version of the progressive alignment method described by Feng & Doolittle, J.Mol.Evol.35:351-360, 1987. The method used is similar to that described by Higgins & Sharp, CABIOS 5:151-153, 1989.Using PILEUP, the reference sequence is compared to other test sequences, and the sequence identity relevance percentage is determined using the following parameters: default gap size (3.00), default gap length (0.10), and weighted end gap. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395, 1984).

[0100] Another example of a suitable algorithm for determining sequence identity and sequence similarity percentages is the BLAST and BLAST 2.0 algorithms (described in Altschul et al., J.Mol.Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res.25:3389-3402, 1977). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses 11 word lengths (W), 50 alignments (B), 10 predictions (E), M=5, N=-4, and comparison of both strands as defaults. The BLASTP program (for amino acid sequences) uses a word length of 3 (W) and a prediction of 10 (E) as defaults, along with the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Oligonucleotides are linear polynucleotide sequences with a nucleotide base length of up to approximately 100.

[0101] As used herein, the term “at least 80% identity” means, with respect to a given reference sequence, “at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or still 100% identity.”

[0102] As used herein, the term “signal peptide” refers to a short amino acid sequence (e.g., approximately 10–35 amino acids in length) that directs a newly synthesized secretory or membrane protein toward or across a membrane (e.g., the endoplasmic reticulum membrane). Signal peptides are typically located at the N-terminus of a polypeptide and are removed by signal peptidases. Signal peptide sequences typically contain three common structural features: an N-terminal polar basic region (n region), a hydrophobic core, and a hydrophilic c region. Exemplary signal peptide sequences are shown in SEQ ID NOs: 1 and 6.

[0103] As used herein, the phrase "specifically binds" refers to the formation of an antibody:antigen-protein complex or a protein:protein complex, or a binding reaction that determines the presence of a target protein, peptide, or polysaccharide (e.g., glycoprotein) in the presence of a heterogeneous population of proteins and other biological products. Therefore, under given conditions, a particular antibody or protein will preferentially bind to a specific target protein, peptide, or polysaccharide (e.g., an antigen present on the surface of a pathogen, e.g., SARS-CoV or SARS-CoV-2 S protein) and will not bind in significant amounts to other proteins or polysaccharides present in the sample of interest. Specific binding can be determined by methods known in the art. The first protein or antibody interacts with approximately 10 -6 Less than molar concentration (for example, about 10) -7 Less than molar concentration, approximately 10 -8 Less than molar concentration, approximately 10 -9 Less than molar concentration, approximately 10 -10 K (less than molar concentration, etc.) D If it has this property, it "specifically binds to the target protein."

[0104] Various immunoassay formats suitable for selecting antibodies or other ligands are immunoreactive specifically to particular proteins. For example, solid-phase ELISA immunoassays are conventionally used to select monoclonal antibodies that are immunoreactive specifically to a protein. For a description of immunoassay formats and conditions that may be used to determine specific immunoreactivity, see Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed., Cold Spring Harbor Publications, New York (2013).

[0105] As used herein, the term “subject” means a living multicellular vertebrate, a category that includes humans and non-human mammals. In one example, the subject is a human. In a specific example, the subject is a human, a camel, or a bat. In a further example, a subject requiring inhibition of SARS-CoV or SARS-CoV-2 infection is selected. For example, the subject is uninfected and at risk of SARS-CoV or SARS-CoV-2 infection, or is infected and requires treatment.

[0106] As used herein, the term “therapeutic dose” means an amount of the drug, such as an antibody, provided herein, sufficient to prevent, treat (including preventive), and / or alleviate (for example, prevent, inhibit, and / or treat) the symptoms and / or underlying cause of a disorder or disease. In some examples, a therapeutic dose is sufficient to reduce or eliminate the symptoms of a disease such as SARS-CoV or SARS-CoV-2 infection. For example, this may be the amount necessary to inhibit or prevent viral replication or to measurably alter the symptoms of a viral infection. Generally, this amount is sufficient to measurably inhibit viral replication or infectivity.

[0107] This description is not intended to be a detailed catalog of all possible ways in which the present invention may be carried out or all possible features that may be added to the present invention. For example, a feature described in one example may be incorporated into another example, and a feature described in a particular example may be omitted from that example. Furthermore, many variations and additions to the various examples suggested herein will be apparent to those skilled in the art in light of this disclosure, which does not depart from the present invention. Accordingly, the following specification is intended to describe some detailed examples of the present invention and will not thoroughly illustrate all permutations, combinations, and variations thereof.

[0108] In one example, the desired response is to inhibit, mitigate, or prevent SARS-CoV or SARS-CoV-2 infection. For the composition to be effective, it is not necessary for SARS-CoV or SARS-CoV-2 infected cells to be completely eliminated, reduced, or prevented. For example, administration of a therapeutically effective dose of the drug may reduce the number of SARS-CoV or SARS-CoV-2 infected cells by a desired amount (e.g., at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or still at least 100% (elimination or prevention of detectable SARS-CoV or SARS-CoV-2 infected cells)) compared to the number of SARS-CoV or SARS-CoV-2 infected cells in the absence of the composition.

[0109] The therapeutically effective dose of the antibody disclosed herein may depend on the subject being treated, the severity and type of the condition being treated, and the mode of administration. The unit dosage of the antibody may be packaged in therapeutic doses or multiples of therapeutic doses, for example, in vials (e.g., with perforated lids) or syringes having sterile components.

[0110] Treatment or prevention of disease: for example, inhibiting the complete onset of a disease or symptoms in a subject at risk of a disease such as SARS-CoV or SARS-CoV-2 infection. "Treatment" refers to a therapeutic intervention that alleviates the signs or symptoms of a disease or condition after the onset of symptoms. In relation to a disease or condition, the term "alleviation" refers to any observable beneficial effect of treatment. Beneficial effects may be demonstrated, for example, by delaying the onset of clinical symptoms of a disease in a susceptible subject, reducing the severity of some or all clinical symptoms of the disease, slower disease progression, reduced viral load, improvement in the subject's overall health and well-being, or other parameters well known in the field that are specific to this particular disease. "Prophylactic" treatment is a treatment administered to a subject who is not showing signs of the disease or is showing only early signs, with the aim of reducing the risk of developing the pathology.

[0111] The terms “to treat,” “to treat,” or “to treat” (or grammatically equivalent terms) mean that the symptoms in question are reduced, at least partially improved, or alleviated, and / or relief, sedation, or reduction is achieved in at least one clinical symptom, and / or there is a delay in the progression of the symptoms.

[0112] As used herein, the terms “prevent,” “prevents,” or “prevention” and “inhibit,” “inhibits,” or “inhibition” (and their grammatical equivalents) encompass any kind of preventive treatment that reduces the onset of symptoms, delays the onset of symptoms, and / or alleviates symptoms associated with symptoms after their onset, without implying the complete elimination of a disease.

[0113] An “effective,” “preventively effective,” or “therapeutic effective” dose, as used herein, is an amount sufficient to provide any improvement or benefit to the subject. In other words, an “effective,” “preventively effective,” or “therapeutic effective” dose is an amount that provides some delay, relief, sedation, or reduction of at least one clinical symptom in the subject. Those skilled in the art will understand that the effect does not need to be complete or curative, as long as it provides some benefit to the subject.

[0114] The terms “reduce” or “mitigate,” as used herein, are relative terms, meaning that the drug reduces a response or symptom if, after administration, the response or symptom is quantitatively reduced or reduced compared to a reference drug. Similarly, the term “prevent” does not necessarily mean that the drug completely eliminates the response or symptom, as long as at least one characteristic of the response or symptom is absent. Therefore, a composition that reduces or prevents an infection or response may completely eliminate such an infection or response, but does not necessarily have to eliminate it completely, as long as such infection or response is measurably reduced by, for example, at least about 50% of the infection or response in the absence of the drug, e.g., at least about 70% or about 80%, or still about 90% (i.e., to less than 10%), or reduced compared to a reference drug.

[0115] As used herein, the term “vector” means a nucleic acid molecule that, upon introduction into a host cell, produces a transformed host cell. A recombinant DNA vector is a vector having recombinant DNA. A vector may include nucleic acid sequences, such as origins of replication, that enable replication within a host cell. A vector may also include one or more selectable marker genes and other gene elements known in the art. A viral vector is a recombinant nucleic acid vector having at least several nucleic acid sequences derived from one or more viruses. A replication-deficient viral vector is a vector that, due to a defect in the function of at least one replication-essential gene, requires the completion of one or more regions of the viral genome necessary for replication. For example, a viral vector in a human patient to whom a viral vector may be transmitted during the course of a therapeutic method, in particular, will not replicate in representative host cells.

[0116] Isolated binding protein of formula I In one aspect, this disclosure relates to an isolated binding protein that specifically binds to an epitope on the SARS-CoV and / or SARS-CoV-2 protein. Specifically, this isolated binding protein that specifically binds to an epitope on the SARS-CoV and / or SARS-CoV-2 protein may neutralize SARS-CoV and / or SARS-CoV-2 infection. Specifically, this isolated binding protein has an amino acid sequence comprising formula I: A-(B) n -C (formula I) During the ceremony, A is a receptor (e.g., angiotensin-converting enzyme 2 (ACE2), DPP4, or a variant thereof) used by SARS-CoV and / or SARS-CoV-2 proteins to mediate cell entry; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; B is a polypeptide linker; C is a fragment crystallization (Fc) domain. These proteins typically dimerize (for example, via an Fc domain).

[0117] The Fc domain used in Formula I may be any human Fc domain, and may be a human IgA, IgM, or IgG Fc domain. Furthermore, the Fc domain may be an optimized Fc domain, such as that described in U.S. Patent Application No. 2010 / 093979. In one aspect of this disclosure, the Fc domain is IgG1. Furthermore, the Fc domain may contain one or more amino acid substitutions (e.g., conservative substitutions) or mutations, for example, to enable enhanced neonatal Fc-receptor (FcRn) binding.

[0118] The ACE2 used in Formula I may be human ACE2. ACE2 or a fragment thereof (a fragment having a length of at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, etc.) may be used in Formula I. In some aspects, the extracellular domain of human ACE2 or a fragment thereof, in particular the extracellular domain excluding the collectrin domain, is used. Figure 15 shows an annotated sequence listing of wild-type human ACE2. This sequence shows the collectrin domain, amino acids 615-740 (enclosed region). A portion of the extracellular region of the ACE2 protein (e.g., amino acids 17, 19, or 19-614 or a portion thereof) may be used as described herein. See Sequence ID No. 11.

[0119] In general, the compositions and methods described herein for Formula I may be used with peptides that are approximately 80% identical to the extracellular region of ACE2, or a portion thereof, excluding the collectrin domain. Furthermore, ACE2 used in Formula I may include one or more amino acid substitutions (e.g., conservative substitutions) or mutations. In one aspect, the mutation eliminates the original enzymatic activity of the ACE2 molecule while protecting / preserving the dimerization domain of the Fc domain. Examples of ACE2 sequences that may be used in this disclosure are SEQ ID NOs: 2, 4, 11, 15, 17, 19, 21, and 23, which provide amino acid sequences of ACE2 including two substitutions or mutations that may be used in the binding proteins described herein. Both SEQ ID NOs: 2 and 4 include H374N and H378N substitutions or mutations.

[0120] Table 1 in Figure 16 describes the amino acid mutations that can be made individually or collectively in the extracellular ACE2 polypeptide sequence. Specifically, one or more (or all) amino acids in these residues may be modified, and the activity of the flexible ACE2 decoys described herein may be conserved (and in some cases enhanced compared to those formed by wild-type extracellular ACE2 polypeptides that do not contain the collectrin domain). For example, one or more amino acids at residue positions 19, 20, 24, 25, 27, 29, 31, 33, 34, 35, 37, 38, 39, 40, 41, 42, 69, 72, 75, 76, 79, 89, 90, 91, 92, 101, 110, 135-136, 160, 169, 192, 219, 239, 271, 273, 309, 312, 324, 324, 325, 330, 338-340, 345, 350, 351, 355, 359, 386, 389, 393, 465-467, 481, 505, 514, 518, and / or 603. Specific modifications in these residues may be those shown in Table 1 or different; in some cases, amino acid modifications may be conservative modifications, for example, based on charge and / or size. For example, SEQ ID NO: 15 shows an example of an extracellular ACE2 polypeptide in which five residues in the collectrin domain are modified: residues K31F, N33D, H34S, E35Q, and H345L. This variant of ACE2 can be linked to the Fc domain via a suitable mobile linker as described herein to form a flexible linked ACE2 decoy (an example of which is shown in SEQ ID NO: 16). SEQ ID NO: 17 shows another example of a variant of ACE2 (extracellular ACE2 with the collectrin domain excluded and residues T27Y, L79T, and N330Y modified). This may be linked to the Fc domain via a suitable mobile linker as described herein to form a flexible linked ACE2 decoy (an example of which is shown in SEQ ID NO: 18). Sequence ID 19 shows an example of another variant of ACE2 (extracellular ACE2 with the collectrin domain removed and residues T20I, H34A, T92Q and Q101H modified).This may be linked to the Fc domain via a suitable mobile linker as described herein to form a flexible ACE2 decoy (an example of which is shown in SEQ ID NO: 20). SEQ ID NO: 21 shows an example of another variant of ACE2 (extracellular ACE2 with the collectrin domain excluded and residues A25V, K31N, E34K and L79F modified). This may be linked to the Fc domain via a suitable mobile linker as described herein to form a flexible ACE2 decoy (an example of which is shown in SEQ ID NO: 22). SEQ ID NO: 23 shows an example of another variant of ACE2 (extracellular ACE2 with residue T27W modified). This may be linked to the Fc domain via a suitable mobile linker as described herein to form a flexible ACE2 decoy (an example of which is shown in SEQ ID NO: 24).

