Recombinant / fusion polypeptides containing mutant angiotensin-converting enzyme 2 (ACE2)

Abstract

The present invention relates to recombinant mutant angiotensin-converting enzyme 2 (ACE2) comprising one or more amino acid substitutions at amino acid positions T27, F28, K31, H34, Y41, and Q42 with improved binding affinity to SARS-CoV-1 and / or SARS-CoV-2. It also relates to combinatorial mutant ACE2 comprising T27Y / K31 H / H34A, T27YZ K31 H / H34V, and T27Y / K31 Y / H34V, and the use of said mutant ACE2 for treating and preventing coronavirus infection.

Description

[Technical field]

[0001] The present disclosure relates generally to recombinant / fusion polypeptides comprising mutant angiotensin-converting enzyme 2 (ACE2) capable of binding to viruses, such as SARS-CoV-1 and SARS-CoV-2. Also provided are compositions comprising the polypeptides, methods of treatment using the polypeptides, and other uses. [Background technology]

[0002] Neutralizing antibodies against certain viruses (including those induced by vaccines) can effectively block infection, but are often too specific to inhibit viral variants that arise through natural mutation during a pandemic. In addition, neutralizing antibodies against a virus often do not cross-react with other viruses that utilize the same entry receptor. For example, most anti-SARS-CoV-1 (SARS) antibodies do not react with SARS-CoV-2 (COVID-19) and vice versa.

[0003] Many coronaviruses, including SARS-CoV-1 and SARS-CoV-2, use the host cell's angiotensin-converting enzyme 2 (ACE2) as an entry receptor to initiate infection. Thus, the extracellular domain of ACE2 could potentially function as a decoy for all ACE2-dependent viruses by competing with host cell ACE2 for binding to the coronavirus spike receptor-binding domain.

[0004] However, native (wild-type) ACE2 often has low affinity for coronavirus spike proteins, and therefore the affinity of ACE2 for multiple coronaviruses and their variants needs to be improved to make it suitable for use as a therapeutic agent. Summary of the Invention

[0005] In one aspect, a recombinant / fusion polypeptide is provided comprising a mutant angiotensin-converting enzyme 2 (ACE2) protein or a fragment thereof and an immunoglobulin fragment.

[0006] In one embodiment, the recombinant / fusion polypeptide comprises an immunoglobulin Fc fragment (region / domain), a protein capable of increasing the half-life of the recombinant / fusion polypeptide (e.g., albumin, human albumin, etc.), a linker capable of increasing binding valency (e.g., a rigid linker or a flexible linker such as GGGGS repeats), or a combination thereof, for example, the immunoglobulin Fc fragment is an IgG Fc fragment, such as a human IgG Fc fragment.

[0007] In one embodiment, the immunoglobulin fragment has the sequence: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23) Includes.

[0008] In one embodiment, the recombinant / fusion polypeptide binds to a viral protein.

[0009] In one embodiment, the viral protein is a coronavirus protein or a Flaviviridae protein.

[0010] In one embodiment, the coronavirus is a severe acute respiratory syndrome virus, such as severe acute respiratory syndrome 1 (SARS-CoV-1) or severe acute respiratory syndrome 2 (SARS-CoV-2).

[0011] In one embodiment, the ACE2 protein or fragment thereof comprises one or more mutations, two or more mutations, three or more mutations, four or more mutations, five or more mutations, or six or more mutations, or seven or more mutations, or eight or more mutations.

[0012] In one embodiment, the ACE2 protein or fragment thereof comprises one or more mutations at amino acid positions selected from the group consisting of 27, 28, 31, 34, 41, and 42, such as positions 27, 28, 31, 34, 41, and 42 as set forth in SEQ ID NO:24.

[0013] In one embodiment, the ACE2 protein or fragment thereof comprises mutations at amino acid positions selected from the group consisting of 27, 31 and 34, such as positions 27, 31 and 34 as set forth in SEQ ID NO:24.

[0014] In one embodiment, the ACE2 protein or fragment thereof comprises: T27A, T27R, T27N, T27D, T27E, T27Q, T27G, T27H, T27I, T27L, T27K, T27M, T27F, T27P, T27S, T27T, T27W, T27Y, T27V; Q42A, Q42R, Q42N, Q42D, Q42E, Q42Q, Q42G, Q42H, Q42I, Q42L, Q42K, Q42M, Q42F, Q42P, Q42S, Q42T, Q42W, Q42Y, Q42V; H34A, H34R, H34N, H34D, H34E, H34Q, H34G, H34H, H34I, H34L, H34K, H34M, H34F, H34P, H34S, H34T, H34W, H34Y, H34V; K31A, K31R, K31N, K31D, K31E, K31Q, K31G, K31H, K31I, K31L, K31K, K31M, K31F, K31P, K31S, K31T, K31W, K31Y, K31V; F28A, F28R, F28N, F28D, F28E, F28Q, F28G, F28H, F28I, F28L, F28K, F28M, F28F, F28P, F28S, F28T, F28W, F28Y, F28V; Y41A, Y41R, Y41N, Y41D, Y41E, Y41Q, Y41G, Y41H, Y41I, Y41L, Y41K, Y41M, Y41F, Y41P, Y41S, Y41T, Y41W, Y41Y, and Y41V The mutations include one or more mutations selected from the group consisting of:

[0015] In one embodiment, the ACE2 protein or fragment thereof comprises: T27A, T27R, T27N, T27D, T27E, T27Q, T27G, T27H, T27I, T27L, T27K, T27M, T27F, T27P, T27S, T27W, T27Y, T27V; Q42A, Q42R, Q42N, Q42D, Q42E, Q42H, Q42I, Q42L, Q42K, Q42M, Q42F, Q42P, Q42S, Q42T, Q42W, Q42Y, Q42V; H34A, H34R, H34N, H34D, H34E, H34Q, H34G, H34I, H34L, H34M, H34F, H34P, H34S, H34T, H34W, H34Y, H34V; K31A, K31R, K31N, K31Q, K31H, K31I, K31K, K31M, K31F, K31W, K31Y, K31V; F28W, F28Y; Y41H and Y41F The mutations include one or more mutations selected from the group consisting of:

[0016] In one embodiment, the ACE2 protein or fragment thereof comprises one or more mutations selected from the group consisting of T27Y, K31Y, K31H, H34A, H34V, and combinations thereof.

[0017] In one embodiment, the ACE2 protein or a fragment thereof is T27Y / K31H / H34A, T27Y / K31Y / H34A, T27Y / K31H / H34V, or T27Y / K31Y / H34V It contains one of the following combinations of mutations:

[0018] In one embodiment, the ACE2 protein or fragment thereof comprises the following combination of mutations: T27Y / K31H / H34A.

[0019] In one embodiment, the recombinant / fusion polypeptide is capable of binding to a virus, and optionally, the recombinant / fusion polypeptide is capable of blocking / interfering / inhibiting / hindering / disrupting the binding of a virus (such as SARS-CoV-2) to a cellular entry receptor.

[0020] In one embodiment, the recombinant / fusion polypeptide is capable of neutralizing or mediating (or initiating) neutralization of a virus such as SARS-CoV-2.

[0021] In one embodiment, the ACE2 protein or fragment thereof comprises one or more mutations that abolish the native peptidase activity of ACE2.

[0022] In one embodiment, the ACE2 protein or fragment thereof comprises a mutation at amino acid position 273 and / or 505, such as at amino acid position 273 and / or 505 as set forth in SEQ ID NO:24.

