WO2022034588A1 - Compositions for diagnosis and treatment of coronavirus infections - Google Patents

Abstract

A composition of matter is provided. The composition comprises a soluble polypeptide comprising an amino acid sequence of an ecto domain of human ACE2, the amino acid sequence comprising a plurality of mutations as compared to the ecto domain of wild type human ACE2 set forth in SEQ ID NO: 1, wherein the plurality of mutations increase binding of the polypeptide to a SPIKE protein of SARS-CoV-2 to a KD below 0.2 nM, as measured by surface plasmon resonance (SPR). Also provided uses of same and methods of producing the same.

Description

COMPOSITIONS FOR DIAGNOSIS AND TREATMENT OF

CORONAVIRUS INFECTIONS

RELATED APPLICATIONS

This application claims priority from Israeli Patent Application No. 276627, filed August 10, 2020 which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 88269 Sequence Listing.txt, created on 9 August 2021, comprising 48,355 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to compositions for diagnosis and treatment of Coronavirus infections.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an ongoing devastating pandemic leading to a substantial global death toll and an unprecedented colossal economic lost. There is an urgent need for anti-viral countermeasures that will help to save lives as well as will shorten the recovery and hospitalization time of sick people. Immunotherapy has the potential to neutralize viruses directly as well as to recruit immune effector functions to clear infected cells, and it makes a promising approach that was already shown to be beneficial in combating other viral diseases. A monoclonal antibody (mAb) against the respiratory syncytial virus is effective and was approved for treating infected children ^1,2. A breakthrough in the field of HIV was the isolation of broadly- neutralizing mAbs ^3,4 and the affirmation of their use in treating ^5,6 and protecting ^7,8 individuals from HIV-1. In addition, antibodies against Lassa ⁹, Junin ¹⁰, Ebola ¹¹ , and even SARS ¹² viruses have been proven effective in animal models. Preliminary reports indicate that also in the case of COVID- 19, sera from convalescent patients help in fighting the disease^13,14. Hence, targeted immunotherapy may become a powerful anti-viral tool for fighting COVID-19.

Immunoadhesins are antibody-like molecules that consist of a binding domain fused to an Fc portion on an antibody ¹⁸. The viral cellular receptor could serve as a binding domain for constructing such immunoadhesins. Due to natural adaptation, though, zoonotic viruses may bind to their animal-derived ortholog cellular receptors at higher affinities than the human cell- surface receptors ¹⁹. Thus, immunoadhesins that are constructed with the host-ortholog receptors can become superior anti-viral therapeutics. This approach was recently demonstrated by constructing Arenacept, which is a powerful immunoadhesin that targets viruses from the Arenaviridae family of viruses ²⁰.

SARS-CoV-2 is a zoonotic virus that utilizes angiotensin-converting enzyme 2 (ACE2) as a cellular receptor ²¹’²³. The genome of SARS-CoV-2 is similar to bat-derived SARS-like coronaviruses ²⁴, but the exact origin of the virus is unknown and other animal species may have served as intermediate reservoirs before SARS-CoV-2 crossed over to the human population ²⁵. It was shown that ACE2 orthologs from various animals can serve as entry receptors for SARS- CoV-2 ²⁶. Furthermore, it was demonstrated that the human-ACE2 is a suboptimal receptor for SARS-CoV-2 ²⁷ (Chan et al. 10.1126/Science.abc0870 (2020).

Additional background art includes:

ClinicalTrials.gov#NCT04287686 teaching the clinical use of an Fc-ACE2 Fusion.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a composition of matter comprising a soluble polypeptide comprising an amino acid sequence of an ecto domain of human ACE2, the amino acid sequence comprising a plurality of mutations as compared to the ecto domain of wild type human ACE2 set forth in SEQ ID NO: 1, wherein the plurality of mutations increase binding of the polypeptide to a SPIKE protein of SARS-CoV-2 to a KD below 0.2 nM, as measured by surface plasmon resonance (SPR).

According to some embodiments of the invention, the decreased KD is in the range of 0.1- 0.01 nM.

According to some embodiments of the invention, the polypeptide inhibits binding of the SPIKE protein to human ACE2 expressing cells.

According to some embodiments of the invention, the plurality of mutations comprise at least one mutation in an N-terminal helix of human ACE2.

According to some embodiments of the invention, the plurality of mutations comprise at least one mutation which improves packing with hydrophobic residues in SARS-CoV-2 receptor binding domain (RBD).

According to some embodiments of the invention, the plurality of mutations comprise at least one mutation at position T27 corresponding to SEQ ID NO: 1.