[0121] Any polypeptide linker, and especially flexible polypeptide linkers, can be used in Formula I to link extracellular ACE2 with the collectrin domain removed to the Fc domain. In some examples, this linker has the sequence GGGGS (SEQ ID NO: 11).

[0122] In some examples, the binding protein may also contain a hinge between the polypeptide linker and the Fc domain in formula I. The location of the hinge in formula I is not important. The hinge region may be anterior to the mobile linker (e.g., between the mobile linker and the extracellular ACE2 domain), posterior to the mobile linker (e.g., (G4S)2-hinge-(G4S)4), or posterior to it (e.g., between the mobile linker and the Fc domain).

[0123] In some further examples, the binding protein of formula I may also include a signal peptide. Examples of signal peptides that may be used are shown in SEQ ID NOs: 1 and 5. Other signal sequences may be used. The position of the signal peptide in formula I is not important.

[0124] In some cases, the binding protein is an antibody or an antibody-binding fragment thereof. When the binding protein is an antibody, this antibody may be a monoclonal antibody, a humanized antibody, a recombinant antibody, a chimeric antibody, a human antibody, a bispecific antibody, or a multispecific antibody. When the binding protein is an antibody-binding fragment, it may be a single-chain antibody, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, an Fsc fragment, an Fv fragment, scFv, sc(Fv)2, or a diabody. Methods for producing antibodies and antibody-binding fragments are well known in the art.

[0125] In certain contexts, amino acid sequence variants of binding proteins provided herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of a binding protein (for example, when the binding protein is an antibody). Amino acid sequence variants of binding proteins (e.g., antibodies) can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the binding protein (e.g., antibody) or by peptide synthesis. Such modifications include, for example, deletions from the amino acid sequence of the binding protein, and / or insertions therein, and / or substitutions of residues therein. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, as long as the final construct has the desired characteristics, such as antigen binding.

[0126] Examples of binding proteins of formula I of this disclosure include those shown in the drawings and those discussed in the following examples. The amino acid sequences of these binding proteins are presented in the sequence listing.

[0127] Isolated bispecific binding protein of formula II Furthermore, bispecific binding proteins that specifically bind to at least one epitope on the SARS-CoV and / or SARS-CoV-2 protein are described herein. Specifically, isolated bispecific binding proteins that specifically bind to at least one epitope on the SARS-CoV and / or SARS-CoV-2 protein can neutralize SARS-CoV and / or SARS-CoV-2 infection. Specifically, the isolated specific binding protein includes at least one heavy chain variable region having an amino acid sequence comprising formula II: X-(Y) n -Z (Formula II) During the ceremony, X is (i) a receptor used to mediate cell entry by SARS-CoV and / or SARS-CoV-2 proteins, such as angiotensin-converting enzyme 2 (ACE2), DPP4, or a variant thereof, or (ii) a variable heavy chain region derived from an antibody that binds to an epitope on SARS-CoV, SARS-CoV-2, or a fragment thereof; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; Y is a polypeptide linker; Z is (i) a receptor used to mediate cell entry by SARS-CoV and / or SARS-CoV-2 proteins, such as angiotensin-converting enzyme 2 (ACE2), DPP4, or a variant thereof, or (ii) a variable heavy chain region derived from an antibody that binds to SARS-CoV, SARS-CoV-2, or a fragment thereof, provided that (a) if X is a receptor used to mediate cell entry by SARS-CoV and / or SARS-CoV-2 proteins, then Z is a variable heavy chain region derived from an antibody that binds to SARS-CoV, SARS-CoV-2, or a fragment thereof, or (b) if X is a variable heavy chain region derived from an antibody that binds to SARS-CoV, SARS-CoV-2, or a fragment thereof, then Z is a receptor used to mediate cell entry by SARS-CoV and / or SARS-CoV-2 proteins. In formula II, if n is 0, there is no polypeptide linker.

[0128] The ACE2 used in Formula II may be human ACE2 or a variant (as already described above). Full-length ACE2 or a fragment thereof (fragments having lengths such as at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, etc.) may be used in Formula II. In some aspects, the extracellular domain of human ACE2 or a fragment thereof may be used. Furthermore, the ACE2 used in Formula II may include one or more amino acid substitutions (e.g., conservative substitutions) or mutations. In one aspect, the mutation eliminates the original enzymatic activity of the ACE2 molecule while protecting / preserving the dimerization domain of the Fc domain. Examples of ACE2 sequences that may be used in this disclosure are SEQ ID NOs: 2, 4, 11, 15, 17, 19, 21, and 23, which provide amino acid sequences of ACE2 including two substitutions or mutations that may be used in the binding proteins described herein. Both SEQ ID NOs. 2 and 4 contain H374N and H378N substitutions or mutations.

[0129] Any polypeptide linker can be used in formula II to link X to Z in formula II. In some examples, this linker has the sequence GGGGS (sequence number 11). Alternatively, in some examples, no linker is present, and X is directly linked to Z (for example, when n is 0).

[0130] In some cases, the binding protein may also contain a hinge between the polypeptide linker and X and Z in formula II. The position of the hinge in formula II is not important.

[0131] In some further examples, the binding protein of formula II may also include a signal peptide. Examples of signal peptides that may be used are shown in SEQ ID NOs: 1 and 5. The position of one peptide in formula II is not important.

[0132] As previously stated, in formula II, if X is ACE2, then Z is a variable heavy chain region derived from a binding protein that specifically binds to at least one epitope on SARS-CoV, SARS-CoV-2, or a fragment thereof (e.g., a fragment of SARS-CoV or a fragment of SARS-CoV-2). Alternatively, if X is a variable heavy chain region derived from a binding protein that specifically binds to at least one epitope on SARS-CoV, SARS-CoV-2, or a fragment thereof, then Z is ACE2. Examples of variable heavy chain regions derived from binding proteins that specifically bind to at least one epitope on SARS-CoV-2 that may be used in binding proteins are the monoclonal antibodies CR3014 or CR3022, which are described in J. ter Meulen, PLoS Medicine, 3(7):1071-1079 (July 2006), and their contents are incorporated herein by reference. The entire variable heavy chain region (e.g., CR3014 or CR3022) or a fragment thereof (a fragment having a length such as at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or at least 100 amino acids) derived from a binding protein that specifically binds to at least one epitope on SARS-CoV or SARS-CoV-2 may be used.

[0133] In some cases, this binding protein is an antibody or an antibody-binding fragment. If this binding protein is an antibody, it may be a bifunctional or heterofunctional antibody. In some aspects, the bispecific antibody may be an scFv. Methods for producing antibodies and antibody-binding fragments are well known in the art.

[0134] In certain contexts, amino acid sequence variants of binding proteins provided herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of a binding protein (for example, when the binding protein is an antibody). Amino acid sequence variants of binding proteins (e.g., antibodies) can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the binding protein (e.g., antibody) or by peptide synthesis. Such modifications include, for example, deletions from the amino acid sequence of the binding protein, and / or insertions therein, and / or substitutions of residues therein. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, as long as the final construct has the desired characteristics, such as antigen binding.

[0135] The bispecific binding protein of formula II may also include other proteins, such as an antibody-variable light chain region derived from another antibody, a variable heavy chain region derived from another antibody, one or more CDRs, one or more light and heavy chain constant regions, a framework region, and an Fc domain derived from another binding protein. If an Fc domain is used, the Fc domain may be any human Fc domain, such as a human IgA, IgMorIgG Fc domain. Furthermore, the Fc domain may be an optimized Fc domain, as described in U.S. Patent Application No. 2010 / 093979. In one aspect of this disclosure, the Fc domain is IgG1. Furthermore, the Fc domain may include one or more amino acid substitutions (e.g., conservative substitutions) or mutations, for example, to enable enhanced neonatal Fc-receptor (FcRn) binding. An example of such other proteins is one or more variable light chain regions derived from an antibody that specifically binds to at least one epitope on SARS-CoV and / or SARS-CoV-2. For example, the light chain variable region of CR3022 having the amino acid sequence in SEQ ID NO: 6 can be used in conjunction with the binding protein of formula II to produce a bispecific antibody.

[0136] An example of the bispecificity-binding protein of formula II of this disclosure is shown in Figure 5. The amino acid sequence for this bispecificity-binding protein is provided in the sequence listing.

[0137] Polynucleotides and expression Also provided are polynucleotides encoding binding proteins of formula I or II that specifically bind to epitopes on SARS-CoV and / or SARS-CoV-2 proteins. These polynucleotides comprise DNA, cDNA, and RNA sequences encoding the binding proteins of formula I or II of this disclosure. Nucleic acids encoding these molecules can be readily generated by those skilled in the art using amino acid sequences presented herein (e.g., CDR and heavy and light chain sequences for antibody production), sequences available in the art (e.g., framework sequences), and the genetic code. Those skilled in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids encoding the same antibody sequence but with different sequences, or nucleic acids encoding conjugate or fusion proteins containing these nucleic acid sequences.

[0138] Polynucleotides encoding the binding protein of the present disclosure of formula I or II may be obtained by any preferred method, for example, a method including the cloning of a suitable sequence, or by the phosphotryster method described by Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method described by Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramide method described by Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; or the solid-phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, e.g., Needham-VanDevanter et al., Nucl Acids It can be prepared using an automated synthesizer as described in Res.12:6159-6168,1984; and by direct chemical synthesis, such as the solid support method described in U.S. Patent No. 4,458,066. Chemical synthesis produces single-stranded oligonucleotides, which can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization using DNA polymerase with this single strand as a template. While chemical synthesis of DNA is generally limited to sequences of about 100 bases, those skilled in the art will recognize that longer sequences can be obtained by ligation of shorter sequences.

[0139] Examples of appropriate cloning and sequencing techniques, as well as sufficient explanations to allow those skilled in the art to perform numerous cloning exercises, are publicly known (see, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed, Cold Spring Harbor, NY, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Product information from manufacturers of biological reagents and laboratory equipment also provides useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., and GIBCO BRL Life. Examples include Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), and Applied Biosystems (Foster City, Calif.), as well as many other market sources known to those skilled in the art.

[0140] Nucleic acids may also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and self-sustaining sequence replication systems (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to those skilled in the art.

[0141] Nucleic acid molecules can be expressed in recombinantly genetically modified cells, such as bacteria, plant cells, yeast cells, insect cells, and mammalian cells. Methods for expressing DNA sequences containing eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archaea, insects, fungi (e.g., yeast), plant cells, and animal cells (e.g., mammalian cells such as human cells). Exemplary cells for use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian myeloma and lymphocyte cell lines. Techniques for culturing mammalian cells are well known (see, for example, Helgason and Miller (Eds.), 2012, Basic Cell Culture Protocols (Method in Molecular Biology), 4th Ed., Humana Press). Examples of commonly used mammalian host cell lines include VERO cells and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, but cell lines such as those specified to provide higher expression, desired glycosylation patterns, or other characteristics may also be used. In some examples, the host cell is HEK293 cell or its derivative, e.g., GnTI - / - cells (ATCC(registered trademark) number CRL-3022) or HEK-293F cells are included.

[0142] The expression of protein-encoding nucleic acids described herein can be achieved by the operable ligation of DNA or cDNA to a promoter (which may be constitutive or inductive) and subsequent incorporation into an expression cassette. The promoter may be any promoter of interest, such as the cytomegalovirus promoter and the human T-cell lymphoproliferative virus promoter (HTLV)-1. Optionally, an enhancer, such as the cytomegalovirus enhancer, is incorporated into the construct. The cassette may be suitable for the replication and integration of either prokaryotes or eukaryotes. Typical expression cassettes include specific sequences useful for regulating the expression of protein-encoding DNA. For example, an expression cassette may include a suitable promoter, enhancer, transcription and translation terminators, start sequences, a start codon (i.e., ATG) preceding the protein-encoding gene, splicing signals for introns, sequences for maintaining the correct reading frame of the gene to enable correct translation of mRNA, and stop codons. The vector may encode selectable markers, such as markers encoding drug resistance (e.g., ampicillin or tetracycline resistance).