[0023] In one embodiment, the ACE2 protein or fragment thereof further comprises an R273Q and / or H505L mutation, for example both R273Q and H505L.

[0024] In one embodiment, the recombinant / fusion protein or fragment thereof comprising the R273Q and / or H505L mutations QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYE DYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGQFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGH IQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFLVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLR LGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22).

[0025] In one embodiment, the recombinant / fusion protein or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-21.

[0026] In one embodiment, the recombinant / fusion protein or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4, for example, SEQ ID NO: 1.

[0027] In one aspect, there is provided a polynucleotide encoding a recombinant / fusion polypeptide as defined above.

[0028] In one aspect, there is provided a vector comprising a polynucleotide as defined above.

[0029] In one aspect, there is provided a host cell transfected with or comprising a vector as defined above.

[0030] In one aspect, there is provided a composition comprising a recombinant / fusion polypeptide as defined above.

[0031] In one aspect there is provided a recombinant / fusion polypeptide or composition as defined above for use as a therapy or medicament.

[0032] In one aspect there is provided a recombinant / fusion polypeptide or composition as defined above for use in the treatment and / or prevention of a viral infection in a subject.

[0033] In one aspect there is provided the use of a recombinant / fusion polypeptide or composition as defined above in the manufacture of a medicament for treating and / or preventing a viral infection in a subject.

[0034] In one aspect, there is provided a method for treating and / or preventing a viral infection in a subject in need thereof, comprising administering to said subject a recombinant / fusion polypeptide or composition as defined above.

[0035] In one embodiment, the viral infection is caused by a coronavirus, particularly a virus from the Coronaviridae family, such as Severe Acute Respiratory Syndrome 1 (SARS-CoV-1), Severe Acute Respiratory Syndrome 2 (SARS-CoV-2), etc. [Brief description of the drawings]

[0036] [Figure 1A]Structural modeling of ACE2 binding to SARS-CoV-1 (Model 1) and SARS-CoV-2 (Model 2) viruses is shown. [Figure 1B] 1 shows a flow chart for computationally predicting higher affinity binding polypeptides. [Diagram 2] Octet BioLayer Interferometry (BLI) Sensor. Kinetic measurements of crude ACE2 polypeptide variants against SARS-CoV-1 or SARS-CoV-2 RBD proteins immobilized on an Octet BioLayer Interferometry (BLI) sensor. [Figure 3A] Figure 1 shows the dissociation rates (koff) of ACE2 variants against SARS-CoV-2 RBD (COVID-19). [Figure 3B] Figure 1 shows the dissociation rates (koff) of ACE2 variants against SARS-CoV-1 RBD (SARS). [Figure 4A] Figure 1 shows affinity measurements of ACE2 variants for the viral RBD of SARS-CoV-1 and SARS-CoV-2. [Figure 4B] Kinetics measurements of ACE2 variants against the viral RBD of SARS-CoV-1 and SARS-CoV-2. [Diagram 5] EC50 values ​​(ng / mL) of ACE2 variants against SARS-CoV-2, its variants, and SARS-CoV-1 in the pseudovirus neutralization assay. Values ​​represent the mean of two independent determinations. [Figure 6] Figure 1 shows the binding affinity of ACE2-YHA-Fc variants and wild-type ACE2 to the RBD of bat SARS-like coronaviruses as measured by BLI assay. Both ACE2-YHA-Fc and WT ACE2-Fc carry the R273Q / H505L mutations. A range of MBP-TEV-RBD concentrations (2-fold dilutions) from 200 nM to 3.125 nM were used to test the binding of immobilized ACE2-Fc to bat RBD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] definition As used herein, the term "recombinant polypeptide" refers to a polypeptide made using any recombinant DNA technique.

[0038] As used herein, the term "fusion polypeptide" refers to a polypeptide comprising at least two domains that are encoded by separate genes and that are joined such that the two domains are transcribed and translated as a single unit, thereby producing a single polypeptide.

[0039] As used herein, the term "polypeptide" refers to a single linear chain of any number of amino acid residues joined through peptide bonds.

[0040] As used herein, the term "angiotensin-converting enzyme 2" or "ACE2" refers to a protein that belongs to the angiotensin-converting enzyme family of dipeptidyl carboxydipeptidases and has significant homology to human angiotensin 1-converting enzyme. ACE2 catalyzes the cleavage of angiotensin I to angiotensin 1-9 and angiotensin II to the vasodilator angiotensin 1-7. ACE2 is known to be expressed in various human organs, and its organ- and cell-specific expression suggests that it may play a role in regulating cardiovascular and renal function, as well as reproductive function. Additionally, the encoded protein is a functional receptor for the spike glycoprotein of the human coronavirus HCoV-NL63 and the human severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2, the latter of which is the causative agent of coronavirus disease-2019 (COVID-19). Multiple splice variants of this gene have been found, with the dACE2 (or MIRb-ACE2) splice variant found to be interferon inducible. Unless specified, the term ACE2 generally refers to human ACE2 (Uniprot ID Q9BYF1 and / or NCBI reference sequence NP_068576.1) or that encoded by nucleotide NM 021804.3.

[0041] As used herein, the term "immunoglobulin" or "antibody" refers to a bivalent Y-shaped molecule that contains two identical heavy chains and two identical light chains. Disulfide bonds link the heavy and light chain pairs and the two heavy chains to each other. Each chain consists of one variable domain that differs in sequence and is responsible for antigen binding, which are V and VD for the heavy and light chains, respectively. H Domain and V L Each chain also consists of at least one constant domain. The light chain contains the constant domain (C L ) in the heavy chain and at least three (C H 1. C H 2, and C H 3), and sometimes 4 depending on the isotype (CH 4) There are five different classes or isotypes of antibodies in humans, including IgA (including IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM.

[0042] As used herein, the term "immunoglobulin fragment" refers to an antibody that may be, but is not limited to, a Fab, a modified Fab, a Fab', a modified Fab', a F(ab')2, a Fv, a Fab-Fv, a Fab-dsFv, a single domain antibody (e.g., VH or VL or VHH), a scFv, a bivalent, trivalent, or tetravalent antibody, a Bis-scFv, a diabody, a triabody, a tetrabody, and an epitope-binding fragment of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). Methods for generating and producing these antibody fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0043] As used herein, the term "viral protein" refers to any protein produced by a virus, such as a viral envelope or viral capsid protein.

[0044] As used herein, the term "coronavirus" refers to a group of enveloped viruses with a positive-sense, single-stranded RNA genome and a nucleocapsid or helical symmetry. Coronaviruses are characterized by club-shaped spikes protruding from their surface, which resemble a star's corona in electron micrographs, hence the name. Examples of coronaviruses include, but are not limited to, SARS-CoV-1, SARS-CoV-2, alphacoronavirus 1, human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), betacoronavirus 1, Middle East Respiratory Syndrome-associated coronavirus (MERS-Cov), murine coronavirus, avian coronavirus, porcine coronavirus HKU15, and the like.

[0045] As used herein, the term "Flaviviridae" refers to a family of positive-sense, single-stranded, enveloped RNA viruses that are transmitted primarily through arthropod vectors such as ticks and mosquitoes. Members of the Flaviviridae family include, but are not limited to, West Nile virus, Dengue virus, tick-borne encephalitis virus, yellow fever virus, Zika virus, and the like.

[0046] As used herein, the term "severe acute respiratory syndrome coronavirus 1" or "SARS-CoV-1" refers to the strain of coronavirus that causes Severe Acute Respiratory Syndrome (SARS).