According to some embodiments of the invention, the at least one mutation is T27L. According to some embodiments of the invention, the plurality of mutations comprise at least one mutation which generates a salt bridge with SARS-CoV-2 RBD.

According to some embodiments of the invention, the at least one mutation is at position D30 and/or Q42 corresponding to SEQ ID NO: 1.

According to some embodiments of the invention, the at least one mutation is D30E and/or Q42R.

According to some embodiments of the invention, the plurality of mutations are at position Glu75 and/or Leu79 which impart improved interaction with Phe486 of SARS-CoV-2 RBD.

According to some embodiments of the invention, the plurality of mutations comprise Gly75R and/or Leu79Y.

According to some embodiments of the invention, the plurality of mutations comprise at least one mutation in N33O for improved packing against Thr500 of SARS-CoV-2 RBD.

According to some embodiments of the invention, the plurality of mutations comprise at least one mutation that abolishes a glycosylation site of the human ACE2.

According to some embodiments of the invention, the glycosylation site comprises an N- X-T glycosylation motif.

According to some embodiments of the invention, the at least one mutation is at positions 89-92 corresponding to SEQ ID NO: 1.

According to some embodiments of the invention, the plurality of mutations comprise at least one mutation that renders the human ACE2 catalytically inactive.

According to some embodiments of the invention, the at least one mutation is at position Glu375 corresponding to SEQ ID NO: 1.

According to some embodiments of the invention, the polypeptide is of a length not exceeding 600 amino acid residues.

According to some embodiments of the invention, the plurality of mutations comprise T27L, D30E, Q42R, E75R, L79Y and N33OF.

According to some embodiments of the invention, the amino acid sequence is as set forth in SEQ ID NO: 2.

According to some embodiments of the invention, the polypeptide is attached to a heterologous moiety.

According to some embodiments of the invention, the heterologous moiety is capable of inducing an antibody dependent cellular-mediated cytotoxicity (ADCC) response. According to some embodiments of the invention, the heterologous moiety is for increasing avidity of the polypeptide.

According to some embodiments of the invention, the heterologous moiety is for multimerization .

According to some embodiments of the invention, the heterologous moiety is a proteinaceous moiety.

According to some embodiments of the invention, the proteinaceous moiety is selected from the group consisting of an immunoglobulin, a galactosidase, a glucuronidase, a glutathione- S-transferase (GST), a carboxy terminal polypeptide (CTP) from chorionic gonadotrophin (CG0), and a chloramphenicol acetyltransferase (CAT).

According to some embodiments of the invention, the proteinaceous moiety is an immunoglobulin .

According to some embodiments of the invention, the immunoglobulin is an IgG Fc.

According to some embodiments of the invention, the composition of matter is as set forth in SEQ ID NO: 4 or 8.

According to some embodiments of the invention, the heterologous moiety is a non- proteinaceous moiety.

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the composition of matter and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present invention there is provided a method of treating a Coronavirus infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of matter of any one of claims 1-32, thereby treating the infection.

According to some embodiments of the invention, the composition of matter is for use in treating a Coronavirus infection in a subject in need thereof.

According to an aspect of some embodiments of the present invention there is provided a method of diagnosing a Coronavirus infection in a subject in need thereof, the method comprising:

(a) contacting a biological sample which may comprise a SPIKE protein of a Coronavirus with the composition of matter of any one of claims 1-32 under conditions which allow complex formation between the composition and the SPIKE; (b) analyzing presence or level of the complex, wherein the presence and/or level is indicative of the Coronavirus infection.

According to some embodiments of the invention, the method is performed ex vivo.

According to some embodiments of the invention, the composition of matter is attached to a detectable moiety.

According to some embodiments of the invention, the coronavirus is SAR-CoV-2, Middle East respiratory syndrome Coronavirus (MERS-CoV) or severe acute respiratory syndrome Coronavirus (SARS-CoV).

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGs. 1A-C show the diversity of the SARS-CoV-2/ACE2 interface, a. Structure of the SARS-CoV-2 RBD (grey surface) in complex with human-ACE2 (orange and yellow ribbon) (PDB: 6M17). The side chains of residues that make the recognition site for SARS-CoV-2 are shown as sticks. The N’ -terminal helix of ACE2 that makes the central part of the binding site is highlighted in orange, b. The sequence diversity of the first N’ -terminal helix of ACE2 in mammals is presented using a WebLogo display ³⁷. The abundance of the amino acid types in each position is represented by the height of their single-letter code. The residues that interact with the RBD of SARS-CoV-2 are indicated by red arrows, c. Interface properties of the various RBS/ACE2-ortholog models. Each dot represents a single model. From left to right, five panels show the calculated total Rosetta energy (using Rosetta energy units), the binding energy (AAG for binding), the buried surface area, the packing statistics, and the shape complementarity of the interface. All the panels are arranged such that the values at the top represent better results. The RBD/human-ACE is indicated with a green dot. The modified ACE2 is indicated with a red dot.