[0143] To obtain high levels of expression of the cloned gene, a construct expression cassette containing a minimal number of strong promoters to support transcription, ribosome-binding sites (internal ribosome-binding sequences) for translation initiation, and transcription / translation terminators is desirable. For Escherichia coli (E. coli), this includes promoters such as T7, trp, lac, or lambda promoters, ribosome-binding sites, and preferably transcription stop signals. For eukaryotic cells, the regulatory sequences may include, for example, immunoglobulin genes, promoters and / or enhancers derived from HTLV, SV40, or cytomegalovirus, as well as polyadenylation sequences, and may further include splicing donor and / or acceptor sequences (e.g., CMV and / or HTLV splicing acceptor and donor sequences). This cassette can be transferred into selected host cells by known methods, such as transformation or electroporation and calcium phosphate treatment for E. coli, and electroporation or lipofection for mammalian cells. Cells transformed by this cassette can be selected based on their resistance to antibiotics conferred by genes contained within the cassette (e.g., AMP, GPT, NEO, and HYG genes).

[0144] When the host is a eukaryote, conventional mechanical procedures such as calcium phosphate coprecipitation, microinjection, and electroporation, as well as DNA transfection methods such as insertion of liposome-encapsulated plasmids or viral vectors, may be used. Eukaryotic cells can also be co-transformed with SARS-CoV or SARS-CoV-2 S, M, N, or E binding proteins or fragments thereof, or polynucleotide sequences encoding antibodies, antibody-binding fragments, or conjugates that specifically bind to SARS-CoV or SARS-CoV-2 S, M, N, or E proteins, and a second exogenous DNA molecule encoding a selectable phenotype, such as the herpesthymidine kinase gene. Another method involves transiently infecting eukaryotic cells with eukaryotic viral vectors, such as Simian virus 40 (SV40) or bovine papillomavirus, i.e., transforming them to express proteins (see, e.g., Viral Expression Vectors, Springer Press, Muzyczka ed., 2011). Those skilled in the art can easily use expression systems such as plasmids and vectors that are useful for protein production in cells including higher eukaryotic cells (e.g., COS, CHO, HeLa, and myeloma cell lines).

[0145] When the binding protein is an antibody or antigen-binding fragment, such antibodies and antigen-binding fragments are individual V H and / or V L The antibody may be expressed as a chain (linked to an effector molecule or detectable marker if necessary) or as a fusion protein. Methods for expressing and purifying antibodies and antigen-binding fragments are known and further described herein (see, for example, Al-Rubeai (ed), Antibody Expression and Production, Springer Press, 2011). The nucleic acid sequence may optionally encode a leader sequence.

[0146] To create scFv, V H and VL V array linked by a movable linker H and V L V H and V L A DNA fragment encoding a linker can be operably linked to another fragment encoding a mobile linker, such as a fragment encoding the amino acid sequence (Gly4-Ser)3 (see, for example, Bird et al., Science 242:423-426, 1988; Huston et al., Proc.Natl.Acad.Sci.USA 85:5879-5883, 1988; McCafferty et al., Nature 348:552-554, 1990; Kontermann and Dubel (Ed), Antibody Engineering, Vols.1-2, 2nd Ed., Springer Press, 2010; Harlow and Lane, Antibodies: A Laboratory Manual, 2nd, Cold Spring Harbor Laboratory, New York, 2013). Optionally, cleavage sites, such as furin cleavage sites, may be included within the linker.

[0147] V H and / or V L The nucleic acid encoding may optionally encode an Fc domain (immunoadhesin). The Fc domain may be an IgA, IgM, or IgG Fc domain. The Fc domain may be an optimized Fc domain, such as that described in U.S. Patent Application Publication 20100 / 093979, which is incorporated herein by reference. In one example, the immunoadhesin is IgG1 Fc.

[0148] Single-chain antibodies are V H and V L When used, it may be unvalent, and two V H and V L When used, it may be divalent, or more than 2 V H and V LIf used, it may be polyvalent. Bispecific or polyvalent antibodies that specifically bind to SARS-CoV S, M, N and / or E proteins and / or other antigens may be prepared.

[0149] Methods for the expression of binding proteins, such as antibodies and antigen-binding fragments, derived from mammalian cells and bacteria such as Escherichia coli, and / or for their folding into appropriate active forms, are described and well known and applicable to the antibodies disclosed herein. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, 2nd, Cold Spring Harbor Laboratory, New York, 2013; Simpson ed., Basic Method in Protein Purification and Analysis: A Laboratory Manual, Cold Harbor Press, 2008; and Ward et al., Nature 341:544, 1989.

[0150] Also provided are populations of cells comprising at least one host cell as described herein. The population of cells may be a heterogeneous population comprising at least one other cell, for example, a host cell not comprising any recombinant expression vector (e.g., a T cell), or a cell other than a T cell, for example, a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc., in addition to a host cell comprising one of the recombinant expression vectors described herein. Alternatively, the population of cells may be substantially homogeneous, and this population mainly comprises (e.g., essentially consists of) host cells comprising recombinant expression vectors. This population may also be a clonal population of cells, where all cells in the population are clones of a single host cell comprising a recombinant expression vector, such that all cells in the population comprise a recombinant expression vector. In one example of this disclosure, the population of cells is a clonal population comprising host cells comprising recombinant expression vectors as described herein.

[0151] Nucleic acids encoding polypeptides described herein can be modified without reducing their biological activity. Some modifications may be made to facilitate cloning, expression, or incorporation of target molecules into fusion proteins. Such modifications are well known to those skilled in the art and include, for example, stop codons, methionine addition at the amino terminus to provide an initiation site, additional amino acids positioned at any terminus to create conveniently located restriction sites, or additional amino acids (e.g., polyHis) to assist in purification steps. In addition to recombination methods, immunoconjugates, effector moieties, and antibodies of this disclosure can also be constructed whole or in part using standard peptide synthesis methods well known in the art.

[0152] In some cases, nucleic acid molecules encode precursors of binding proteins of the Disclosure that, when expressed in appropriate cells, can be processed into SARS-CoV or SARS-CoV-2 proteins or fragments thereof. For example, a nucleic acid molecule may encode a binding protein of the Disclosure that includes an N-terminal signaling sequence for entry into the cellular secretory system, which is proteolytically cleaved during the intracellular processing of SARS-CoV or SARS-CoV-2 proteins or fragments thereof.

[0153] The polynucleotides encoding the binding proteins of this disclosure may include recombinant DNA that is incorporated within a vector, an autologous plasmid or virus, within prokaryotic or eukaryotic or genomic DNA, or as a separate molecule (e.g., mRNA or cDNA) independent of other sequences. This nucleotide may be a ribonucleotide, a deoxyribonucleotide, or a modified form of any of these nucleotides. The term includes single-stranded and double-stranded forms of DNA. In one non-limiting example, the immunoglobulins of this disclosure are expressed using the pVRC8400 vector (described in Barouch et al., J. Virol, 79, 8828-8834, 2005, which is incorporated herein by reference).

[0154] Once expressed, the binding proteins or antibodies, or antibody-binding fragments of this disclosure, which specifically bind to epitopes on the SARS-CoV or SARS-CoV-2 proteins, can be purified according to standard procedures in the field, including ammonium sulfate precipitation, affinity columns, and column chromatography (see, in general, Simpson ed., Basic Method in Protein Purification and Analysis: A Laboratory Manual, Cold Harbor Press, 2008). The SARS-CoV or SARS-CoV-2 proteins or fragments thereof, or the antibodies or antibody-binding fragments that specifically bind to epitopes on SARS-CoV or SARS-CoV-2, do not need to be 100% pure.

[0155] In many cases, functional heterologous proteins from E. coli or other bacteria need to be isolated from inclusion bodies, solubilized with a strong denaturant, and then refolded. During the solubilization process, a reducing agent must be present to separate the disulfide bonds, as is well known in the art. An exemplary buffer containing a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of disulfide bonds can occur in the presence of low molecular weight thiol reagents in reducing and oxidizing forms, as described in Saxena et al., Biochemistry 9:5015-5021, 1970, and in particular by Buchner et al., as mentioned above.

[0156] In addition to recombination methods, binding proteins containing any antibody or antigen-binding fragment can be constructed whole or partially using standard peptide synthesis. Solid-phase synthesis of polypeptides can be achieved by attaching the C-terminal amino acid of the sequence to a soluble support, and then sequentially adding the remaining amino acids in the sequence. Techniques for solid-phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol.2: Special Method in Peptide Synthesis, Part A. pp.3-284; Merrifield et al., J.Am.Chem.Soc.85:2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Longer proteins can be synthesized by condensation of the amino and carboxyl terms of shorter fragments. Methods for forming peptide bonds by activating the carboxyl terminus (for example, by using the coupling reagent N,N'-dicyclohexylcarbodiimide) are well known in the field.

[0157] Composition and administration The binding proteins of formula I or II are often included in pharmaceutical compositions (including therapeutic and prophylactic formulations) in combination with one or more pharmaceutically acceptable vehicles and optionally other therapeutic components (e.g., antibiotics or antiviral agents). These compositions are useful, for example, for the treatment or detection of SARS-CoV or SARS-CoV-2 infection, or for inducing an immune response to SARS-CoV or SARS-CoV-2 infection in a subject.

[0158] This composition may be prepared in unit dose form for administration to a subject. The amount and timing of administration are determined at the discretion of the treating clinician to achieve the desired objective. The binding proteins of this disclosure, or polynucleotides encoding such molecules, may be formulated for systemic or topical administration. In one example, the binding proteins of this disclosure, or polynucleotides encoding such molecules, that specifically bind to an epitope on SARS-CoV or SARS-CoV-2, may be formulated for parenteral administration, for example, intravenous administration.

[0159] The binding proteins of this disclosure, or polynucleotides encoding such molecules, or compositions comprising such molecules, as well as further agents, may be administered to a subject by various means, including local and systemic administration, such as by subcutaneous, intravenous, intra-arterial, intranasal, intraperitoneal, intramuscular, intradermal, or intrathecal injection. In one example, the therapeutic agent is administered by one subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection once daily. The therapeutic agent may also be administered by direct injection at or near the site of the disease.

[0160] In some cases, this composition is administered by inhalation (e.g., by aerosol delivery) using a nebulizer, such as a vibrating mesh nebulizer. In other cases, this composition may be used with a dry powder inhaler or a fixed-dose inhaler.

[0161] Further administration methods may involve an osmotic pump (e.g., an Alzet pump) or minipump (e.g., an Alzet mini-osmotic pump) that enables controlled, continuous, and / or sustained-release delivery of the therapeutic agent or pharmaceutical composition over a predetermined period of time. The osmotic pump or minipump may be implanted subcutaneously or near the target site.

[0162] Therapeutic agents or compositions thereof may also be administered by other means. Determining the most effective mode of administration of a therapeutic agent or composition thereof is within the scope of the art of those skilled in the art. Therapeutic agents may be administered, for example, as pharmaceutical formulations suitable for oral (including buccal and sublingual) administration, rectal administration, nasal administration, topical administration, pulmonary administration, vaginal administration or parenteral administration, or in a form suitable for administration by inhalation or inhalation. Depending on the intended mode of administration, pharmaceutical formulations may be in solid, semi-solid or liquid dosage forms, such as tablets, suppositories, pills, capsules, powders, liquids, suspensions, emulsions, creams, ointments, lotions, etc.

[0163] In some cases, the composition may be provided in unit dosing form for use to induce an immune response in a subject, for example, to prevent, inhibit, or treat SARS-CoV or SARS-CoV-2 infection in the subject. The unit dosing form includes a pre-selected single-dose form suitable for administration to the subject, or two or more pre-selected unit dosing forms that are suitablely marked or quantified, and / or a measuring mechanism for administering a unit dose or a multiple thereof. In other examples, the composition further comprises an adjuvant.

[0164] Typical compositions for intravenous administration of binding proteins of formula I or II contain approximately 0.01 to approximately 30 mg / kg per patient per day. Practical methods for preparing administerable compositions are known and obvious to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa. (1995).

[0165] For the preparation of pharmaceutical compositions, the conjugate proteins of this disclosure, or polynucleotides encoding such molecules, which specifically bind to epitopes on SARS-CoV or SARS-CoV-2 proteins, may be combined with a variety of pharmaceutically acceptable additives, as well as a base or vehicle for dispersing the conjugate. Desired additives include, but are not limited to, pH regulators such as arginine, sodium hydroxide, glycine, hydrochloric acid, and citric acid. Furthermore, local anesthetics (e.g., benzyl alcohol), isotonic agents (e.g., sodium chloride, mannitol, sorbitol), absorption inhibitors (e.g., TWEEN® 80), absorption enhancers (e.g., cyclodextrins and their derivatives), stabilizers (e.g., serum albumin), and reducing agents (e.g., glutathione).