[0047] As used herein, the term "severe acute respiratory syndrome coronavirus 2" or "SARS-CoV-2" refers to the coronavirus that causes novel coronavirus disease 2019 (COVID-19).

[0048] Both SARS-CoV-1 and SARS-CoV-2 are enveloped, positive-sense, single-stranded RNA viruses that infect epithelial cells in the lungs. The viruses enter host cells by binding to ACE2 and are known to infect a variety of organisms, including but not limited to humans, bats, and cats.

[0049] The term "large spike glycoprotein", used interchangeably with "spike protein" and "surface glycoprotein", abbreviated as "S protein", as used herein, refers to the spike protein of SARS-CoV-2. The S protein is a 180-200 kDa trimeric class I fusion protein consisting of two subunits, called the S1 and S2 subunits. The S1 subunit (symbol: CoV_S1, Pfam: PF01600, InterPro: IPR002551) is composed of 672 amino acids (residues 14-685 of the S protein) and is involved in mediating binding to host cells via interaction with the human receptor angiotensin-converting enzyme 2 (ACE2). The S2 (symbol: CoV_S2, Pfam: PF01601, InterPro: IPR002552) subunit is composed of 588 amino acids (residues 686-1273) and mediates fusion of the viral cell membrane by forming a six-helix bundle via two heptad repeat domains. Thus, the S protein plays an important role in binding to and invasion of host cells. The amino acid sequence of the S protein is set forth in GenBank No. QHD43416.1.

[0050] As used herein, the term "mutation" refers to a change in the amino acid sequence of a peptide, polypeptide, or protein, or a change in the nucleic acid sequence encoding a peptide, polypeptide, or protein. The four main causes of mutations are spontaneous mutations, mutations due to errors during replication, mutations introduced during DNA repair, and induced mutations caused by mutagens. Mutations can be further classified into large-scale mutations, such as gene duplications, polyploidization, and deletions of large chromosomal regions, and small-scale mutations, such as insertions, deletions, and substitutions, that usually affect one or a few nucleotides.

[0051] As used herein, the term "neutralization" refers to reducing viral infectivity, typically by binding to the surface of the viral particle, such that the binding viral receptor, thereby blocking a step in the viral replication cycle that precedes viral transcription or synthesis.

[0052] As used herein, the term "subject" includes patients and non-patients. The term "patient" refers to an individual who is suffering from or likely to suffer from a medical condition, such as a Flavivirus infection, while "non-patient" refers to an individual who is not suffering from or unlikely to suffer from a medical condition. "Non-patient" includes healthy individuals, non-diseased individuals, and / or individuals who do not have a medical condition. The term "subject" includes humans and animals. Animals include rodents and the like. "Rodent" refers to any mammal of the Murine family, such as mice, rats, and the like.

[0053] The terms "treatment," "treat," and "therapy," and their equivalents, as used herein, refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (attenuate) medical conditions, including, but not limited to, diseases (such as flavivirus infections), symptoms, and disorders. A medical condition also includes the body's response to a disease or disorder, such as inflammation. Those in need of such treatment include those who already have a medical condition, as well as those who are prone to a medical condition or those for whom a medical condition is desired to be prevented.

[0054] As used herein, the term "therapeutic agent" refers to a drug, protein, peptide, gene, chemical compound, or other pharma- ceutical active ingredient.

[0055] The term "and / or", e.g., "X and / or Y", should be understood to mean either "X and Y" or "X or Y" and should be interpreted as providing explicit support for both meanings or either meaning.

[0056] Further, in the description herein, the term "substantially", whenever used, is understood to include but is not limited to "entirely" or "completely", etc. Furthermore, terms such as "comprising", "comprise", etc., whenever used, are intended to be open-ended descriptive language in that they broadly include the elements / components listed after such term in addition to other components not expressly listed. For example, when "comprising" is used, a reference to "a" feature is also intended to be a reference to "at least one" of that feature. Terms such as "consisting", "consist", etc., may be considered subsets of terms such as "comprising", etc., in appropriate context. Thus, in embodiments disclosed herein using terms such as "comprising", it will be understood that these embodiments provide teachings of corresponding embodiments using terms such as "consisting", etc. Additionally, terms such as "about," "approximately," and the like, whenever used, typically refer to a reasonable variation, such as ±5% variation of the disclosed value, or a 4% variation of the disclosed value, or a 3% variation of the disclosed value, or a 2% variation of the disclosed value, or a 1% variation of the disclosed value.

[0057] Further, in the description herein, certain values ​​may be disclosed in ranges. The values ​​at the end of the range are intended to indicate the preferred range. Whenever a range is described, it is intended that the range encompasses and teaches all possible subranges in addition to the individual values ​​within the range. That is, the end points of the range should not be interpreted as invariant limits. For example, a description of a range of 1% to 5% is intended to specifically disclose subranges such as 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., in addition to values ​​within the range such as 1%, 2%, 3%, 4%, and 5%, respectively. It is also to be understood that the individual values ​​within the range include integers, fractions, and decimals. Furthermore, whenever a range is described, it is also intended that the range encompasses and teaches values ​​from the end points of the numerical values ​​set forth to a further decimal point or to two significant digits (where appropriate). For example, a description of a range of 1% to 5% is intended to specifically disclose the range of 1.00% to 5.00%, as well as the range of 1.0% to 5.0% and all intermediate values ​​therein (e.g., 1.01%, 1.02%...4.98%, 4.99%, 5.00% and 1.1%, 1.2%...4.8%, 4.9%, 5.0%, etc.). The above specific disclosure is applicable to ranges of any depth / breadth.

[0058] As used herein, "at least 95% identical" is intended to refer to an amino acid sequence that is 95% or more identical to a reference sequence over its entire length, for example, 96%, 97%, 98%, or 99% identical. Software programs can be used to calculate the percent identity.

[0059] Furthermore, when describing some embodiments, the present disclosure may disclose a method and / or process as a particular sequence of steps. However, it will be understood that unless specifically required, the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of steps disclosed herein should not be construed as unduly limiting. Unless specifically required, the method and / or process disclosed herein should not be limited to steps performed in the order described. The sequence of steps may be varied and still be within the scope of the present disclosure.

[0060] Furthermore, while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, it will be understood that one or more of these features / characteristics may be denied in other alternative embodiments, and the present disclosure provides support for such negations and these related alternative embodiments.

[0061] Description of the embodiments Those skilled in the art will appreciate that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the present disclosure as broadly described. For example, in the description herein, features of different illustrative embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, subsumed, etc. among the different illustrative embodiments. The present embodiments are therefore considered in all respects to be illustrative and not restrictive.

[0062] In one aspect, a recombinant / fusion polypeptide is provided comprising a mutant angiotensin-converting enzyme 2 (ACE2) protein or a fragment thereof and an immunoglobulin fragment.

[0063] Advantageously, the polypeptides disclosed herein have improved binding affinity to viral spike proteins, such as the receptor binding domain (RBD) of SARS-CoV-1 and SARS-CoV-2. Additionally, the disclosed polypeptides have enhanced neutralizing capacity against SARS-CoV-1, SARS-CoV-2, and SARS-CoV-2 variant pseudoviruses. Thus, the polypeptides disclosed herein are likely to be used in therapy, for example, in the treatment of viral infections, such as SARS and COVID-19.