FIGs. 2A-F show optimized design of the ACE2 interface for improved binding of SARS- CoV-2. The interfaces of SARS-CoV-2 RBD/human-ACE2 (grey and orange/yellow, respectively) and of the SARS-CoV-2 RBD/modified-ACE2 (pink and light blue, respectively) are shown, a. Leucine, instead of threonine in position 27, makes better Van der Waals interactions with hydrophobic residues on the SARS-CoV-2 RBD. b. Glutamic acid in position 30 of ACE2 can make a salt bridge with Lys417 of SARS-CoV-2, but not an aspartic acid that is present in the human-ACE2. c. Arginine in position 42 can form a salt-bridge with Asp38 of ACE2 to stabilize it in a configuration that allows it to make a hydrogen bond with the hydroxyl of Tyr449 from SARS-CoV-2 RBD. An arginine in this position can also assume a different rotamer that will allow it to form electrostatic interaction with the main-chain carbonyl oxygen of Gly447 of SARS-CoV-2 RBD. d. A double replacement of Leu79 and Glu75 with tyrosine and arginine respectively allows favorable interaction between Phe486 of SARS-CoV-2 RBD and Tyr79 that is stabilized through a hydrogen bond by Arg75. e. Phenylalanine in position 330 of ACE2 is predicted to pack better against the aliphatic portion of Thr500 from SARS-CoV-2 RBD. f. A replacement of Thr92 with arginine abrogates the glycosylation site on Asn90, which bears a glycan that can sterically interfere with the binding of SARS-CoV-2 RBD. An arginine in position 92 of ACE2 can form a hydrogen bond with the nearby Gln388.

FIGs. 3A-C show that ACE2^mod-Fc is a superior binder of SARS-CoV-2, as well as SARS-CoV-1. Figure 3A. Coomassie- stained SDS-PAGE showing ACE2-Fc and ACE2^mod-Fc. Figures 3B-C. SPR analyses of SARS-CoV-2 RBD or SARS-CoV-1 RBD interaction with ACE2-Fc and ACE2^mod-Fc. Both ACE2-Fc and ACE2^mod-Fc were immobilized to a protein-A sensor chip and SARS-CoV-2 RBD was injected at the indicated concentration series in a singlecycle kinetics experiment. The kinetic parameters are indicated. In the case of SARS-CoV-2 RBD binding to ACE2^mod-Fc these parameters are derived from a simple 1:1 binding model. In the case of SARS-CoV-2 RBD binding to ACE2-Fc, these parameters are derived from heterogeneous-ligand binding model, and reflect the first component. These experiments were repeated twice and a representative sensorgram is shown for each ligand.

FIGs. 4A-E is a graph showing that ACE2^mod-Fc is endowed with improved biological activity. Neutralization of pseudotyped viruses by ACE2-Fc and ACE2^mod-Fc is shown. The calculated IC50 and ICso values are indicated. Error bars represent standard deviations. Experiments were repeated three times, and representative graphs are shown.

FIG. 5 is a graph showing the enzymatic activity of ACE2. Enzymatic activity of ACE2- Fc and ACE2^mod-Fc was evaluated using an activity reporter assay. Activity of lOng of ACE2-Fc, as well as lOng and lOOng of ACE2^mod-Fc was measured and compered to blank control.

FIGs.6A-B show that the addition of a rich GGSG linker between ACE2^mod and Fc improves its neutralization activity. Figure 3A. Schematic representation of ACE2^mod-Fc and its linker-added version, ACE2^mod GS3-Fc , and a coomassie- stained SDS-PAGE showing both of them. Figure 3B. Box plot of IC50 values for neutralization of pseudotyped SARS-CoV2 viruses by ACE2^mod-Fc and ACE2^mod GS3-Fc. Data points represent 4 different experiments and error bars represent standard error.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to compositions for diagnosis and treatment of Coronavirus infections.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

The binding of SARS-CoV-2 to the human ACE2 is characterized by somewhat compromised affinity which may affect the use of this interaction in designing treatment and diagnostic modalities to combat the disease.