[0166] Compositions for administration may include solutions of the binding protein of the Disclosure or polynucleotides encoding such molecules, dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. Various aqueous carriers, such as buffered saline, may be used. Since the composition needs to approximate a physiological state, it may also contain pharmaceutically acceptable adjuvants or excipients, such as pH adjusters and buffers, toxicity modifiers, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the binding protein of the Disclosure, or polynucleotides encoding such molecules, that specifically binds to epitopes on SARS-CoV or SARS-CoV-2 proteins, in these formulations can vary widely and is selected first based on liquid volume, viscosity, body weight, etc., according to the needs of a selected specific mode of administration and target population.

[0167] The binding proteins of this disclosure, or polynucleotides encoding such molecules, may be provided in lyophilized form and rehydrated with sterile water before administration, or they may be provided in sterile solutions of known concentrations.

[0168] Movable linker A mobile linker may be included in any of the flexibly linked ACE2 decoys described herein. Any suitable mobile linker may be used between each ACE2 region and Fc region, and more specifically, one having a length longer than 5 nm (so that the total length between the two ACE2 regions is greater than approximately 14 nm) may be used. When used herein, the mobile linker may provide some degree of movement between each ACE2 region and Fc region. This mobile linker may generally consist of small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. These small-sized amino acids may provide flexibility and allow movement of the binding region. The incorporation of Ser or Thr may maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules and may reduce undesirable interactions between the linker and protein portions.

[0169] This mobile linker may have a sequence mainly consisting of Gly and Ser residues ("GS" linker). An example of a mobile linker is (Gly-Gly-Gly-Gly-Ser). n It has the sequence (KESGSVSSEQLAQFRSLD). By adjusting the copy number "n", the length of this GS linker is adjusted to achieve proper separation of the functional region (e.g., the ACE2 region or other binding regions). Other mobile linkers may be mostly small or polar amino acids, such as Gly and Ser, but may also contain additional amino acids such as Thr and Ala to maintain flexibility, and polar amino acids such as Lys and Glu to improve solubility. Other types of mobile linkers include KESGSVSSEQLAQFRSLD and EGKSSGSGSESKST. These linkers are (KESGSVSSEQLAQFRSLD) n Or (EGKSSGSGSESKST) n This may be repeated. Another movable linker is GSAGSAAGSGEF, or (GSAGSAAGSGEF) nIn any of these linkers, the linker length can be adjusted by selecting the number of repetitions n (for example, n is 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, etc.). The length of each linker, for example, the linker between the ACE2 region and the Fc region, can be selected for complete isolation of the ACE2 domain (or, in some examples, the ACE2 domain and another COV).

[0170] Mucus trap As used herein, the term “trapping ability” means the ability of a binding protein (e.g., a binding protein described herein) to specifically bind to a target pathogen and inhibit the movement of the pathogen through the mucus. Traping ability can be measured by methods known in the art and by methods disclosed herein. Traping ability can be quantified, for example, as the amount of binding protein (e.g., the concentration of binding protein in the mucus) required to reduce the motility of pathogens in a mucus gel by at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%) and to at least half (e.g., one-quarter, one-tenth) of the natural motility in a solution (e.g., physiological saline) and / or mucus. Motility in the mucus can be measured using techniques known in the art and by techniques described herein. Alternatively, trapping ability may be quantified as a percentage reduction in the amount of pathogens penetrating the mucus.

[0171] The term "enhancing trapping ability" means an increase compared to a protein (e.g., the Fc domain in a flexibly linked ACE2 decoy described herein). Furthermore, any binding protein described herein may be selected or further configured to enhance mucin crosslinking by including a glycosylation pattern comprising a branched coreglycan structure Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4G1cNAcβ1 having terminal N-acetylglucosamines at each branch. This glycosylation pattern may be on the Fc region of the protein (e.g., a flexibly linked ACE2 decoy). Alternatively, or further, the composition of the constructs described herein may be selected or configured such that at least x% of the constructs (e.g., dimerized flexible linked ACE2 decoy-binding proteins) have a glycosylation pattern comprising a branched coreglycan structure Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4G1cNAcβ1 having terminal N-acetylglucosamines on each branch, where x% is 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or substantially all of the constructs. For example, compositions containing more than 20% (more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, etc.) of the constructs described herein, which provide increased mucin crosslinking, may be particularly beneficial for mucus trapping of targets (e.g., SARS-like CoV) once they have bound to them.

[0172] The binding proteins, compositions, and methods comprising flexible linked ACE2 decoys described herein may include methods for inhibiting and / or treating infection by SARS-like CoV (and more specifically SARS-CoV-2), and / or removing pathogens from mucosal surfaces. More specifically, the subject of this disclosure relates to constructs and compositions that enable the aggregation of pathogens (e.g., SARS-CoV) and / or the restraining of pathogen replication, and / or the trapping of pathogens in mucus, thereby inhibiting the delivery of pathogens via mucus secretion, which may result in the destruction and / or natural elimination of these pathogens.

[0173] The binding protein constructs described herein (including flexibly linked ACE2 decoys) can generally diffuse rapidly through mucus, slowed only slightly by weak, transient adhesive interactions with mucin in the mucus. This rapid diffusion allows the construct to collect pathogens. When multiple constructs are bound to a pathogen, the adhesive interactions between the multiple constructs and the mucus may be sufficient to trap the bound pathogen in the mucus, thereby preventing or mitigating infection. Pathogens trapped in the mucus cannot reach target cells in the body and are instead swept away and / or inactivated by spontaneous thermal degradation and further protective factors in the mucus, such as defensins. As disclosed herein, this pathogen aggregation and / or trapping activity provides protection without neutralization and can effectively inhibit infection even at relatively low doses. The low-affinity interactions that the constructs described herein may form with mucin may also be influenced by glycosylation.

[0174] Therefore, the constructs described herein may contain oligosaccharides at the glycosylation site (more specifically, on the Fc domain), which contain or consist of (or, in some examples, are essentially thereto) a pattern related to (providing) an increased trapping ability of the binding protein in mucus. The binding protein specifically binds to a target (e.g., a SARS-like CoV target such as SARS-CoV-2). The glycosylation pattern / oligosaccharide component of the binding protein (e.g., a flexibly linked ACE2 decoy protein) can maximize the trapping ability of the binding protein once it has complexed with one or more targets (e.g., pathogens such as SARS-CoV-2), without unduly hindering the ability of the unbound construct to rapidly diffuse through mucus to rapidly bind the target. In certain cases, the constructs described herein, once complexed with one or more targets, exhibit mucus motility reduced to about 50% or less of the natural motility in a solution (e.g., mucus, saline, or water), for example, to about 40%, 30%, 20%, 10%, or 5% or less, and effectively trap target pathogens in the mucus (e.g., at least 50% of the targets are slowed to at least half their motility). In some cases, the constructs described herein reduce the motility of at least 50% of the targets, for example, at least 50%, 60%, 70%, 80%, or 90% or more of the targets, by at least 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) or more. In other examples, the structures described herein reduce the percentage of mucus-penetrating targets (e.g., pathogens) by at least 10%, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.For example, the constructs described herein may have a sufficient binding rate to target epitopes to trap target pathogens in mucus within one hour (e.g., within 30 minutes or 15 minutes) at mucus construct concentrations of less than 10 mg / ml (e.g., less than 5 mg / ml, less than 1 mg / ml, less than 0.1 mg / ml, less than 50 μg / ml, less than 30 μg / ml, less than 20 μg / ml, less than 10 μg / ml, less than 5 μg / ml, less than 2.5 μg / ml, less than 1 μg / ml, less than 0.5 μg / ml, less than 0.1 μg / ml, etc.).

[0175] In some examples, the constructs described herein may include oligosaccharide components bound to an N-linked glycosylation site in the Fc region of the construct. The N-linked glycosylation site may be an asparagine residue on the Fc region, for example, the Asn297 asparagine residue. Amino acid numbering is based on the standard amino acid structure of human / humanized IgG molecules. As described above, Fc regions derived from IgM, IgD, IgG, IgA, and IgE, or modified variants thereof, may be used.

[0176] The N-glycan structure may be in the G0 / G0F form or in the pure GnGn form (for example, having terminal N-acetylglucosamine on each chain and not having terminal galactose or sialic acid). In some examples, this oligosaccharide component, i.e., the glycan bound to the construct, contains, is essentially, or consists of a core structure that has no fucose residues. In some examples, this oligosaccharide component contains fucose on the side chains. In other examples, this glycan does not contain any galactose residues. In some examples, this glycan does not contain galactose.

[0177] The constructs described herein may comprise a mixture of constructs having different oligosaccharide components. In some examples, this mixture comprises at least about 30%, for example, at least about 40%, 50%, 60%, 70%, 80%, 90% or more, of G0 / G0F coreglycan structures (e.g., with or without fucose residues).

[0178] In some examples, constructs produced in human cell lines (e.g., 293 cell line, e.g., 293T cell line), other mammalian cell lines (e.g., CHO), plants (e.g., Nicotiana), or other microorganisms (e.g., Trichoderma) are described herein.

[0179] The constructs described herein may be useful for binding to a target and trapping it in the mucus, thereby inhibiting infection by that target. The constructs described herein may be used to treat, prevent, or mitigate infection by any virus that binds to ACE2, such as coronaviruses (e.g., SARS-CoV-2), which can infect a target through the mucous membrane.

[0180] The terms virus, pathogen and viral pathogen may be used interchangeably herein and further refer to any virus that binds to ACE2, such as coronavirus (e.g., SARS-CoV-2).

[0181] composition As will be recognized by those skilled in the art, the constructs described herein may also be formed into suitable compositions, for example, pharmaceutical compositions for administration to a subject, for treating or preventing infection or disease or disorder caused by infection with a target pathogen (e.g., a virus that binds to ACE2, such as a coronavirus like SARS-CoV-2). The composition may comprise, essentially consist of, or consist of, a preventive or therapeutic dose of the constructs described herein and a pharmaceutically acceptable carrier.

[0182] Pharmaceutical compositions comprising the constructs described herein may be formulated in combination with any suitable pharmaceutical vehicle, excipient, or carrier commonly used in the art (including conventional materials for this purpose, e.g., saline, dextrose, water, glycerol, ethanol, and combinations thereof). As those skilled in the art will recognize, the specific vehicle, excipient, or carrier used will vary depending on the subject or state of the subject, and various modes of administration will be suitable for the compositions described herein. Preferred methods of administration of any pharmaceutical composition disclosed herein include, but are not limited to, topical, oral, intranasal, buccal, inhalation, rectal, and vaginal administration. Herein, such administration achieves the delivery of the binding protein to the target mucous membrane.

[0183] The composition may be any type of composition suitable for delivering the constructs described herein to a mucosal surface, and may be in various forms known in the art (solid, semi-solid or liquid form, or lotion form, oil in water or water in oil emulsion, or in an aqueous gel composition). The composition may be, but is not limited to, a gel, paste, suppository, irrigation, vaginal suppository, foam, film, spray, ointment, pessary, capsule, tablet, jelly, cream, milk, dispersion, liposome, powder / talc or other solid, suspension, solution, emulsion, microemulsion, nanoemulsion, liquid, aerosol, microcapsule, time-delayed release capsule, controlled-release formulation, sustained-release formulation or bioadherent gel (e.g., a mucosal-adherent thermosensitive gelling composition), or other forms encapsulated in a matrix for delayed or controlled release of the composition to a surface to which it is applied or in contact.

[0184] When topical administration is preferred, the composition may be formulated in a suitable form as needed (e.g., ointment, cream, gel, lotion, dropper (e.g., eye drops and ear drops) or solution (e.g., mouthwash)). The composition may contain conventional additives such as preservatives, solvents to promote penetration, and emollients. Topical formulations may also contain a cream or ointment base and conventional carriers such as ethanol or oleyl alcohol. Other formulations for administration, including intranasal administration, are intended for use relating to the subject matter of this disclosure. All formulations, devices, and methods known to those skilled in the art that are suitable for delivering the constructs described herein or compositions containing the constructs described herein to one or more mucous membranes of interest may be used relating to the subject matter of this disclosure.

[0185] Any of the compositions described herein may include mixtures of the constructs described herein.

[0186] The compositions used in the methods described herein may include antibodies and antiviral agents, as well as other agents that do not adversely affect or otherwise inhibit the efficacy of the components of the composition. For example, pharmaceutically acceptable carriers, diluents, vehicles, or excipients, which are solids, liquids, or mixtures of solids and liquids, may be used in the pharmaceutical composition. Suitable, physiologically acceptable, and substantially inert carriers include water, polyethylene glycol, mineral oil or petrolatum, propylene glycol, hydroxyethylcellulose, carboxymethylcellulose, cellulosic derivatives, polycarboxylic acids, conjugated polyacrylic acids, e.g., carbopols; and other polymers such as poly(lysine), poly(glutamic acid), poly(maleic acid), poly(lactic acid), thermopolyaspartate, and aliphatic-aromatic resins; glycerin, starch, lactose, calcium sulfate dihydrate, clay, sucrose, talc, gelatin, pectin, acacia, magnesium stearate, stearic acid, syrup, peanut oil, olive oil, and physiological saline solution.