[0064] In one embodiment, the recombinant / fusion polypeptide comprises an immunoglobulin Fc fragment (region / domain), a protein capable of increasing the half-life of the recombinant / fusion polypeptide (e.g., albumin, human albumin, etc.), a linker capable of increasing binding valency (e.g., a rigid linker or a flexible linker such as GGGGS repeats), or a combination thereof, for example, the immunoglobulin Fc fragment is an IgG Fc fragment, such as a human IgG Fc fragment.

[0065] In various embodiments, the immunoglobulin fragment may include, but is not limited to, an Fc region / domain, one or more CH regions / domains, Fab, Fab', F(ab')2, single chain Fv (ScFv) and / or Fv fragments, a hinge region / domain, and fragments or portions thereof.

[0066] In some embodiments, the recombinant / fusion polypeptides may also contain portions of immunoglobulin molecules or antibodies, including, but not limited to, all or a portion of the constant heavy chain, variable heavy chain, constant light chain, variable light chain, hinge region, and / or Fc domain of Ig, and variants thereof. For example, the recombinant / fusion polypeptides described herein may be combined with a portion of human IgG. In various embodiments, the immunoglobulin type may include one or more types, such as, but not limited to, IgG, IgE, IgM, IgD, IgA, and IgY. In various embodiments, the immunoglobulin type may be of the classes IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or subclasses thereof.

[0067] In one embodiment, the immunoglobulin fragment is an IgG Fc fragment. In one embodiment, the immunoglobulin fragment is a human IgG Fc fragment, such as an Fc fragment comprising SEQ ID NO: 23. Thus, in one embodiment, the immunoglobulin fragment comprises the sequence: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23) Includes.

[0068] In one embodiment, the recombinant / fusion polypeptide binds to a viral protein.

[0069] In one embodiment, the viral protein is a coronavirus protein or a Flaviviridae protein.

[0070] In one embodiment, the coronavirus is a severe acute respiratory syndrome virus, such as severe acute respiratory syndrome 1 (SARS-CoV-1), severe acute respiratory syndrome 2 (SARS-CoV-2), etc. Thus, in one embodiment, the recombinant / fusion polypeptide binds to SARS-CoV-1 and / or SARS-CoV-2.

[0071] In various embodiments, SARS-CoV-2 includes various variants known in the art, such as alpha variants, beta variants, gamma variants, epsilon variants, eta variants, iota variants, kappa variants, mu variants, zeta variants, delta variants, etc. In various embodiments, the viruses described herein are selected from the group consisting of SARS-CoV-2, CoV-2 V483A, CoV-2 G476S, CoV-2 L455I / F456V, CoV-2 V483I, CoV-2 N439K, CoV-2 S494P, CoV-2 D614G, CoV-2 S477N, CoV-2 A222V, CoV-2 Y453F, CoV-2 N501Y, CoV-2 E406W, CoV-2 Q493F, CoV-2 Q493K, CoV-2 L455F, CoV-2 E484K, CoV-2 G485D, CoV-2 F486K, CoV-2 F486V, CoV-2 F486A, CoV-2 F486L, CoV-2 This includes, but is not limited to, SARS-CoV-1 K417N, CoV-2 N501Y / D614G, CoV-2 E406W / D614G, CoV-2 K417N / E484K / N501Y, alpha (B.1.1.7), beta (B.1.351), gamma (P.1), kappa (B.1.617.1), delta (B.1.617.2), epsilon (B.1.429), and other variants.

[0072] In various embodiments, the ACE2 protein or fragment thereof may comprise one mutation, or two mutations, or three mutations, or four mutations, or five mutations, or six mutations, or seven mutations, or eight mutations. In various embodiments, the ACE2 protein or fragment thereof may comprise at least one mutation, or at least two mutations, or at least three mutations, or at least four mutations, or at least five mutations, or at least six mutations, or at least seven mutations, or at least eight mutations. In various embodiments, the ACE2 protein or fragment thereof may comprise no more than one mutation, or no more than two mutations, or no more than three mutations, or no more than four mutations, or no more than five mutations, or no more than six mutations, or no more than seven mutations, or no more than eight mutations.

[0073] In various embodiments, mutations can include, but are not limited to, substitutions, insertions, deletions, duplications, frameshifts, and the like.

[0074] In one embodiment, the mutation is a substitution.

[0075] In various embodiments, the substitution replaces an amino acid with a naturally occurring amino acid, such as, but not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In various embodiments, the substitution does not include replacement with cysteine. Thus, in various embodiments, the substitution replaces an amino acid with a naturally occurring amino acid, such as, but not limited to, alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0076] In various embodiments, the substitution may be a conservative substitution, substituting another with similar properties. For example, replacing one amino acid with another amino acid of the same group. In various embodiments, the substitution may include substitution with an amino acid with different properties. In various embodiments, the substitution is made without affecting the biological activity of the recombinant polypeptides described herein. It will be understood that the mutations described herein provide mutant ACE2 proteins with substantially the same structure as the native ACE2 protein. Thus, the mutant ACE2 proteins described herein are capable of binding to viral proteins or fragments thereof. In various embodiments, the mutant ACE2 proteins still retain the ability to bind to viral proteins or fragments thereof, but have lost or reduced native peptidase activity. In various embodiments, the substitution increases the binding affinity to viral proteins.

[0077] In various embodiments, the mutations may be present in a compact region of ACE2 that binds to the spike glycoprotein of SARS-CoV and / or SARS-CoV-2. For example, the mutations may be present at positions shown in PDB structures 2AJF (SARS-CoV, ACE2 Uniprot ID Q9BYF1; Spike GP Uniprot ID P59594, DOI 10.2210 / pdb2AJF / pdb) and 6M0J (SARS-CoV-2, ACE2 Uniprot ID Q9BYF1, Spike GP Uniprot ID P0DTC2; DOI10.2210 / pdb6M0J / pdb).

[0078] In various embodiments, the designated positions may be selected to increase binding stability between ACE2 and the SARS-CoV-2 spike glycoprotein. In various embodiments, the designated positions may be determined using free energy calculations. Without wishing to be bound by theory, the mutations in the present disclosure increase interface complementarity by increasing hydrophobicity and / or interdigitation within the contact regions while minimizing clashes between residues.

[0079] In various embodiments, ACE2 as used herein refers to the extracellular domain of ACE2. In various embodiments, ACE2 as used herein refers to amino acids 18-740 of human ACE2 (Uniprot ID Q9BYF1 and / or NCBI reference sequence NP_068576.1), or encoded by nucleotide NM 021804.3. Thus, in one embodiment, the recombinant / fusion polypeptide comprises a mutant extracellular domain of ACE2.

[0080] In various embodiments, the recombinant / fusion polypeptide may / may not include the C-terminal domain of ACE2. In various embodiments, the recombinant / fusion polypeptide may include a domain that interacts with SARS-CoV-1 and / or SARS-CoV-2. In various embodiments, the recombinant / fusion polypeptide may include a domain that interacts with the spike glycoprotein of SARS-CoV-1 and / or SARS-CoV-2. In various embodiments, the recombinant / fusion polypeptide may include a truncated ACE2 that is capable of binding to SARS-CoV-1 and / or SARS-CoV-2 (such as the spike glycoprotein of SARS-CoV-1 and / or SARS-CoV-2). In various embodiments, the recombinant / fusion polypeptide does not include a truncated ACE2 that does not have binding affinity to SARS-CoV-1 and / or SARS-CoV-2 (such as the spike glycoprotein of SARS-CoV-1 and / or SARS-CoV-2).