To identify advantageous alterations of ACE2 that may enhance the binding to SARS- CoV-2, 70 orthologous of ACE2 genes were selected having sequence identity to the human- ACE2 which is greater than 80%. These orthologs were modeled by Rosetta to identify their interaction with SARS-CoV-2 based on a crystal structure of SARS-CoV-2 RBD in complex with human- ACE2 (PDB: 6VW1)³⁰. These models were analyzed using five different metrics that measure the interfaces' energy and structure properties (Figure 1C). For in-depth analysis the top- 20 models in terms of computed binding energy (AAG of binding) were selected. These models showed broad distributions of their total Rosetta scores, buried surface areas, packing statistics, and shape complementarities (Figure 1C). All top-20 models were visually inspected and specific residues predicted to make better contacts with the SARS-CoV-2 RBD compared with the human-ACE2 counterparts were identified. Based on these analyses a soluble polypeptide was designed that comprises selected mutations in the human ACE2 sequence that impart the polypeptide with improved binding to the SPIKE protein as evidenced by decreased KD. Such a polypeptide can be advantageously used as is or to generate a treatment modality to combat COVID19.

Thus, according to an aspect of the invention there is provided a composition of matter comprising a soluble polypeptide comprising an amino acid sequence of an ecto domain of human ACE2, said amino acid sequence comprising a plurality of mutations as compared to said ecto domain of wild type human ACE2 set forth in SEQ ID NO: 1 (of which the ecto domain according to a specific embodiment, is set forth in SEQ ID NO: 7), wherein said plurality of mutations increase binding of said polypeptide to a SPIKE protein of SARS-CoV-2 to a KD below 0.2 nM as measured by surface plasmon resonance (SPR).

As used herein “composition of matter” refers to the polypeptide per se or the polypeptide mixed or attached to another agent (such as in the case of a fusion protein or chemically modified polypeptide or any of the above mixed with another agent such as a buffer).

As used herein “soluble” refers to the portion of ACE2 which is devoid of a transmembrane domain (as defined by coordinates 741-761 of the human ACE2 protein) and the membrane proximal extracellular portion 616-741, and optionally also the intracellular domain (762 till end 805). Amino acid coordinates correspond to SEQ ID NO: 1.

As used herein “ACE2” refers to Angiotensin-converting enzyme 2 (ACE2) E.C. 3.4.17.23 (GenBank Accession No. NP_068576), which is encoded in human by the ACE2 gene. ACE2 is an enzyme attached to the cell membranes of cells in the lungs, arteries, heart, kidney, and intestines. ACE2 lowers blood pressure by catalysing the hydrolysis of angiotensin II (a vasoconstrictor peptide) into angiotensin (1-7) (a vasodilator). ACE2 counters the activity of the related angiotensin-converting enzyme (ACE) by reducing the amount of angiotensin-II and increasing Ang(l-7) ACE2 also serves as the entry point into cells for Coronaviruses, including HCoV-NL63, SARS-CoV, and SARS-CoV-2. The human version of the enzyme is often referred to as hACE2.

According to a specific embodiment, the ACE2 is human ACE2.

As used herein “an ecto domain of ACE2” refers to the extracellular portion of the ACE2 protein i.e., amino acid coordinates 1-740 e.g., 19-615 of the human protein (i.e., SEQ ID NO: 1).

The “N terminal helix of hACE2” is defined by coordinates 21-53 of SEQ ID NO: 1.

According to a specific embodiment, the ecto domain is a fragment of the extracellular portion but is capable of binding the SPIKE protein of SARS-CoV-2 or any other Coronavirus. The ecto domain of ACE2 has been subjected to site directed mutagenesis in order to select those amino acid coordinates and optionally critical substitutions therein which increase the binding affinity to the RBD-SPIKE protein, as described hereinbelow.

The terms "peptide" and “polypeptide” which are interchangeably used herein encompass native peptides backbone (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body, more capable of penetrating into cells improving clearance, biodistribution and/or pharmacokinetics. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.

Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by N- methylated amide bonds (-N(CH3)-CO-), ester bonds (-C(=O)-O-), ketomethylene bonds (-CO- CH2-), sulfinylmethylene bonds (-S(=O)-CH2-), a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl (e.g., methyl), amine bonds (-CH2-NH-), sulfide bonds (-CH2-S-), ethylene bonds (-CH2- CH2-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), fluorinated olefinic double bonds (-CF=CH-), retro amide bonds (-NH-CO-), peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally present on the carbon atom.

These modifications can occur at any of the bonds along the peptide chain and even at several (e.g. 2-3) bonds at the same time.

Natural aromatic amino acids, Tyr and Phe, may be substituted by non-natural aromatic amino acids such as l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O-methyl-Tyr.

According to a specific embodiment, the polypeptide comprises naturally occurring Trp residues.