[0187] Pharmaceutical compositions useful in the methods of the present invention, as described herein, may further comprise diluents, fillers, binders, colorants, stabilizers, fragrances, gelling agents, antioxidants, humectants, preservatives, acids, and other elements known to those skilled in the art. For example, suitable preservatives are well known in the art and include, for example, methylparaben, propylparaben, butylparaben, benzoic acid, and benzyl alcohol.

[0188] The carrier for injection may typically be a liquid, such as sterile pyrogen-free water, pyrogen-free buffered saline solution, bacteriostatic water, or CremophorEL® (BASF, Parsippany, NJ). Carriers for other administration methods may be solid or liquid.

[0189] For oral administration, the constructs described herein may be administered in solid dosage forms, e.g., capsules, tablets, and powders, or in liquid dosage forms, e.g., elixirs, syrups, and suspensions. The compositions may be encapsulated in gelatin capsules together with inert components and powdered carriers, e.g., glucose, lactose, sucrose, mannitol, starch, cellulose, or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate, etc. Examples of further inert components that can add desirable color, taste, buffering capacity, dispersion, or other known desirable characteristics include red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink. Similar diluents may be used to prepare compressed tablets. Both tablets and capsules may be manufactured as sustained-release products to provide continuous release of the pharmaceutical over several hours. Compressed tablets may be sugar-coated or film-coated to mask any unpleasant taste and protect the tablets from the environment, or they may be enterically coated for selective disintegration in the gastrointestinal tract. Liquid formulations for oral administration may contain colorants and flavorings to increase patient acceptance.

[0190] Suitable compositions for oral (sublingual) administration include tablets or lozenges containing the binding protein in a flavoring base (usually sucrose and acacia or tragacanth); and lozenges containing the binding protein in an inert base (gelatin and glycerin or sucrose and acacia). This composition may include compositions that dissolve or disintegrate in the mouth. Alternatively, this composition may include a powder, aerosolized or granulated solution or suspension containing the binding protein. When dispersed, such powdered, aerosolized or granulated compositions preferably have an average particle size or average droplet size in the range of about 0.1 to about 200 nanometers.

[0191] Compositions of the constructs described herein, suitable for parenteral administration, include sterile aqueous and sterile non-aqueous injectable solutions of the constructs described herein, which are preferably isotonic with the blood of the intended recipient. These preparations may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the composition isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may contain suspending agents and thickeners. The compositions may be provided in unit / dose containers or multi-dose containers (e.g., sealed ampoules and sealed vials) and may be stored in a freeze-dried state requiring only the addition of a sterile liquid carrier (e.g., physiological saline or water for injection) immediately before injection.

[0192] Solutions and suspensions for immediate injection may be prepared from the sterile powders, granules, and tablets of the types described above. For example, in one aspect, an injectable, stable, sterile composition containing the constructs described herein in unit dose form is provided in a sealed container. The constructs described herein may be provided in a lyophilized form, which is reconstituted by forming a liquid composition suitable for injection into a subject with a suitable pharmaceutically acceptable carrier.

[0193] Compositions suitable for rectal administration may be provided as unit-dose suppositories. These can be prepared by mixing the constructs described herein with one or more conventional solid carriers (e.g., cocoa butter) and then molding the resulting mixture.

[0194] More specifically, alternatively, the constructs described herein may be formulated for nasal administration or otherwise administered to the target lung by any suitable means, for example, by an aerosol suspension of respiratory particles containing the construct described herein, which is inhaled by the patient. The respiratory particles may be liquid or solid. The term “aerosol” includes any gaseous suspension phase which can be inhaled into the bronchi or nasal cavity. Specifically, an aerosol includes a gaseous suspension of droplets which may be produced in a constant-dose inhaler or nebulizer or mist atomizer. An aerosol also includes a dry powder composition suspended in air or another carrier gas which may be delivered, for example, by a blowing method from an inhalation device. See Ganderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Harwood (1987); Gonda (1990), Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth. 27:143 (1992). Liquid particle aerosols containing the constructs described herein can be produced by any suitable means, such as a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, which are known to those skilled in the art. See, for example, U.S. Patent No. 4,501,729. Solid particle aerosols containing the constructs described herein can similarly be produced by any solid particle pharmaceutical aerosol manufacturer using techniques known in the pharmaceutical field.

[0195] Alternatively, the constructs described herein may be administered topically rather than systemically, for example, in a depot formulation or a sustained-release formulation.

[0196] The structures described herein may be coated onto or impregnated onto a device (or a composition containing the structures described herein may be coated onto or impregnated onto a device). The device may be for the delivery of the compositions containing the structures and synthetic binders described herein to mucous membranes (including the lungs, nose, mouth, etc.).

[0197] As described herein, the constructs described herein are diffusible through mucus when unbound and can be bound to a target (e.g., a pathogen) at a desired rate. It is also desirable that, once the constructs described herein are bound to a target, the cumulative effect of binding protein-mucin interactions effectively traps the pathogen in the mucus and / or aggregates the target.

[0198] In some examples, the pharmaceutical composition may further contain additional active agents, such as prophylactic or therapeutic agents. Suitable antiviral agents include, for example, virus inactivators, such as nonionic, anionic and cationic surfactants, and C31G (amine oxides and alkyl betaines), polybiguanides, docosanol, acylcarnitine analogs, octyl glycol, and antimicrobial peptides, such as magainin, gramicidine, protegurin, and retrocycline. Mild surfactants, such as sorbitan monolaurate, may be usefully used as antiviral agents in the compositions described herein. Other antiviral agents that may be usefully used in the compositions described herein include nucleotide or nucleoside analogs, such as tenofovir, acyclovir, amantadine, didanosine, foscarnet, ganciclovir, ribavirin, vidarabine, zalcitabine, and zidovudine. Further antiviral agents that may be used include non-nucleoside reverse transcriptase inhibitors, such as UC-781 (thiocarboxyanilide), pyridinone, TIBO, nevaripine, delavirbin, karanolide A, caplavirine, and efavirenz. Drugs and their analogues from these reverse transcriptase inhibitors, which have shown poor oral bioavailability, are particularly suitable for administration to mucosal tissues.

[0199] The subject matter of this disclosure further includes constructs described herein or compositions comprising constructs described herein, and kits comprising devices for optionally administering such constructs or compositions. [Examples]

[0200] The family of viruses that bind to ACE2 includes SARS-CoV-2, SARS-CoV-1, and NL63-CoV. These viruses enter cells when the receptor-binding domain (RBD) of their spike protein (S) binds to angiotensin-converting enzyme 2 (ACE2) on the surface of the target cell. This ACE2 tropism may be used to develop ACE2-Fc decoys capable of neutralizing viruses. One strategy is to ligate the entire ACE2 molecule (residues 18-740, including the self-dimerizing collectrin domain) to human IgG1-Fc, or simply to ligate the extracellular segment of ACE2 lacking the C-terminal collectrin domain (residues 18-614) to human IgG1-Fc. See, for example, Figures 6A and 6B. However, because the S-protein only binds to ACE2 with moderate affinity, the neutralizing ability of such ACE2-decoys based on wild-type ACE2 is limited; typical binding affinity (EC50) and neutralizing ability (IC50) range from several hundred ng / mL to several tens of μg / mL. Such ability is at least approximately 1-2 log worse than that of monoclonal antibodies undergoing EUA or active clinical development. See, for example, Figures 1B and 2A showing ACE-Fc dimers.

[0201] To overcome the limited affinity of wild-type ACE2 for the S protein, higher-affinity ACE2 variants have been genetically engineered using random mutagenesis and selection with yeast surface display (see, e.g., Table 1, Figures 16A-16B). This controlled evolutionary strategy leads to the possibility of escape viruses that bind to wild-type ACE2 but not to mutated ACE2. Therefore, while binding affinity can be improved, another approach that still utilizes naturally occurring ACE2 may be beneficial. Cryo-electron microscopy of SARS-CoV-1 shows that approximately 50-100 S proteins are present on the virus surface, spaced about 15 nm apart on average. The trimer morphology of the S protein spike also results in large distances between any two of the three S proteins on each individual spike. In both cases, this distance limits the simultaneous binding of two Fab domains on the antibody to two distinct S proteins. Assuming high sequence homology between viruses that bind to ACE2 (e.g., SARS-CoV-1 and SARS-CoV-2), the presence of S proteins on such viruses is likely to be similar. Therefore, the methods and compositions described herein may improve the ability of ACE2 decoys by adjusting the presence of two ACE2 domains to maximize the likelihood of achieving bivalent binding to the viral surface.

[0202] Cryo-EM analysis suggests that most spike proteins have one or two RBDs in the "upper" configuration, while a small number of spike proteins appear to have all three RBDs of the same spike in the "upper" configuration. Natural ACE2 is a homodimer with a collectrin domain contributing as the primary dimerization domain. The studies conducted herein suggest that this geometry may prevent the molecule from achieving optimal intraspike binding. To overcome this drawback, the Fc domain (such as, but not limited to, the VH-CH1 domain of standard IgG1 Fab) can be combined with the extracellular domain of ACE2 from which the collectrin domain (residues 18-614) has been removed, and further include an extended (e.g., 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, etc.) amino acid, a mobile linker between the ACE2 fragment and the Fc domain (Fc constant heavy chain domain, CH2) (designed to extend the distance the molecule can reach and thereby increase binding affinity). See, for example, Figure 6C (and Figures 1A and 2B).

[0203] As described herein, a flexible, linked ACE2 decoy construct (e.g., ACE2-(G4S)6-Fc as shown in Figure 6C) binds to different variants of the SARS-CoV-2 S protein, neutralizes SARS-CoV-2 pseudoviruses at picomolar concentrations, effectively traps SARS-CoV-2 virus-like particles in human respiratory mucus, can be stably sprayed, and effectively mitigates SARS-CoV-2 infection in hamsters.

[0204] To determine the distance between ACE molecules bound to the same S protein, the spike protein structure reported from 7A98 was constructed in the “upper three” RBD configuration. This model was used to determine whether ACE2-Fc could bind to two RBDs on the same S protein trimer (e.g., Figures 1B and 2A). See, for example, Figure 7A. As shown in Figure 7A, if one of the two ACE2 domains on ACE2-Fc binds to any one of the three RBDs, the remaining ACE2 domain aligns upward away from the S protein due to the lack of flexibility and length in the hinge of IgG1 binding ACE2 to Fc. Therefore, it is unlikely that ACE2-Fc can efficiently bivalently bind to either two RBDs on the same spike protein or to RBDs between different spike proteins on the virus. As shown in Figure 6A, since collectrin domain dimerization directly limits the reach of adjacent ACE2 fragments, conventional ACE2-Fc containing a collectrin domain faces the same limitation.

[0205] When these three RBDs are in a “top three” configuration, the estimated distance between RBDs on the same spike protein ranges from 60 to 100 angstroms. To bridge this distance and add flexibility to the molecule, as shown in an example of a flexibly linked ACE2 decoy construct (e.g., ACE2-(G4S)6-Fc in Figure 6C), a mobile linker approximately 10 nm long (such as, but not limited to, a (GGGGS)6 mobile linker) was added between the extracellular ACE2 fragment and the Fc region (e.g., IgG1-Fc). Since the mobile linker is present on each of the two heavy chains, the two ACE2 fragments on the flexibly linked ACE2 decoy (e.g., ACE2-(G4S)6-Fc) can theoretically be nearly twice this length, i.e., approximately 20 nm. The modeling suggests that, when any two RBDs are oriented in an "upper" configuration, a movable linker has sufficient length to flexibly link an ACE2 decoy (e.g., ACE2-(G4S)6-Fc, but this is just one example) to achieve divalent linking, as shown in Figure 7B.

[0206] This relationship was examined in mammalian cultures for both ACE2-Fc and ACE2-(G4S)6-Fc, and both molecules were purified using standard Protein A chromatography. These molecules were tested in undenatured PAGE (see Figure 7C); they electrophoresed at a molecular weight of approximately 350 kDa, and the presence of a single band for both confirmed their monomeric existence. Their molecular weights were examined using size exclusion chromatography / multi-angle light scattering (SEC / MALS). As shown in Figure 7D, ACE2-Fc and ACE2-(G4S)6-Fc have MWs of approximately 208 kDa and 212 kDa, respectively, which are in good agreement with the theoretical MW. Both molecules are found primarily in monomeric form: after simple Protein A purification, ACE2-Fc and ACE2-(G4S)6-Fc were approximately 85% and 91% monomers, respectively. The remainder corresponds to protein oligomers and aggregates. Clearly, a significantly higher yield was consistently obtained for ACE2-(G4S)6-Fc product, averaging approximately 86 mg per 500 mL of culture. This is more than double the typical yield achieved by ACE2-Fc production under the same conditions (producing approximately 36 mg of protein per 500 mL of culture). See, for example, Figure 13.