[0081] In one embodiment, the ACE2 protein or fragment thereof comprises one or more mutations at amino acid positions selected from the group consisting of 27, 28, 31, 34, 41, and 42. Advantageously, the inventors have found that these designated amino acid positions may alter the interaction of ACE2 with viral spike proteins, for example affecting the interface stability between ACE2 and viral spike proteins. Thus, introducing a mutation at one or more of these positions is likely to increase the binding affinity of ACE2 to viral spike proteins.

[0082] In one embodiment, the ACE2 protein or fragment thereof comprises mutations at amino acid positions selected from the group consisting of 27, 31, 34, and 42. In another embodiment, the ACE2 protein or fragment thereof comprises mutations at amino acid positions selected from the group consisting of 27, 31, and 34. In one embodiment, the ACE2 protein or fragment thereof comprises mutations at amino acid positions 27, 31, and 34. Surprisingly, introduction of mutations at these designated amino acid positions slowed the rate of dissociation from the viral spike protein.

[0083] In some embodiments, the recited amino acid positions are calculated from position 1 with reference to ACE2 according to human ACE2 (Uniprot ID Q9BYF1 and / or NCBI reference sequence NP_068576.1) or as encoded by nucleotide NM 021804.3.

[0084] In various embodiments, the human ACE2 has the sequence: (SEQ ID NO:24) Includes.

[0085] Thus, in one embodiment, the mutations at amino acid positions 27, 28, 31, 34, 41, and 42 refer to the mutations at amino acid positions 27, 28, 31, 34, 41, and 42 as set forth in SEQ ID NO:24. Thus, in one embodiment, the ACE2 protein or a fragment thereof comprises one or more mutations at amino acid positions selected from the group comprising 27, 28, 31, 34, 41, and 42 as set forth in SEQ ID NO:24. In one embodiment, the ACE2 protein or a fragment thereof comprises one or more mutations at amino acid positions selected from the group comprising 27, 31, 34, and 42 as set forth in SEQ ID NO:24. In one embodiment, the ACE2 protein or a fragment thereof comprises one or more mutations at amino acid positions selected from the group comprising 27, 31, and 34 as set forth in SEQ ID NO:24. In one embodiment, the ACE2 protein or a fragment thereof comprises one or more mutations at amino acid positions selected from the group comprising 27, 31, and 34 as set forth in SEQ ID NO:24.

[0086] In one embodiment, the ACE2 protein or fragment thereof comprises: T27A, T27R, T27N, T27D, T27E, T27Q, T27G, T27H, T27I, T27L, T27K, T27M, T27F, T27P, T27S, T27T, T27W, T27Y, T27V; Q42A, Q42R, Q42N, Q42D, Q42E, Q42Q, Q42G, Q42H, Q42I, Q42L, Q42K, Q42M, Q42F, Q42P, Q42S, Q42T, Q42W, Q42Y, Q42V; H34A, H34R, H34N, H34D, H34E, H34Q, H34G, H34H, H34I, H34L, H34K, H34M, H34F, H34P, H34S, H34T, H34W, H34Y, H34V; K31A, K31R, K31N, K31D, K31E, K31Q, K31G, K31H, K31I, K31L, K31K, K31M, K31F, K31P, K31S, K31T, K31W, K31Y, K31V; F28A, F28R, F28N, F28D, F28E, F28Q, F28G, F28H, F28I, F28L, F28K, F28M, F28F, F28P, F28S, F28T, F28W, F28Y, F28V; Y41A, Y41R, Y41N, Y41D, Y41E, Y41Q, Y41G, Y41H, Y41I, Y41L, Y41K, Y41M, Y41F, Y41P, Y41S, Y41T, Y41W, Y41Y, and Y41V The mutations include one or more mutations selected from the group consisting of:

[0087] In one embodiment, the ACE2 protein or fragment thereof comprises: T27A, T27R, T27N, T27D, T27E, T27Q, T27G, T27H, T27I, T27L, T27K, T27M, T27F, T27P, T27S, T27W, T27Y, T27V; Q42A, Q42R, Q42N, Q42D, Q42E, Q42H, Q42I, Q42L, Q42K, Q42M, Q42F, Q42P, Q42S, Q42T, Q42W, Q42Y, Q42V; H34A, H34R, H34N, H34D, H34E, H34Q, H34G, H34I, H34L, H34M, H34F, H34P, H34S, H34T, H34W, H34Y, H34V; K31A, K31R, K31N, K31Q, K31H, K31I, K31K, K31M, K31F, K31W, K31Y, K31V; F28W, F28Y; Y41H and Y41F The mutations include one or more mutations selected from the group consisting of:

[0088] In one embodiment, the ACE2 protein or fragment thereof comprises one or more mutations selected from the group consisting of T27Y, K31Y, K31H, H34A, H34V, and combinations thereof. In one embodiment, the ACE2 protein or fragment thereof comprises ...

[0089] Advantageously, the inventors have demonstrated that these designated mutations result in mutants that exhibit the slowest dissociation rates for viral spike proteins, such as the RBD of SARS-CoV-1 and / or SARS-Cov-2.

[0090] In one embodiment, the ACE2 protein or a fragment thereof is T27Y / K31H / H34A, T27Y / K31Y / H34A, T27Y / K31H / H34V, or T27Y / K31Y / H34V It contains one of the following combinations of mutations:

[0091] Advantageously, recombinant / fusion polypeptides containing these triple mutation combinations exhibit significantly improved binding affinity to spike proteins, such as the RBD of SARS-CoV-1 and / or SARS-Cov-2, compared to polypeptides carrying single mutations.

[0092] In one embodiment, the ACE2 protein or fragment thereof comprises the following combination of mutations: T27Y / K31H / H34A. Advantageously, recombinant / fusion polypeptides comprising T27Y / K31H / H34A showed the best overall binding affinity to viral spike proteins such as the RBD of SARS-CoV-1 and / or SARS-Cov-2. Surprisingly, such triple mutant polypeptides also showed significantly improved neutralization potency across a broad range of SARS-CoV-2 variants (more than 30 variants) and SARS-CoV-1 compared to polypeptides with single T27Y, K31H, or H34A mutations. Furthermore, the triple mutant polypeptides showed high binding affinity to at least five bat SARS-like coronavirus strains.

[0093] In one embodiment, the recombinant / fusion polypeptide is capable of binding to a virus, and optionally, the recombinant / fusion polypeptide is capable of blocking / interfering / inhibiting / hindering / disrupting the binding of a virus (such as SARS-CoV-2) to a cellular entry receptor.

[0094] In various embodiments, the recombinant / fusion polypeptides bind strongly to viruses, such as SARS-CoV-2. In various embodiments, the recombinant / fusion polypeptides bind strongly to proteins (such as spike protein) of viruses, such as SARS-CoV-2. In various embodiments, the recombinant / fusion polypeptides have a strong blocking / interfering / inhibiting / hindering / disrupting effect on viral binding, such as SARS-CoV-2 binding to cell entry receptors. In various embodiments, the recombinant / fusion polypeptides bind strongly to the relevant EC 50 The value is from about 0.1 nM to about 360 nM, from about 0.5 nM to about 100 nM, from about 0.5 nM to about 50 nM, from about 0.5 nM to about 25 nM, from about 0.5 nM to about 10 nM, from about 0.5 nM to about 9 nM, or from about 0.5 nM to about 8 nM. In some embodiments, the associated EC 50The values ​​are about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 9 nM or less, or about 8 nM or less, or about 7 nM or less, or about 6 nM or less, or about 5 nM or less, or about 4 nM or less, or about 3 nM or less, or about 2 nM or less, or about 1 nM or less.