The polypeptides of some embodiments of the invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc). The term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phospho threonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids (stereoisomers).

Tables 1 and 2 below list naturally occurring amino acids (Table 1), and non-conventional or modified amino acids (e.g., synthetic, Table 2) which can be used with some embodiments of the invention. Table 1

Table 2

The amino acids of the polypeptides of some embodiments of the present invention may be substituted either conservatively or non-conservatively.

The term “conservative substitution” as used herein, refers to the replacement of an amino acid present in the native sequence in the polypeptide with a naturally or non-naturally occurring amino or a peptidomimetics having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).

As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be easily determined bearing in mind the fact that in accordance with the invention replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions. For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner.

When affecting conservative substitutions the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

The phrase "non-conservative substitutions" as used herein refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or -NH-CH[(-CH2)5-COOH]-CO- for aspartic acid. Those non- conservative substitutions which fall under the scope of the present invention are those which still constitute a polypeptide capable of binding SPIKE.

The polypeptides of some embodiments of the invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with polypeptide characteristics, cyclic forms of the polypeptide can also be utilized.

Since the present peptides are preferably utilized in therapeutics which requires the peptides to be in soluble form, the peptides of some embodiments of the invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing polypeptide solubility due to their hydroxylcontaining side chain.

The polypeptide is shorter than the full length hACE2. According to some embodiments, by referring to the length, the skilled artisan would appreciate that the reference is made to the ACE2 portion of the polypeptide and not to the length of the polypeptide when attached to a proteineceous heterologous moiety.

According to specific embodiments, the polypeptide is less than 740 amino acids in length.

According to specific embodiments, the polypeptide is less than 720 amino acids in length.

According to specific embodiments, the polypeptide is less than 700 amino acids in length.

According to specific embodiments, the polypeptide is less than 650 amino acids in length.

According to specific embodiments, the polypeptide is less than 600 amino acids in length.

According to specific embodiments, the polypeptide is up to 740, 720, 700, 650, 600 amino acids in length, each possibility represents a separate embodiment of the present invention.

Following is a description of amino acid substitutions, which may be preferably employed. Although additional substitutions can be made measures are taken to minimize the number of mutations as compared to the human sequence in order to decrease immunogenicity.

As used herein plurality of mutations refer to 2-20, 2-15, 2-10, 2-8, 2-6, 2-5, 2-4, 2-3 mutations (with respect to the human sequence) which affect the KD of the polypeptide to SPIKE.

According to a specific embodiment, the plurality of mutations refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mutations.

As used herein “mutation” refers to any mutation (e.g., amino acid substitution, deletion, insertion). According to a specific embodiment, the mutation is a point mutation. According to a specific embodiment, the mutation is substitution. The definition of mutation is with reference to SEQ ID NO: 1.

According to a specific embodiment, the level of identity of the polypeptide to the human ecto domain of ACE2 (SEQ ID NO: 7) is at least 80-99.9999999 % or 100 % identity (e.g., at least 99 %, 99.1 %, 99.2 %, 99.3 %, 99.4 %, 99.5 %, 99.6 %, 99.7 %, 99.8 %, 99.91 %). Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm such as Blast, ClustalW, and MUSCLE.

Thus, according to an embodiment of the invention, the plurality of mutations comprise at least one mutation in an N-terminal helix of human ACE2.

According to an embodiment of the invention, the plurality of mutations comprise at least one mutation which improves packing with hydrophobic residues in SARS-CoV-2 receptor binding domain (RBD). Improved packing is defined by shape complementarity between the two interacting proteins and by small gaps between their surfaces.

As used herein “SARS-CoV-2 receptor binding domain (RBD)” refers to the receptor (ACE2) binding domain of SARS-CoV-2 of SPIKE, residues Arg319-Phe541 of SPIKE (i.e., SEQ ID NO: 6).

The overall ACE2-binding mode to the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor According to an embodiment of the invention, the plurality of mutations comprise at least one mutation at position T27 corresponding to SEQ ID NO: 1.

According to an embodiment of the invention, the at least one mutation is T27L/M/A/D/K/H/WW/Y/F/C .

According to an embodiment of the invention, the at least one mutation is T27L.

According to an embodiment of the invention, the plurality of mutations comprise at least one mutation which generates a salt bridge with SARS-CoV-2 RBD.

A salt bridge between amino acids can be generated between the following pairs of amino acids.

According to an embodiment of the invention, the at least one mutation is at position D30 and/or Q42 corresponding to SEQ ID NO: 1.

According to an embodiment of the invention, the at least one mutation is D30E and/or Q42R.