[0207] method ACE2 decoys described herein (including flexibly ligated ACE2 decoys) were cloned from a plasmid (pAce2-mFc) containing ACE2 without a CD domain fused to the monomeric Fc domain. gblocks®, a double-strand DNA product containing a (GGGGS)6-Fc fusion, was purchased from IDT DNA. To construct a plasmid for ACE2-(G4S)6-Fc (pAce2-LdFc), pAce2-mFc was digested with BamH and XhoI, and (GGGGS)6-Fc was inserted by Gibson assembly. This reaction product was chemically transformed into Competent TOP10® (Thermo Fisher) and spread on LB+ carbenicillin plates. Assembly was confirmed using Sanger sequencing. To construct a plasmid for ACE2-Fc (pAce2-dFc), Fc was amplified from (GGGGS)6-Fcgblock® using primers pF1 and pR1 and high-fidelity Phusion polymerase. The PCR product was then cloned into pAce2-mFc digested with BamH and XhoI by Gibson assembly, as described above.

[0208] Cloning for soluble expression of SARS-CoV-2 wild-type and mutant S proteins was performed from the plasmid nCov-2.sol, which encodes the SARS-CoV-2 wild-type S protein with the 2P mutation (mutant furin site, C-terminal foldon, and hexahistidine tag). This plasmid was used for soluble expression of the S protein. To generate the S protein encoding the South African strain mutation of SARS-CoV-2 SA in nCov2.sol, the plasmid was digested with AgeI and NheI. PCR primers Pf2, Pr2, Pf3, and pR3 were designed to amplify two fragments from the S protein with mutations K417N, E484K, and N501Y. These two fragments were cloned into the digested nCov2.sol by Gibson assembly to generate the full-length S protein with the SA mutation. The correct assembly of the protein was confirmed by Sanger sequencing (Genewiz). nCov2.sol was amplified using primers Pf5 and Pr5 (which amplified the vector and S protein residues 1-816 and 943-1208). The Hexapro mutation, intended to stabilize the soluble protein, was then inserted into wild-type and SA-nCov2.sol by inserting a DNA fragment encoding S protein residues 817-942 containing the hexa-pro mutation via Gibson assembly. The resulting vectors are hereafter referred to as WT-hexapro-nCov2.sol and SA-hexapro-nCov2.sol. The fragment containing the hexa-pro mutation was amplified from the plasmid UK-hexapro-nCoV2.xdna. This plasmid encodes the S protein of the UK strain containing the hexa-pro mutation and was purchased from Twist Bioscience.

[0209] Plasmids necessary for generating SARS-CoV-2 pseudotype infectious lentiviruses were constructed as follows: Plasmid pUC57-2019-nCoV-S, containing human codon-optimized spike DNA, was purchased from Genscript Molecular Cloud. This DNA was amplified using primers (P6, P6), a C-terminal shortening was performed, and then cloned into the mammalian expression vector pAH to produce pAH-S-CoV-2-ΔCt. To produce pAH-S-CoV-2-ΔCt with the SA mutation, a fragment of the SA S protein was amplified from SA-nCoV1.sol using primers Pf7 and Pr7. Another fragment of the WT S protein was amplified from WT pAH-S-CoV-2-ΔCt using Pf8 and Pr8. This fragment was then cloned by Gibson assembly into a pAH cloning vector digested with KpnI and XhoI. Lentiviruses were generated using a third-generation packaging system that employs four plasmids, pMDLg / pRRE(Addgene)+pRSV-Rev(Addgene)+pAH-S-CoV-2-ΔCt, and a transfer plasmid (pLL7.0 GFP) containing the EGFP gene used to track infection.

[0210] Plasmids necessary for generating non-replicating SARS-CoV-2 wild-type and SAVLPS were prepared as follows: gblock®, which encodes the C-terminal domain of SARS-CoV-1, was purchased from IDT DNA. WT-hexapro-nCov2.sol, SA-hexapro-nCov2.sol, and UK-hexapro-nCov2.sol were digested with BamHI and XhoI to remove the foldon domain and his tag, and then gblock®, which contains the C-term of SARS-CoV-1, was introduced by Gibson assembly.

[0211] For transfection, endotoxin-free pAce2-dFc and pAce2-LdFc were purified using NucleoBond Xtra Midi Plus EFkit (Macherey-Nagel). ACE2-Fc and ACE2-(G4S)6-Fc were produced in Expi293T® cells by transient transfection using ExpiFectamine® TransfectionKit (Thermo Fisher). Cells were harvested from 500 mL of culture before viability fell below approximately 75%. For purification by protein A chromatography, the cell culture supernatant was concentrated using tangent flow (Sartorius Vivaflow 50 crossflow cassette system with a 100,000 MWCO cassette equipped with a polyethersulfone membrane). Three 5 mL HiTrap protein A columns (Cytivia) were directly connected to an NGC Quest 10 FPLC (BioRad). This column was equilibrated with 5 column volumes (CV) of 10 mM sodium phosphate buffer (pH 7.0). Proteins were loaded into the column at 0.5 mL / min, followed by a 10 CV washing step using phosphate buffer, and then eluted by a 5 CV isotonic elution step using 100% 0.2 M glycine buffer (pH 2.0). A 3 mL fraction was collected in a tube filled with 300 μL of 1 M Tris buffer (pH 8) containing 0.2% polysorbate 80. The purity of each fraction was evaluated by SDS-PAGE, and fractions without extraneous bands were combined. The buffer was then changed using a Spin-X® UF 50k MWCO PES spin column (Corning) with 20 mM His, mg / mL sucrose, 0.2% polysorbate 80, and 130 mM NaCl (pH 6.2) (standard buffer). After buffer exchange, the protein was filtered through a 0.22 μm filter, then flushed and frozen in liquid nitrogen, and stored at -80°C.

[0212] Endotoxin-free ncov2.sol, WT-hexapro-nCov2.sol, SA-hexapro-nCov2.sol, and UK-hexapro-nCov2.sol were purified using the NucleoBond Xtra Midi Plus EF kit. These plasmids were transfected into Expi293T® cells using the ExpiFectamine® Transfection Kit. Cells were harvested from 500 mL of culture before viability fell below approximately 45%. The cell culture supernatant was concentrated 10-fold using tangent flow (Sartorius Vivaflow 50 cross-flow cassette system with a 100,000 MWCO cassette equipped with a polyethersulfone membrane). The concentrated supernatant was incubated overnight with 1 mL of Ni-Nta agarose resin (Qiagen) and then collected using a gravity flow column (Bio-Rad). Next, the resin was washed with several column volumes of PBS containing 20 mM imidazole, and then eluted with PBS containing 500 mM imidazole. Subsequently, the protein was buffer-changed using a Spin-X® UF50kMWCOPES spin column with PBS or 20 mM tris (pH 7) containing 120 mM sucrose and 20 mM sodium chloride. After buffer exchange, the protein in the tris-sucrose buffer was flushed and frozen in liquid nitrogen, and then stored at -80°C.

[0213] Fluorescent VLPs were prepared by simultaneous transfection of a 1:1 ratio of pGAG-mcherry plasmid (kindly provided by Gummuluru lab) and Cov2S protein plasmid. Non-replicating lentiviral pseudotypes with SARS-CoV-2UK spike protein were prepared using the following plasmids in a 1:1:1:2 ratio: pMDLg / pRRE, pRSV-REV, SARS-CoV-2UK spike, and pLL7 GFP. Non-replicating lentiviral pseudotypes with SARS-CoV-2 South Africa spike were prepared using the same plasmid / ratio as above, replacing SARSCov2UK spike with SARSCov2 South Africa spike. All plasmids were purified using the NucleoBond Xtra Midi Plus EF kit. Plasmids were transfected into LVMaxx using the LVMaxx Transfection kit. Each VLP was generated in 60 mL of culture and collected after 48 hours. VLPs were purified using a 25% sucrose (25 mM Hepes / 130 mM NaCl) cushion spin protocol. 3 mL of 25% sucrose solution was added to each Beckman-Coulter ultracentrifuge tube, resulting in 7 mL of cell culture supernatant gently layered at the top. These tubes were then centrifuged at 36,000 rpm for 2.5 hours at 4°C. The sucrose / supernatant was then aspirated and discarded, and 20 μL of 10% sucrose solution was added to the top of the VLP pellet. After 24 hours at 4°C, the VLPs were dispensed and stored at -80°C.

[0214] The 3D models described herein were created using UCSF Chimera 1.14 to generate all protein models, and UCSF Chimera X1.1 was used to create the models for publication. ACE2-Fc and ACE2-(G4S)6-Fc were constructed using model 6M17 for ACE2, 1HZH for human IgG, and 1EIB for the GGGGS linker. ACE2-Fc and ACE2-(G4S)6-Fc bound to S proteins were created by matching the ACE2 of ACE2-Fc and the ACE2 of ACE2-(G4S)6-Fc with the RBD-bound ACE2 in the "all upper" S protein model 7A98. A predictive 3D model of ACE2-Fc with a collectrin domain was then modified from this model.

[0215] SEC-MALS analysis and undenatured PAGE of purified proteins were performed using solutions containing 1.0 mg / mL ACE2-(G4S)6-Fc or Ace2-Fc prepared in standard buffer. Then, 100 μL of these solutions were loaded into a Superdex 200 Increase 10 / 300 GL (Cytivia) column mounted on an NGC Quest 10 FPLC (BioRad). This column was pre-equilibrated with PBS, and all runs were performed at 0.5 mL / min. The molecular weight of the proteins eluted from the column was determined using a Mini Dawn multi-angle diffuse light detector and its comparison software, Astra8 (Wyatt), assuming an elongation coefficient of 1.92. Molecular weights were calculated for two independent batches of ACE2-(G4S)6-Fc and two batches of Ace2-Fc. For undenatured PAGE, 5 μg of protein was loaded onto a 3-12% Bis-Tris gel (Invitrogen), and this gel was electrophoresed as described in the manufacturer's protocol.

[0216] For scanning differential fluorescence quantification, the melting point of ACE2-LFC was determined using nanoDSF with Promethius NT.48 (Nanotemper Technologies). The sample was heated from 25°C to 95°C at a rate of 1°C / min. The sample was measured in triplicate. The reported data are the average of three independent replicates.

[0217] The ELISA binding assay was performed using a 96-well half-area plate (Fisher Scientific, Costar 3690) coated with 0.5 μg / mL of S protein, and incubated overnight at 4°C. Subsequently, the ELISA plate was blocked at room temperature for 1 hour with 5% (w / v) milk (LabScientific MSPP-M0841) containing Tween 20 (Fisher Scientific BP337-100) at a 1:2000 dilution. After initiating blocking, the 5% milk was discarded, and the sample was spread on the plate diluted in 1% (w / v) milk containing Tween 20 at a 1:10,000 dilution. The sample was incubated at room temperature for 1 hour, and the solution was discarded after 1 hour of incubation. The plate was then washed four times with PBS containing Tween 20 at a 1:2000 dilution. Peroxidase-conjugated goat anti-human IgG Fc antibody (Rockland 709-1317) was diluted 1:5000 in milk (1%) containing Tween 20, spread on a plate, and incubated at room temperature for 1 hour. The solution was then discarded, and the plates were washed twice with PBS containing Tween 20, followed by two more washes with plain PBS. The plates were developed with TMB solution (ThermoFisher 34029), and development was stopped by adding 2N HCl (Sigma-Aldrich 320331). Absorbance at 450 nm and 595 nm was then measured using a microplate photodetector (Fisher Scientific, accuSkan FC).

[0218] For the neutralization assay described herein, a series of 10 quadruple serial dilutions were prepared with ACE2-linker-Fc or ACE2-Fc or IgG, starting at 20 μg / ml in OptiMEM. Each 10 μl dilution was added in triplicate to the wells of a 96-well plate. To each of these dilutions, 0.5 μl of SARS-CoV-2 pseudotyped lentivirus (diluted to 10 μl in OptiMEM, MOI 1, and titer estimated using infection with HEK293-ACE2 cells) was added and incubated at room temperature for 30 minutes. Three wells contained 20 μl of OptiMEM, and three wells contained 19.5 μl of OptiMEM + 0.5 μl of pseudovirus, for normalization and IC. 50 This serves as a control for calculation. After 30 minutes, 5000 HEK-ACE2 cells were added to each well of a plate in 100 μl of DMEM + 10% FBS and incubated at 37°C with 5% CO2 for 72 hours. After 72 hours, the medium was carefully removed without destroying the cells, and the cells were trypsinized and analyzed by flow cytometry (Attune NxT, ThermoFisher), and EGFP fluorescence was recorded for each well.

[0219] The MFI for EGFP fluorescence in the triple-well system was averaged and plotted against the ACE2 / mAb concentration, and the IC50 of neutralization was estimated using a four-parameter nonlinear regression.