[0095] In some embodiments, the recombinant / fusion polypeptide is capable of neutralizing or mediating the neutralization of the entry of a virus, such as SARS-CoV-2, into a cell, optionally a human cell, and further optionally an ACE2 expressing / presenting cell.

[0096] In various embodiments, the recombinant / fusion polypeptide has a strong neutralizing effect on the cell entry of a virus such as SARS-CoV-2. In various embodiments, the relevant EC50 value of the recombinant / fusion polypeptide is about 1 ng / mL to about 17000 ng / mL, about 5 ng / mL to about 1000 ng / mL, about 5 ng / mL to about 100 ng / mL, about 5 ng / mL to about 60 ng / mL, about 5 ng / mL to about 30 ng / mL, or about 5 ng / mL to about 12 ng / mL. In some embodiments, the relevant EC50 value of the recombinant / fusion polypeptide is about 500 ng / mL or less, about 100 ng / mL or less, about 50 ng / mL or less, about 40 ng / mL or less, about 30 ng / mL or less, about 20 ng / mL or less, or about 10 ng / mL or less.

[0097] Thus, in one embodiment, a recombinant / fusion polypeptide is provided that is capable of recognizing / interacting / binding to Severe Acute Respiratory Syndrome 2 (SARS-CoV-2) and / or Severe Acute Respiratory Syndrome 1 (SARS-CoV-1).

[0098] In one embodiment, the ACE2 protein or fragment thereof comprises one or more mutations that abolish the natural peptidase activity of ACE2. The advantage of including such mutations is that the risk of undesirable side effects caused by the enzymatic activity of ACE2 is reduced.

[0099] In one embodiment, the ACE2 protein or fragment thereof comprises a mutation at amino acid position 273 and / or 505, such as at amino acid position 273 and / or 505 according to SEQ ID NO: 24. Advantageously, the mutation at position 273 and / or 505 can affect the natural peptidase activity of ACE2.

[0100] In one embodiment, the ACE2 protein or fragment thereof further comprises an R273Q and / or H505L mutation, for example both R273Q and H505L.

[0101] In one embodiment, the recombinant / fusion protein or fragment thereof comprising the R273Q and / or H505L mutation has the sequence: QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYE DYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGQFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGH IQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFLVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLR LGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22).

[0102] In one embodiment, the recombinant / fusion protein or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-21, as shown in Table 1 below.

[0103] [Table 1-1]

[0104] [Table 1-2]

[0105] [Table 1-3]

[0106] [Table 1-4]

[0107] [Table 1-5]

[0108] [Table 1-6]

[0109] [Table 1-7]

[0110] In one embodiment, the recombinant / fusion protein or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4, e.g., SEQ ID NO: 1. Thus, in one embodiment, the recombinant / fusion protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the recombinant / fusion protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the recombinant / fusion protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the recombinant / fusion protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0111] In one aspect, there is provided a polynucleotide encoding a recombinant / fusion polypeptide as defined above.

[0112] In various embodiments, the polypeptides and / or polynucleotides described herein may include polypeptides and / or polynucleotides having similarity or identity to the sequences described herein. For example, the polypeptides and / or polynucleotides described herein may include polypeptides and / or polynucleotides having at least 70%, at least 75%, 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 100% identity compared to the sequences described herein. Percent sequence identity can be determined using methods known in the art, for example, using programs such as BLAST, NBLAST, XBLAST, etc.

[0113] In one aspect, there is provided a vector comprising a polynucleotide as defined above.

[0114] In some embodiments, the vector is selected from the group consisting of plasmids, viral particles, phages, baculoviruses, yeast plasmids, lipid-based vehicles, polymeric microspheres, liposomes, and cell-based vehicles, colloidal gold particles, lipopolysaccharides, polypeptides, polysaccharides, viral vehicles, adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, foamy viruses, cytomegaloviruses, Semliki Forest viruses, pox viruses, pseudorabies viruses, RNA viral vectors, DNA viral vectors, and vectors derived from combinations of plasmids and phage DNA, and further optionally, the polynucleotide is operably linked to expression control sequence(s) to direct peptide synthesis, and even further optionally, the vector comprises one or more selectable marker genes to provide a phenotypic trait for selecting transformed host cells.

[0115] In one aspect, there is provided a host cell transfected with or comprising a vector as defined above.

[0116] Any suitable host cell / vector system can be used for expression of the DNA sequences encoding the molecules of the invention. Bacterial, such as E. coli and other microbial systems, as well as eukaryotic, such as mammalian, host cell expression systems can be used. Suitable mammalian host cells include CHO cells, myeloma cells, or hybridoma cells.

[0117] In one embodiment, the host cell may include a bacterial cell, a yeast cell, an animal cell, such as a mammalian cell, and / or a plant cell. In various embodiments, the host cell may be a mammalian cell, such as, but not limited to, a Chinese Hamster Ovary (CHO) cell.

[0118] In one aspect, there is provided a composition comprising a recombinant / fusion polypeptide as defined above. In one embodiment, the composition comprises a therapeutic composition or a pharmaceutical composition. Thus, in one aspect, there is provided a pharmaceutical and / or therapeutic composition comprising a recombinant / fusion polypeptide as defined above.

[0119] In one embodiment, the composition further comprises a pharma- ceutically acceptable excipient, buffer, or carrier.

[0120] Pharmaceutically acceptable salts may be used, for example mineral acid salts such as hydrochloride, hydrobromide, phosphate, and sulfate; or organic acid salts such as acetates, propionates, malonates, and benzoates.

[0121] Pharmaceutically acceptable carriers in therapeutic compositions may further contain liquids such as water, saline, glycerol, ethanol, etc. In addition, auxiliary substances such as wetting or emulsifying agents, or pH buffering substances, may be present in such compositions. Such carriers allow the pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, and suspensions for ingestion by a patient. A thorough discussion of pharma-ceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ 1991).

[0122] In one aspect there is provided a recombinant / fusion polypeptide or composition as defined above for use as a therapy or medicament.

[0123] In one aspect there is provided a recombinant / fusion polypeptide or composition as defined above for use in the treatment and / or prevention of a viral infection in a subject.

[0124] In one aspect there is provided the use of a recombinant / fusion polypeptide or composition as defined above in the manufacture of a medicament for treating and / or preventing a viral infection in a subject.

[0125] In one aspect, there is provided a method for treating and / or preventing a viral infection in a subject in need thereof, comprising administering to the subject a recombinant / fusion polypeptide or composition as defined above.

[0126] In one aspect, there is provided a method of neutralising a virus in a subject in need thereof, comprising administering a recombinant / fusion protein and / or composition as defined above, wherein for example the virus is a coronavirus such as SARS-CoV-1 and / or SARS-CoV-2.

[0127] In one embodiment, the recombinant / fusion polypeptide or composition is administered to a subject by any mode of administration known in the art, including but not limited to, intramuscular, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intranasal, parenteral, etc.

[0128] In one embodiment, the subject is a mammal, such as a monkey, rabbit, mouse, rat, pig, or dog, hi one embodiment, the subject is a human.

[0129] In one embodiment, the viral infection is caused by a coronavirus, particularly a virus from the Coronaviridae family, such as Severe Acute Respiratory Syndrome 1 (SARS-CoV-1), Severe Acute Respiratory Syndrome 2 (SARS-CoV-2), etc.