According to an embodiment of the invention, the at least one mutation is D30E/I/V.

According to an embodiment of the invention, the at least one mutation is Q42/R/M/L/VV/K/H/C .

According to an embodiment of the invention, the plurality of mutations are at position Glu75 and/or Leu79 which impart improved interaction with Phe486 of SARS-CoV-2 RBD (numbering corresponds to full length SPIKE).

According to an embodiment of the invention, the at least one mutation is Glu75R/L/I/V.

According to an embodiment of the invention, the at least one mutation is Lcu79Y/M/I/V/T/R/W/F/P.

According to an embodiment of the invention, the plurality of mutations comprise at least one mutation in N33O for improved packing against Thr500 of SARS-CoV-2 RBD.

According to an embodiment of the invention, the plurality of mutations comprise at least one mutation that abolishes a glycosylation site of said human ACE2.

According to an embodiment of the invention, the glycosylation site comprises an N-X-T glycosylation motif. This is an N-linked glycosylation site which belongs to the classical N- glycosylation motif N-X-S/T (where N is asparagine, X is any amino acid except proline, S is serine, T is threonine).

According to an embodiment of the invention, the at least one mutation is at positions 90- 92 corresponding to SEQ ID NO: 1. It will be appreciated that other approaches can be used to eliminate glycosylation at this site and the skilled in the art of biochemistry and/or protein expression would know which to select. These include but are not limited to, bacterial expression, chemical modification for the removal of glycosylation post expression or the use of cell expression systems which are engineered with a modified glycosylation pathway which prevents this glycosylation.

According to an embodiment of the invention, the plurality of mutations comprise at least one mutation that renders said human ACE2 catalytically inactive.

The catalytic domain of human ACE2 comprises residues His505, His345. In addition Arg273 is critical for substrate binding. Glu375 is important for zinc binding. Many additional residues will likely abrogate activity (see PMID: 16008552).

Various mutations can be introduced to render the protein catalytically inactive. These include, but are not limited to, any of the abovementioned residues.

ACE2 activity can be evaluated using commercially available kits, for example, the SensoLyte® 390 ACE2 Activity Assay Kit (ANASPEC; cat# 72086) according to the manufacturer's protocol.

According to an embodiment of the invention, the at least one mutation is at position Glu375 corresponding to SEQ ID NO: 1.

According to an embodiment of the invention, the polypeptide is of a length not exceeding 600 amino acid residues.

According to an embodiment of the invention, the plurality of mutations comprise T27L, D30E, Q42R, E75R, T92R, L79Y and N33OF.

According to an embodiment of the invention, the amino acid sequence is as set forth in SEQ ID NO: 2.

According to another embodiment, the polypeptide comprises a protecting moiety or a stabilizing moiety.

The term "protecting moiety" refers to any moiety (e.g. chemical moiety) capable of protecting the polypeptide from adverse effects such as proteolysis, degradation or clearance, or alleviating such adverse effects.

The term “stabilizing moiety” refers to any moiety (e.g. chemical moiety) that inhibits or prevents a polypeptide from degradation.

The addition of a protecting moiety or a stabilizing moiety to the polypeptide typically results in masking the charge of the polypeptide terminus, and/or altering chemical features thereof, such as, hydrophobicity, hydrophilicty, reactivity, solubility and the like. Examples of suitable protecting moieties can be found, for example, in Green et al., "Protective Groups in Organic Chemistry", (Wiley, 2. sup. nd ed. 1991) and Harrison et al., "Compendium of Synthetic Organic Methods", Vols. 1-8 (John Wiley and Sons, 1971-1996).

The protecting moiety (or group) or stabilizing moiety (or group) may be added to the N- ( amine) terminus and/or the C- (carboxyl) terminus of the polypeptide.

Representative examples of N-terminus protecting/stabilizing moieties include, but are not limited to, formyl, acetyl (also denoted herein as “Ac”), trifluoroacetyl, benzyl, benzyloxycarbonyl (also denoted herein as "CBZ"), tert-butoxycarbonyl (also denoted herein as "BOC"), trimethylsilyl (also denoted "TMS"), 2-trimethylsilyl-ethanesulfonyl (also denoted "SES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (also denoted herein as "FMOC"), nitro-veratryloxycarbonyl (also denoted herein as "NVOC"), t- amyloxycarbonyl, adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl, 2- chlorobenzyloxycarbonyl and the like, nitro, tosyl (CH3C6H4SO2-), adamantyloxycarbonyl, 2, 2, 5, 7, 8-pentamethylchroman-6-sulfonyl, 2,3,6-trimethyl-4-methoxyphenylsulfonyl, t-butyl benzyl (also denoted herein as “BZL”) or substituted BZL, such as, p-methoxybenzyl, p- nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, 2,6-dichlorobenzyl, t-butyl, cyclohexyl, cyclopentyl, benzyloxymethyl (also denoted herein as “BOM”), tetrahydropyranyl, chlorobenzyl, 4-bromobenzyl, and 2,6-dichlorobenzyl.