[0220] For mucus trapping, multi-particle tracking analysis of fluorescent SARS-CoV-2 VLPs in human airway mucus (AM) was performed. In short, a solution of fluorescent VLPs and ACE2-Fc or ACE2-(G4S)6-Fc was added to approximately 10 μL of fresh, undiluted airway mucus in a custom-made glass chamber. The samples were then incubated at 37°C for approximately 30 minutes and subsequently examined under a microscope. PBS and antibody CR3022 (final concentration 10 μg / ml) were used as negative and positive controls, respectively. The same AM was used for all runs to directly compare samples. Video of VLP diffusion in the AM was recorded at a time resolution of 66.7 ms using MetaMorph software (Molecular Devices, Sunnyvale, CA). The video was analyzed using NetTracker from AI Tracking Solutions, and the raw video data was converted into particle tracking data. The time-averaged mean squared displacement (MSD) and effective diffusion coefficient were calculated by transforming the particle centroid coordinates (Equation < Δr 2 (τ)>=[x(t+τ)-x(t)] 2 +[y(t+τ)-y(t)] 2 The time was then converted to MSD (where τ is the elapsed time or time lag).

[0221] Hamster studies for the evaluation of ACE2-(G4S)6-Fc, as referenced herein, were conducted in a golden Syrian hamster model of SARS-CoV-2 infection, with some modifications as previously described. Four groups (n=8 per group) received intranasal administration of ACE2-(G4S)6-Fc either prophylactically (4 hours before exposure) or therapeutically (4, 24, or 48 hours after challenge). One group received PBS as a negative control. Viral challenge was performed by intranasal inoculation of mice with 100 μL of SARS-CoV-2 diluted in Dulbecco's modified Eagle medium. Hamsters were then administered ACE2-(G4S)6-Fc daily until sacrifice 4 days after viral exposure. Viral load was quantified in the nasal turbinates by qRT-PCR and normalized by β-actin (internal gene control).

[0222] For nebulization studies, ACE2-(G4S)6-Fc (10 mg / mL) in standard buffer was sprayed using a Phillips Innospire Go vibrating mesh nebulizer. The aerosol was collected in a glass impinger mechanism with upper and lower chambers, according to the protocol guidelines in European Pharmacopoeia 5.0. The nebulizer was operated until visually dry. Then, buffer was added to the different chambers of the glass impinger, and the deposited antibody was collected. Aggregate formation in the upper chamber, lower chamber, and residual ("dead volume") samples was evaluated using SEC with an EN-Rich 650 size exclusion column (Bio-Rad) mounted on an NGC Quest 10 FPLC (BioRad), and the aforementioned undenatured PAGE. The binding affinity of the sprayed molecules was evaluated by the S-protein ELISA described above.

[0223] result The molecular stability was examined using differential scanning calorimetry. The melting point T of ACE2-(G4S)6-Fc was determined. M The temperature was approximately 52 ± 0.6°C (see, for example, Figure 14).

[0224] The capabilities of different ACE2 decoys were first determined by measuring the binding affinity of these different ACE2 decoys to the spike protein of the WT strain USA-WA1 / 2020 using ELISA. In addition to ACE2-Fc and ACE2-(G4S)6-Fc, the full-length ACE2 decoy (i.e., ACE2(740)-Fc (abbreviated as 208)) was also examined. Of the three ACE2 decoys, ACE2-(G4S)6-Fc consistently showed the highest binding affinity, as shown in Figure 8A. Across multiple independently generated batches, ACE2-(G4S)6-Fc showed the highest EC2-222 binding affinity at picomolar concentrations. 50 (Average: 490 pM, or 96 ng / mL, see Figure 8B, for example) consistently showed the central EC of the highest-capacity batch of ACE2-(G4S)6-Fc. 50The values ​​were as low as 136 pM, or 27 ng / mL. In contrast, the mean EC2 of ACE2-Fc and ACE2(740)-Fc at 3.6 nM (680 ng / mL) and 1.6 nM (370 ng / mL) were low. 50 It was approximately 7.3 times and 3.3 times worse than the ACE2-(G4S)6-Fc.

[0225] ACE2-(G4S)6-Fc was converted to EC equivalent to ACE2-(G4S)6-Fc produced in Expi293 cells, using Chinese hamster ovary (CHO) cells, which are most commonly used for high-volume biopharmaceutical production. 50 (See, for example, Figure 8C) was used for production.

[0226] In general, the flexible annealed ACE2 decoys described herein are likely to bind more readily to different SARS-CoV-2 variants than conventional monoclonal antibodies (mAbs). As shown in Figure 8C, binding affinity experiments confirmed that ACE2-(G4S)6-Fc does indeed bind to different SARS-CoV-2 variants using ELISA with B.1.1.7(UK) and B.1.351(SA) spike proteins. Across five independently produced batches of ACE2-(G4S)6-Fc, its binding affinities to the WT, UK, and SA variants were well-competitive (Figure 8D). For comparison, the binding of RGN10989, an mAb developed by Regeneron (part of an mAb cocktail that received an EUA from the FDA), was also tested. While RGN10989 was able to bind to the WT and UK S proteins with comparable binding affinity, this mAb failed to achieve detectable binding to the SA S protein. These results highlight the utility of the flexible ACE2 decoys described herein (including, but not limited to, ACE2-(G4S)6-Fc) across all viruses that bind to ACE2, such as SARS-CoV-2 variants.

[0227] The significantly increased binding affinity of ACE2-(G4S)6-Fc also correlates with greater neutralizing activity. The neutralizing abilities of ACE2-(G4S)6-Fc, ACE2-Fc, and ACE2(740)-Fc were measured via a standard pseudovirus assay in which HEK cells overexpressing ACE2 were infected with a lentivirus encoding an eGFP transgene pseudotype having the D614G variant of the SARS-CoV-2 spike protein. The infectivity of the pseudovirus at different ACE2-decoy concentrations could be determined by measuring the eGFP fluorescence of cells incubated with varying amounts of ACE2 decoy using flow cytometry. In this assay mechanism, ACE2-(G4S)6-Fc showed an affinity for the SARS-CoV-2 pseudovirus at a picomolar concentration of 52 ng / mL, with an average IC50. 50 Neutralization was performed with [method]. In contrast, the neutralization capacity of ACE2-Fc and ACE2(740)-Fc was approximately 5 and 6 times lower, respectively, with IC2 of about 240 ng / mL and about 310 ng / mL. 50 The ACE2-(G4S)6-Fc is also an IC that is about twice as large as the ACE2-Fc. 90 The results showed that ACE2-(G4S)6-Fc actually possesses greater binding, affinity, and neutralizing ability than conventional ACE2-decoys.

[0228] Flexible annealed ACE2 decoys described herein, such as ACE2-(G4S)6-Fc, can effectively trap and stably spray ACE2-bound viruses, such as SARS-CoV-2 and VLPs, in human respiratory mucus. SARS-CoV-2, like SARS-CoV-1, NL63, and HKU1 coronaviruses, strictly infects via the apical side of the respiratory epithelium (i.e., the respiratory lumen), and as the infection spreads from the upper to the lower respiratory tract, it sheds progeny viruses primarily into the respiratory mucus (AM), with no apparent basic shedding or cell-to-cell diffusion. This mechanism of viral nucleic acid means that the virus must spread across the AM in order to transmit the infection within the respiratory tract. Similarly, preventing the virus from spreading across the AM by cross-linking it to the mucin matrix of the AM can help stop the spread of infection and facilitate rapid clearance from the respiratory tract via the natural mucosal ciliary clearance mechanism. This could enable robust trapping of the virus within human amniotic fluid (AM), leading to rapid clearance of VLPs from the airways.

[0229] To evaluate whether the flexible ACE2 decoys described herein (such as, but not limited to, ACE2-(G4S)6-Fc) can trap SARS-CoV-2 in human amniotic membranes (AMs), fluorescent SARS-2 VLPs were prepared by co-expression of the S protein with a GAG-mCherry fusion construct, and their motility was visualized in fresh human AMs isolated from extubated endotracheal tubes. As shown in Figures 10A and 10B, ACE2-(G4S)6-Fc effectively trapped SARS-2 VLPs in AMs, reducing the rapidly moving viral population (defined as having sufficient diffusivity to spread across a layer of approximately 50 μm in approximately 1 hour), even at a concentration of 1 μg / mL in AMs, by approximately 14-fold compared to a saline control. In contrast, neither ACE2-Fc nor CR3022, which are high-affinity mAbs for the S protein from JNJ / Cru cells, could reduce viral motility to a similar degree, even at 10-fold higher concentrations.

[0230] The most direct method for achieving therapeutic concentrations of mAbs in the respiratory system, particularly the pulmonary airways, is direct delivery of mAbs via inhalation. Vibrating mesh nebulizers allow for the spraying of protein therapeutics without causing localized heating and shearing that can degrade proteins. The flexible linked ACE2 decoys described herein can be sprayed stably. For example, ACE2-(G4S)6-Fc was sprayed using a Philip's Innospire Go vibrating mesh nebulizer, and the resulting aerosol was collected in a two-chamber glass impinger mechanism designed to capture aerosols greater than 6 mm (upper chamber) and aerosols less than 6 mm (lower chamber) according to European Pharmacopoeia 5.0. The binding affinity of the recovered sprayed ACE2-(G4S)6-Fc was measured via S-protein ELISA. The results are shown in Figure 11. No significant loss in binding affinity was observed in ACE2-(G4S)6-Fc recovered from either the upper or lower chamber compared to unsprayed ACE2-(G4S)6-Fc. Unmodified PAGE also confirmed the absence of heavy chain separation or detectable aggregation (see, for example, Figures 12A-12B). These results highlight the ability to stably spray a flexible, linked ACE2 decoy for direct inhalation delivery into the respiratory tract.

[0231] Furthermore, intranasal delivery of flexible ligated ACE2 decoys reduces viral load in the nasal turbinates. For example, hamsters infected with SARS-CoV-2 showed a reduction in viral load after treatment with flexible ligated ACE2 decoys. As an in vivo proof of concept, the efficacy of intranasal delivery of ACE2-(G4S)6-Fc in live SARS-CoV-2 infected golden Syrian hamsters was assayed. The hamsters showed clinical signs of weight loss and histopathological changes due to high viral load in the lungs, which makes these hamsters a suitable model for testing mAb-based approaches, despite differences in respiratory anatomical diagrams. Most previous studies evaluated mAbs against SARS-CoV-2 administered within 2–6 hours post-infection. Here, daily administration of ACE2-(G4S)6-Fc was evaluated pre-infection or at 4, 24, and 48 hours after infection. ACE2-(G4S)6-Fc treatment provided an approximately tenfold reduction in viral load in nasal turbinate tissue by 96 hours, even when delayed up to 48 hours after infection. This is interpreted as a substantial reduction in weight loss over exactly two days (p=.03).

[0232] Despite the remarkable capabilities of many mAbs that have progressed to clinical trials, viral escape variants can readily arise for any individual mAb, and these escape variants still retain ACE2 binding. To prevent escape variants, many groups have focused on combining two distinct antibody-targeted structural epitopes. While the risk of viral escape can be greatly reduced through the use of mAb cocktails, the possibility remains that viral escape variants could escape from both mAbs in the cocktail simultaneously. Given the concerns about viral escape variants, as well as the enormous costs and time required to advance mAb molecules to Phase 3 clinical trials, developing binding proteins without the risk of viral escape is extremely beneficial. Furthermore, given that there are already at least three human coronaviruses that target ACE2 as the primary host entry receptor, including two with pandemic potential (SARS-CoV-1 and SARS-CoV-2), it is likely only a matter of time before another respiratory virus that also targets ACE2 will exhibit pandemic potential. For these reasons, the flexible annealed ACE2 decoy described herein may enable immunotherapy against all ACE2-targeted viruses. In fact, the soluble flexible annealed ACE2 decoy described herein can block infection by both SARS-CoV-1 and SARS-CoV-2 and can bind to the S protein derived from WT, UK, and SA strains of SARS-CoV-2 with comparable affinity (see, for example, Figures 8C and 8D).

[0233] The flexible ACE2 decoys described herein may use wild-type (WT) ACE2 fragments to reduce the potential risk of escape virus variants that bind to WT ACE2 but are not captured by ACE2 variants, although in some cases mutated ACE2 may be used as described herein. Furthermore, the Fc domain may be wild-type (e.g., IgG1-Fc) or modified. Generally, the collectrin domain may be omitted, and the linkage between the extracellular fragment of ACE2 and the Fc (e.g., IgG1-Fc) domain may be optimized so that the length of the linker region allows for multivalent binding. These binding molecules exhibit substantially better binding affinity and neutralizing ability compared to full-length ACE2 with a collectrin domain or ACE2-Fc conjugates without a mobile linker, with picomolar binding affinity and inhibitory concentration (IC) comparable to or exceeding that of ACE2-decoys lacking a sufficiently long mobile linker described herein. 50 It contains approximately 52 ng / mL.