[0130] In one aspect, there is provided a protein, composition, therapy, product, or method as described herein. EXAMPLES

[0131] Exemplary embodiments of the present disclosure will be better understood and readily apparent to those skilled in the art from the following discussion and, where applicable, in conjunction with the drawings. It should be understood that other modifications may be made without departing from the scope of the present invention. Exemplary embodiments are not necessarily mutually exclusive, as some may be combined with one or more embodiments to form new exemplary embodiments. Example embodiments should not be construed as limiting the scope of the present disclosure.

[0132] Example 1 - Development of higher affinity ACE2 polypeptide variants The affinity improvement process began with structural modeling and computational prediction of amino acid positions that, when mutated, may alter interactions with the viral spike protein. Figure 1A shows structural modeling of ACE2 binding to SARS-CoV-1 (model 1) and SARS-CoV-2 (model 2) viruses. Figure 1B shows a flow chart of the process for computational prediction of higher affinity binding ACE2 polypeptide variants.

[0133] ACE2 variants with mutations R273Q / H505L, which abolish its native peptidase activity, were cloned as fusion proteins with human IgG1 Fc. Amino acids at positions 27, 28, 31, 34, 41, and 42 were selected by computational prediction for mutational studies. Amino acids at each position were replaced with all possible natural amino acids except cysteine ​​by site-directed mutagenesis, and ACE2 variants were expressed in ExpiCHO-S cells by transient transfection. Crude recombinant ACE2 variants were then subjected to binding analysis by biolayer interferometry (BLI) to determine dissociation from the receptor binding domain (RBD) of SARS-CoV-1 or SARS-CoV-2.

[0134] The analytical results are shown in Figure 2 and Figures 3A and 3B. ACE2 variants that showed slower dissociation rates for one or both antigens were selected and combined to form combinatorial mutants for further analysis.

[0135] Example 2 - Development of combinatorial ACE2 polypeptide variants Four combinatorial ACE2 variants were generated: T27Y / K31H / H34A (YHA), T27Y / K31H / H34V (YHV), T27Y / K31Y / H34A (YYA), and T27Y / K31Y / H34V (YYV). All of these contain two additional mutations R273Q / H505L that abolish the enzymatic activity of ACE2. The affinity of these four ACE2 variants along with wild-type ACE2 (also containing R273Q / H505L) was measured by biolayer interferometry (BLI). ACE2 protein was captured on an anti-human IgG Fc (AHC) sensor and binding to the receptor binding domain (RBD) was measured at two-fold dilutions starting from 200 nM to 3.125 nM. The RBDs of SARS-CoV-2 or SARS-CoV-1 used in affinity measurements were fused to the C-terminus of maltose binding protein (MBP). The results showed that the triple mutant combinations T27Y / K31H / H34A (YHA), T27Y / K31H / H34V (YHV), T27Y / K31Y / H34A (YYA), and T27Y / K31Y / H34V (YYV) all showed significantly improved binding affinity to the RBDs of both SARS-CoV-1 and SARS-CoV-2 (see Figure 4A). The optimal variant ACE2-YHA bound to the SARS-CoV-1 RBD and SARS-CoV-2 RBD with KD values ​​of 0.75 nM and 1.69 nM, respectively, which were 55- and 15-fold lower than WT ACE2. The K31H mutation improved binding affinity to the SARS-CoV-1 RBD over K31Y, and the H34A mutation improved SARS-CoV-2 RBD binding over H34V. Affinity and kinetic results are shown in Figures 4A and 4B.

[0136] The neutralizing capacity of wild-type and mutant ACE2 against SARS-CoV-1 and SARS-CoV-2, as well as a panel of SARS-CoV-2 variants, was then measured using a pseudovirus neutralization assay. 50 The values ​​are summarized in Figure 5.

[0137] In general, binding affinity was observed to correlate well with neutralization potency. Three single amino acid mutants (ACE2-T27Y, ACE2-K31H, and ACE2-H34A) and WT (wild-type) ACE2-Fc were able to neutralize all pseudotyped viruses, but their neutralizing efficacy against different mutants varied. In contrast, ACE2-YHA retained high neutralizing potency across a broad range of SARS-CoV-2 variants and SARS-CoV-1, with an IC 50 was in the range of 5 ng / mL–60 ng / mL in most cases. It showed significantly improved potency against SARS-CoV-1 and several SARS-CoV-2 variants, such as E406W, E406W / D614G, and F486A, with IC50s >9-fold lower than that of WT ACE2.

[0138] Example 3 - Binding affinity of ACE2-YHA variants to bat coronaviruses We next tested the binding affinity of the ACE2-YHA variants to five bat SARS-like coronavirus strains: LYRa11, Rs4084, Rs4231, Rs7327, and RsSCH014.

[0139] The RBD sequences of LYRa11 and Rs7327 are known to be highly similar to SARS-CoV-1, whereas Rs4231, RsSHC014, and Rs4084 showed greater genetic divergence from SARS-CoV-1 in the RBD region. Pseudotyped viruses expressing the spikes of Rs7327, Rs4231, and RsSHC014 were able to replicate in human ACE2-expressing cells. Therefore, the RBDs of these coronaviruses were generated and fused to the C-terminus of the MBP protein for kinetic binding assays. The results of the assays are shown in Figure 6.

[0140] ACE2-YHA showed 1.9- to 2.7-fold improved binding affinity compared to WT ACE2. Recombinant WT ACE2 was previously shown to block cell entry of pseudotyped viruses of these five strains, suggesting that ACE2-YHA variants with improved binding affinity should also have cross-CoV neutralizing activity.

[0141] Purpose The present inventors have developed novel recombinant / fusion polypeptides containing mutant angiotensin-converting enzyme 2 (ACE2) with enhanced affinity for the spike proteins of SARS-CoV-1 and SARS-CoV-2 coronaviruses. Specifically, the present disclosure demonstrates that the recombinant polypeptide, known as ACE-YHA2, has the ability to neutralize pseudotyped viruses expressing the spike proteins of over 30 existing SARS-CoV-2 variant strains.

[0142] Thus, the polypeptides disclosed herein may enhance neutralizing potency against SARS-CoV-1, SARS-CoV-2, and SARS-CoV-2 variant pseudoviruses, and thus may function as universal therapeutic agents against viruses such as SARS-CoV-1 and SARS-CoV-2.

Claims

1. A recombinant / fusion polypeptide comprising a mutant angiotensin-converting enzyme 2 (ACE2) protein or a fragment thereof and an immunoglobulin fragment.

2. 2. The recombinant / fusion polypeptide of claim 1, comprising an immunoglobulin Fc fragment (region / domain), a protein capable of extending the half-life of the recombinant / fusion polypeptide (e.g., albumin, human albumin, etc.), a linker capable of increasing valency (e.g., a rigid linker or a flexible linker such as GGGGS repeats), or a combination thereof, wherein, for example, the immunoglobulin Fc fragment is an IgG Fc fragment, such as a human IgG Fc fragment.

3. The immunoglobulin fragment has the sequence: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23) 2. The recombinant / fusion polypeptide of claim 1, comprising:

4. The recombinant / fusion polypeptide of claim 1 which binds to a viral protein.

5. The recombinant / fusion polypeptide described in claim 1, wherein the recombinant / fusion polypeptide binds to a viral protein, and the viral protein is a coronavirus protein or a Flaviviridae protein.