According to one embodiment of the invention, the protecting/stabilizing moiety is an amine protecting moiety.

According to a specific embodiment, the protecting/stabilizing moiety is a terminal cysteine residue.

Representative examples of C-terminus protecting/stabilizing moieties are typically moieties that lead to acylation of the carboxy group at the C-terminus and include, but are not limited to, benzyl and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers, allyl ethers, monomethoxytrityl and dimethoxy trityl. Alternatively the -COOH group of the C-terminus may be modified to an amide group.

Other modifications of polypeptides include replacement of the amine and/or carboxyl with a different moiety, such as hydroxyl, thiol, halide, alkyl, aryl, alkoxy, aryloxy and the like.

According to a specific embodiment, the protecting/stabilizing moiety is an amide.

According to a specific embodiment, the protecting/stabilizing moiety is a terminal cysteine residue.

According to one embodiment, the protecting/stabilizing moiety comprises at least one, two, three or more cysteine residues at the N- or C- termini of the polypeptide. Also included in the scope of the present invention are "chemical derivative" of a polypeptide or analog. Such chemical derivates contain additional chemical moieties not normally a part of the polypeptide. Covalent modifications of the polypeptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Many such chemical derivatives and methods for making them are well known in the art, some are discussed hereinbelow.

Also included in the scope of the invention are salts of the polypeptides and analogs of the invention. As used herein, the term “salts” refers to both salts of carboxyl groups and to acid addition salts of amino groups of the polypeptide molecule. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as those formed for example, with amines, such as triethanolamine, arginine, or lysine, piperidine, procaine, and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid. Such chemical derivatives and salts are preferably used to modify the pharmaceutical properties of the polypeptide insofar as stability, solubility, etc., are concerned.

According to one embodiment of the invention, the isolated polypeptide capable of binding the SPIKE protein (i.e., the polypeptide described herein) is attached to a heterologous moiety.

As used herein the phrase "heterologous moiety" refers to an amino acid sequence which does not endogenously form a part of the isolated polypeptide’s amino acid sequence. Preferably, the heterologous moiety does not affect the biological activity of the isolated polypeptide (e.g. capability of binding a Coronavirus).

The heterologous moiety may thus serve to ensure stability of the isolated polypeptide of the present invention without compromising its activity. For example, the heterologous polypeptide may increase the half-life of the isolated polypeptide or molecule in the serum.

The heterologous moiety of the present invention may be capable of inducing an antibody dependent cellular-mediated cytotoxicity (ADCC) response as discussed in detail hereinbelow.

According to one embodiment, the heterologous moiety does not induce an immune response. Thus, for instance, in the case of Ig, it may contain human sequences that do not produce an immune response in a subject administered therewith. According to one embodiment, the heterologous moiety is for increasing avidity of the polypeptide.

According to one embodiment, the heterologous moiety is for multimerization of the isolated polypeptide (e.g. at least for dimerization of the isolated polypeptides).

According to one embodiment, the heterologous moiety is a proteinaceous moiety.

Examples of heterologous amino acid sequences that may be used in accordance with the teachings of the present invention include, but are not limited to, immunoglobulin, galactosidase, glucuronidase, glutathione-S-transferase (GST), carboxy terminal polypeptide (CTP) from chorionic gonadotrophin (CGb) and chloramphenicol acetyltransferase (CAT) [see for example U.S. Publication No. 20030171551].

According to a specific embodiment, the heterologous amino acid sequence is an immunoglobulin .

Generally the heterologous amino acid sequence is localized at the amino- or carboxylterminus (N-ter or C-ter, respectively) of the isolated polypeptide of the present invention. The heterologous amino acid sequence may be attached to the isolated polypeptide amino acid sequence by any of polypeptide or non-polypeptide bond. Attachment of the isolated polypeptide amino acid sequence to the heterologous amino acid sequence may be effected by direct covalent bonding (polypeptide bond or a substituted polypeptide bond) or indirect binding such as by the use of a linker having functional groups. Functional groups include, without limitation, a free carboxylic acid (C(=O)OH), a free amino group (NH2), an ester group (C(=O)OR, where R is alkyl, cycloalkyl or aryl), an acyl halide group (C(=O)A, where A is fluoride, chloride, bromide or iodide), a halide (fluoride, chloride, bromide or iodide), a hydroxyl group (OH), a thiol group (SH), a nitrile group (C=N), a free C-carbamic group (NR”-C(=O)-OR’, where each of R’ and R” is independently hydrogen, alkyl, cycloalkyl or aryl).