[0234] ACE2 dimerizes via its collectrin domain on the cell surface. The flexible ligated ACE2 decoys described herein explicitly remove the collectrin domain of ACE2 (which allows for the grafting of extracellular fragments of ACE2 onto wild-type Fc cells using the well-defined linkers described herein). In an example of a flexible ligated ACE2 decoy described herein using wild-type Fc cells (e.g., ACE2-(G4S)6-Fc), the construct can also promote other cell-mediated immunizations. Surprisingly, the flexible ligated ACE2 decoys described herein also yielded greater yield and stability; for example, the yield of ACE2-(G4S)6-Fc was comparable to other high-expression IgGs produced under similar conditions and had a regenerative monomer profile (in contrast to other ACE2 decoys prone to aggregation).

[0235] ACE2 can also bind only to the S protein having its RBD in the "upward" conformation. As a result, to achieve a divalent bond, two RBD domains need to be in the "upward" conformation. The S protein having the RBD domains in the "two upward" conformation has not been observed during imaging of the SARS-CoV-2 S protein; however, it has been suggested that binding to the RBD can trigger the transition of the S protein to the "three upward" state (a mechanism conserved among Coronaviridae). Mutations in different regions of the S protein can increase the proportion of S proteins having RBD domains in the "two upward" or "three upward" conformations. For example, the S protein with D614G showed a higher proportion of molecules in the "two upward" and "three upward" conformations than D614. The Hexa-pro mutation also increases the number of S proteins having two RBDs in the upward direction. Other mutations that increase the ratio of "two upward" to "one upward" S proteins have been reported. As a result, spike-in binding to SARS-CoV-2 can be achieved, and higher neutralization of SARS-CoV-2 with mutations that increase RBD exposure such as D614G is predicted.

[0236] Local inhalation delivery offers significant advantages. Firstly, it maximizes local concentration in the lungs and minimizes the total required dose of the drug (e.g., mAbs) compared to systemic administration, as typically systemically administered ABs are distributed to the airways in very small proportions. For the same amount of drug (e.g., a binder such as mAbs), local delivery is likely to treat 4 to 10 times more patients compared to systemic delivery, while achieving higher concentrations in the lungs. Both of these reduce the cost burden and, more importantly, allow for the treatment of more patients. This is a critical concern given the near-unprecedented scale of COVID-19. Secondly, as is common with all antiviral drugs, early treatment is highly desirable. Unfortunately, systemically administered drugs or small molecule drugs, even when given rapidly after diagnosis, experience a considerable delay before the drug can reach Cmax in the lungs. For example, oseltamivir takes 3 days with twice-daily dosing to reach stable concentrations in the lungs. In contrast, the spray delivers ACE2-(G4S)6-Fc, a flexibly linked ACE2 decoy described herein, directly into the airway, thereby enabling rapid attainment of local Cmax. The spray also avoids the need for an infusion chair and post-infusion monitoring, allowing treatment to be administered directly in the comfort of the patient's home. This greatly reduces the burden on healthcare infrastructure structures for administering treatment compared to IV delivery. This typically lasts for about 1-2 hours of infusion followed by a period comparable to that of post-infusion observation.

[0237] AM is secreted continuously into the pulmonary airways daily, which is carried by the natural mucociliary or cough-driven clearance from the lower airways (bronchioles) to the trachea and then unconsciously swallowed in the esophagus for sterilization by the acidic and degradative gastric environment. Natural mucus clearance rapidly removes any exogenous particles deposited along the pulmonary airways. Respiratory viruses must diffuse through AM and have specifically evolved to do this efficiently. By cross-linking the virus to the mucin using a binding protein, it is established that not only can the virus not diffuse through the mucus, but it also directly removes the virus and associated antigens from the airways. This, in turn, minimizes the potential for inflammation, as well as antigen-directed immune responses that can occur by macrophages and neutrophils that can infiltrate into the lungs. The flexibly linked ACE2 decoy described herein may be used once daily, for example, once a day.

[0238] Sequence Listing SEQ ID NO: 1 (Signal Peptide) MSSSSWLLLSLVAVTAA SEQ ID NO: 2 (ACE2 with H374N+H378N) QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFV SVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHNEMGNIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFK GEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD Sequence ID 3 (G4S6+hinge+Fc) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* Sequence ID 4 (Hinge + Fc) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* Sequence ID 5 (Signal peptide) MKWVTFISLLFLFSSAYSGS Sequence ID 6 (CR3022 light chain) DIQLTQSPDSLAVSLGERATINCKSSQSVLYSSINKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* Sequence ID 7 (ACE2+ Linker) QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSM LTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEP VPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGS Sequence ID 8 (CR3022VH+CH1+Fc) QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGLEWMGIIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAGGSGISTPMDVWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* Sequence ID 9 (CR3022VH+CH1+Fc) QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGLEWMGIIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAGGSGISTPMDVWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP Sequence ID 10 (Linker + ACE2) * Sequence ID 11 (ACE2 WT, aa19~615, collector domain excluded) STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWES WRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFV SVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFK GEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD Sequence ID 12 (Fc heavy chain 1) PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence ID 13 (Fc heavy chain 2) PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence ID 14 (Hinge) EPKSCDKTHTCP Sequence ID 15 313-(G4S)6-Fc (ACE2 with the collectrin domain removed and residues K31F, N33D, H34S, E35Q, and H345L modified) QSTIEEQAKTFLDFFDSQAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWE SWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFV SVGLPNMTQGFWENSMLTDPGNVQKAVCLPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFK GEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD Sequence ID 16 313-(G4S)6-Fc (ACE2+(G4S)6 linker+hinge+Fc with the collectrin domain excluded and residues K31F, N33D, H34S, E35Q, and H345L modified.)Optionally, a GS may be included before the linker. QSTIEEQAKTFLDFFDSQAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMS TIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFE EIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCLPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTI VGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQK LFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence ID 17 sACE2.v2.4-8h-(G4S)6-Fc (ACE2 with the collectrin domain removed and residues T27Y, L79T, and N330Y modified) QSTIEEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTTAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWE SWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFV SVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFK GEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD Sequence ID 18 sACE2.v2.4-8h-(G4S)6-Fc (ACE2+(G4S)6 linker+hinge+Fc with the collectrin domain excluded and residues T27Y, L79T, N330Y modified.)Optionally, a GS may be included before the linker. QSTIEEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTTAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMS TIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFE EIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTI VGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQK LFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence ID 19 3J320v2-(G4S)6-Fc (ACE2 with the collectrin domain removed and residues T20I, H34A, T92Q, and Q101H modified) QSIIEEQAKTFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLQVKLQLQALHQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYA Sequence number 20 3J320v2-(G4S)6-Fc (ACE2+(G4S)6 linker+hinge+Fc with the collectrin domain excluded and residues T20I, H34A, T92Q, and Q101H modified. Optionally, GS may be included before the linker.) QSIIEEQAKTFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLQVKLQLQALHQNGSSVLSEDKSKRLNTILNTMS TIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFE EIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGN VQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALT IVGTLPTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQ KLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence ID 21 3N39v2-(G4S)6-Fc (ACE2 with the collectrin domain removed and residues A25V, K31N, E34K and L79F modified) QSTIEEQVKTFLDNFNHKAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTFAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWE SWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFF VSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVF KGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYA Sequence ID 22 3N39v2-(G4S)6-Fc (ACE2+(G4S)6 linker+hinge+Fc with the collectrin domain excluded and residues A25V, K31N, E34K and L79F modified.)Optionally, a GS may be included before the linker. QSTIEEQVKTFLDNFNHKAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTFAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMS TIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFE EIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGN VQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALT IVGTLPTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQ KLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence ID 23 ACE2615-foldon-T27W-(G4S)6-Fc (ACE2 modified from residue T27W) QSTIEEQAKWFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWE SWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFV SVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFK GEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD Sequence ID 24 ACE2615-foldon-T27W-(G4S)6-Fc (ACE2+(G4S)6 linker+hinge+Fc with the collectrin domain excluded and residue T27W modified.)Optionally, a GS may be included before the linker. QSTIEEQAKWFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMS TIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFE EIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTI VGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQK LFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence ID 25 LCB1 DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAEERLLEEVER Sequence ID 26 LCB1-(G4S)6-Fc Optionally, a GS may be included before the linker. DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVERGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence ID 27 LCB3 Optionally, a GS may be included before the linker. NDDELHMLMTDLVYEALHFAKDEEIKKRVFQLFELADKAYKNNDRQKLEKVVEELKELLERLLS Sequence ID 28 LCB3-(G4S)6-Fc Optionally, a GS may be included before the linker. NDDELHMLMTDLVYEALHFAKDEEIKKRVFQLFELADKAYKNNDRQKLEKVVEELKELLERLLSGGGGSGGGGSGGGGSGGGGSGGGGSGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Claims

1. Isolated binding proteins that bind to ACE2-targeted viruses, having the following amino acid sequence: A-(B) ｎ -C (Formula I) And in the formula, A is the extracellular component of angiotensin-converting enzyme 2 (ACE2) with the collectrin domain removed, or a variant thereof; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; B is a polypeptide mobile linker; The isolated binding protein is a fragment crystallization (Fc) domain, wherein C is a fragment crystallization (Fc) domain, the isolated binding protein dimerizes, the extracellular portion of ACE2 or its variant has 90% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 11, n is selected such that the average distance between the A domains of the dimer is greater than 14 nm, and the polypeptide mobile linker is configured to enable the isolated binding protein to bivalently bind to the S protein of the ACE2 target virus.

2. The binding protein according to claim 1, wherein the Fc domain is a human IgA, IgM, or IgG Fc domain.

3. The binding protein according to claim 1, wherein the Fc domain is a human IgG1 Fc domain.

4. The binding protein according to any one of claims 1 or 2, wherein the Fc domain comprises a YTE mutation, an LS mutation, or a LALA-PG mutation.

5. The binding protein according to any one of claims 1 to 4, wherein the extracellular portion of ACE2 is the extracellular portion of human ACE2.

6. The binding protein according to any one of claims 1 to 5, wherein the extracellular portion of ACE2 has an amino acid sequence having up to 10 amino acid differences within the amino acid of SEQ ID NO:

11.

7. The binding protein according to any one of claims 1 to 5, wherein the extracellular portion of ACE2 contains at least one mutation.

8. The binding protein according to claim 7, wherein the ACE2 comprises two or more mutations.

9. The binding protein according to any one of claims 1 to 8, wherein the polypeptide mobile linker has the sequence GGGGS.

10. The binding protein according to any one of claims 1 to 9, further comprising a hinge between the mobile linker and the Fc domain.

11. The binding protein according to any one of claims 1 to 10, wherein the Fc domain has an oligosaccharide, and at least 30% of the oligosaccharide has a G0 glycosylation pattern.

12. The binding protein according to any one of claims 1 to 11, wherein the Fc domain comprises an oligosaccharide having at least 30% G0 glycosylation pattern, comprising a branched coglycan structure Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 having terminal N-acetylglucosamines in each branch, which enhances the ability of the binding protein to be trapped in the mucus.

13. A pharmaceutical composition comprising a binding protein according to any one of claims 1 to 12 and a pharmaceutically acceptable excipient.

14. The pharmaceutical composition according to claim 13, wherein the excipient, diluent, or carrier is configured for inhalation.

15. The pharmaceutical composition according to claim 13, comprising one or more of the following: oral administration, parenteral administration, intraperitoneal administration, transmucosal administration, transdermal administration, rectal administration, inhalation administration, and topical administration.

16. A pharmaceutical composition according to any one of claims 13 to 15 for use in a method of treating a subject infected with SARS-CoV-2, wherein the method comprises administering a pharmaceutically acceptable amount of the pharmaceutical composition according to any one of claims 13 to 15.

17. The pharmaceutical composition according to claim 16, wherein the administration includes systemically applying the pharmaceutical composition to the patient.

18. The pharmaceutical composition according to claim 16, wherein the administration includes applying the pharmaceutical composition to the mucous membrane of a patient.

19. The pharmaceutical composition according to claim 16, wherein the administration includes spraying or providing the powder of the pharmaceutical composition.

20. A pharmaceutical composition according to any one of claims 13 to 15 for use in a method for treating or inhibiting a viral infection by an ACE2-targeted virus, wherein the method comprises administering a binding protein according to any one of claims 1 to 12 via an inhalation route.

21. The pharmaceutical composition according to claim 20, wherein the ACE2-targeted virus is SARS-CoV-2 or NL63-CoV.

22. Isolated binding proteins that bind to ACE2-targeted viruses, having the following amino acid sequence: A-(B) ｎ -C (Formula I) And in the formula A is the extracellular component of angiotensin-converting enzyme 2 (ACE2) with the collectrin domain excluded, having more than 90% amino acid sequence identity with the amino acid sequence of Sequence ID No. 11; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; B is a polypeptide mobile linker; C is a fragment crystallization (Fc) domain, The isolated binding protein is dimerized, Furthermore, n is the isolated binding protein selected such that the average distance between the A domains of the dimer is greater than 14 nm.

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