6. The recombinant / fusion polypeptide of claim 1, wherein the recombinant / fusion polypeptide binds to a coronavirus protein, and the coronavirus is a severe acute respiratory syndrome virus such as severe acute respiratory syndrome 1 (SARS-CoV-1) or severe acute respiratory syndrome 2 (SARS-CoV-2).

7. The recombinant / fusion polypeptide of claim 1, wherein the ACE2 protein or fragment thereof comprises one or more mutations, two or more mutations, three or more mutations, four or more mutations, five or more mutations, six or more mutations, seven or more mutations, or eight or more mutations, for example three mutations.

8. 2. The recombinant / fusion polypeptide of claim 1, wherein the ACE2 protein or fragment thereof comprises one or more mutations at amino acid positions selected from the group consisting of positions 27, 28, 31, 34, 41, and 42, such as positions 27, 28, 31, 34, 41, and 42 set forth in SEQ ID NO:

24.

9. The recombinant / fusion polypeptide of claim 1, wherein the ACE2 protein or fragment thereof comprises mutations at amino acid positions selected from the group consisting of positions 27, 31, and 34, for example positions 27, 31, and 34 set forth in SEQ ID NO:

24.

10. The ACE2 protein or a fragment thereof is T27A, T27R, T27N, T27D, T27E, T27Q, T27G, T27H, T27I, T27L, T27K, T27M, T27F, T27P, T27S, T27T, T27W, T27Y, T27V; Q42A, Q42R, Q42N, Q42D, Q42E, Q42Q, Q42G, Q42H, Q42I, Q42L, Q42K, Q42M, Q42F, Q42P, Q42S, Q42T, Q42W, Q42Y, Q42V; H34A, H34R, H34N, H34D, H34E, H34Q, H34G, H34H, H34I, H34L, H34K, H34M, H34F, H34P, H34S, H34T, H34W, H34Y, H34V; K31A, K31R, K31N, K31D, K31E, K31Q, K31G, K31H, K31I, K31L, K31K, K31M, K31F, K31P, K31S, K31T, K31W, K31Y, K31V; F28A, F28R, F28N, F28D, F28E, F28Q, F28G, F28H, F28I, F28L, F28K, F28M, F28F, F28P, F28S, F28T, F28W, F28Y, F28V; Y41A, Y41R, Y41N, Y41D, Y41E, Y41Q, Y41G, Y41H, Y41I, Y41L, Y41K, Y41M, Y41F, Y41P, Y41S, Y41T, Y41W, Y41Y, and Y41V 2. The recombinant / fusion polypeptide of claim 1, comprising one or more mutations selected from the group consisting of:

11. The ACE2 protein or a fragment thereof is T27A, T27R, T27N, T27D, T27E, T27Q, T27G, T27H, T27I, T27L, T27K, T27M, T27F, T27P, T27S, T27W, T27Y, T27V; Q42A, Q42R, Q42N, Q42D, Q42E, Q42H, Q42I, Q42L, Q42K, Q42M, Q42F, Q42P, Q42S, Q42T, Q42W, Q42Y, Q42V; H34A, H34R, H34N, H34D, H34E, H34Q, H34G, H34I, H34L, H34M, H34F, H34P, H34S, H34T, H34W, H34Y, H34V; K31A, K31R, K31N, K31Q, K31H, K31I, K31K, K31M, K31F, K31W, K31Y, K31V; F28W, F28Y; Y41H and Y41F 2. The recombinant / fusion polypeptide of claim 1, comprising one or more mutations selected from the group consisting of:

12. 2. The recombinant / fusion polypeptide of claim 1, wherein the ACE2 protein or fragment thereof comprises one or more mutations selected from the group consisting of T27Y, K31Y, K31H, H34A, H34V, and combinations thereof.

13. The ACE2 protein or a fragment thereof is selected from the group consisting of: T27Y / K31H / H34A, T27Y / K31Y / H34A, T27Y / K31H / H34V, or T27Y / K31Y / H34V 2. The recombinant / fusion polypeptide of claim 1, comprising one of the following combinations of mutations:

14. The ACE2 protein or a fragment thereof is selected from the group consisting of: T27Y / K31H / H34A 2. The recombinant / fusion polypeptide of claim 1, comprising a combination of mutations:

15. 2. The recombinant / fusion polypeptide of claim 1, which is capable of binding to a virus and optionally blocking / interfering / inhibiting / hindering / disrupting the binding of a virus (such as SARS-CoV-2) to a cell entry receptor.

16. 2. The recombinant / fusion polypeptide of claim 1, which is capable of neutralizing or mediating (or initiating) the neutralization of a virus such as SARS-CoV-2.

17. 2. The recombinant / fusion polypeptide of claim 1, wherein the ACE2 protein or fragment thereof comprises one or more mutations that abolish the native peptidase activity of ACE2.

18. 2. The recombinant / fusion polypeptide of claim 1, wherein the ACE2 protein or fragment thereof comprises a mutation at amino acid positions 273 and / or 505, for example, at amino acid positions 273 and / or 505 according to SEQ ID NO:

24.

19. The recombinant / fusion polypeptide of claim 1, wherein the ACE2 protein or fragment thereof further comprises the mutations R273Q and / or H505L, e.g., both R273Q and H505L.

20. The ACE2 protein or fragment thereof further comprises a R273Q and / or H505L mutation, for example both R273Q and H505L, and the recombinant / fusion protein or fragment thereof comprising the R273Q and / or H505L mutation has the sequence: QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQS TLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDN PQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYE DYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYIS PIGCLPAHLLGDMWGQFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVS VGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMG HIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEIN FLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYC DPASLFLVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISSNSTEAGQKLFNML RLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSI KVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPR ISFNFFVTAPKNVSDIIPRTEEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPV SEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22) 20. The recombinant / fusion polypeptide of claim 19, comprising:

21. 2. The recombinant / fusion polypeptide of claim 1, wherein the recombinant / fusion protein or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-21.

22. The recombinant / fusion polypeptide of claim 1, wherein the four recombinant / fusion polypeptides or fragments thereof comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4, e.g., SEQ ID NO:

1.

23. A polynucleotide encoding the recombinant / fusion polypeptide of claim 1.

24. A vector comprising a polynucleotide encoding the recombinant / fusion polypeptide of claim 1.

25. A host cell transfected with or containing a vector comprising a polynucleotide encoding the recombinant / fusion polypeptide of claim 1.

26. A composition comprising the recombinant / fusion polypeptide of claim 1.

27. A pharmaceutical composition comprising the recombinant / fusion polypeptide of claim 1.

28. A pharmaceutical composition for treating and / or preventing viral infections, comprising the recombinant / fusion polypeptide of claim 1.

29. 27. Use of a recombinant / fusion polypeptide according to claim 1 or a composition according to claim 26 in the manufacture of a medicament for treating and / or preventing a viral infection in a subject.

30. 29. The pharmaceutical composition of claim 28, wherein the viral infection is caused by a coronavirus, particularly a virus of the Coronaviridae family, such as Severe Acute Respiratory Syndrome 1 (SARS-CoV-1), Severe Acute Respiratory Syndrome 2 (SARS-CoV-2), etc.

31. The use according to claim 29, wherein the viral infection is caused by a coronavirus, particularly a virus of the Coronaviridae family, such as Severe Acute Respiratory Syndrome 1 (SARS-CoV-1) or Severe Acute Respiratory Syndrome 2 (SARS-CoV-2).

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