An example of a heterologous amino acid sequence which may be used in accordance with this aspect of the present invention is an immunoglobulin amino acid sequence, such as the hinge and Fc regions of an immunoglobulin heavy domain (see U.S. Pat. No. 6,777,196). The immunoglobulin moiety in the molecules of this aspect of the present invention may be obtained from IgGl, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM, as further discussed hereinbelow.

According to a specific embodiment the Ig-fusion is as set forth in SEQ ID NO: 4 or 8.

Typically, in such fusions the chimeric molecule will retain at least functionally active hinge and CH2 and CH3 domains of the constant region of an immunoglobulin heavy chain. Fusions can also be generated to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CHI of the heavy chain or the corresponding region of the light chain.

Though it may be possible to conjugate the entire heavy chain constant region to the isolated polypeptide amino acid sequence of the present invention, it is preferable to fuse shorter sequences. For example, a sequence beginning at the hinge region upstream of the papain cleavage site, which defines IgG Fc chemically; residue 216, taking the first residue of heavy chain constant region to be 114, or analogous sites of other immunoglobulins, may be used in the fusion. In a particular embodiment, the isolated polypeptide’s amino acid sequence is fused to the hinge region and CH2 and CH3, or to the CHI, hinge, CH2 and CH3 domains of an IgGl, IgG2, or IgG3 heavy chain (see U.S. Pat. No. 6,777,196).

As mentioned, the immunoglobulin sequences used in the construction of the chimeric molecules of this aspect of the present invention may be from an IgG immunoglobulin heavy chain constant domain. Such IgG immunoglobulin sequence can be purified efficiently on, for example, immobilized protein A. Selection of a fusion partner may also take into account structural and functional properties of immunoglobulins. Thus, for example, the heterologous polypeptide may be IgG3 hinge which is longer and more flexible, so it can accommodate larger amino acid sequences that may not fold or function properly when fused to IgGl. Another consideration may be valency; IgG are bivalent homodimers, whereas Ig subtypes like IgA and IgM may give rise to dimeric or pentameric structures, respectively, of the basic Ig homodimer unit. Other considerations in selecting the immunoglobulin portion of the chimeric molecules of this aspect of the present invention are described in U.S. Pat. No. 6,777,196.

The molecules of the present invention can be generated using recombinant techniques such as described by Bitter et al. (1987) Methods in Enzymol. 153:516-544; Studier et al. (1990) Methods in Enzymol. 185:60-89; Brisson et al. (1984) Nature 310:511-514; Takamatsu et al. (1987) EMBO J. 6:307-311; Coruzzi et al. (1984) EMBO J. 3:1671-1680; Brogli et al. (1984) Science 224:838-843; Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

The heterologous moiety may also be chemically linked to the isolated polypeptide following the independent generation of each. Thus, the two polypeptides may be covalently or non-covalently linked using any linking or binding method and/or any suitable chemical linker known in the art. Such linkage can be direct or indirect, as by means of a polypeptide bond or via covalent bonding to an intervening linker element, such as a linker polypeptide or other chemical moiety, such as an organic polymer. Such chimeric peptides may be linked via bonding at the carboxy (C) or amino (N) termini of the peptides, or via bonding to internal chemical groups such as straight, branched, or cyclic side chains, internal carbon or nitrogen atoms, and the like. The exact type and chemical nature of such cross-linkers and cross linking methods is preferably adapted to the type and nature of the peptides used.

Any linker known in the art can be used with specific embodiments of the invention.

According to specific embodiments, the linker may be derived from naturally-occurring multi-domain proteins or is an empirical linker as described, for example, in Chichili et al., (2013), Protein Sci. 22(2): 153-167, Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357- 1369, the entire contents of which are hereby incorporated by reference. In some embodiments, the linker may be designed using linker designing databases and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369 and Crasto et al (2000), Protein Eng. 13(5):309-312, the entire contents of which are hereby incorporated by reference.

According to specific embodiments, the linker is a synthetic linker such as PEG.

According to specific embodiments, the linker is a peptide linker.

A peptide linker (e.g., 1-20, 1-20, 1-6 amino acids), for example, may comprise repetitive units. For example the linker may comprise several units of GG; GGS; GSG, or SGG and combinations thereof. As mentioned, single amino acid linkers may also be included, e.g., glycine. The peptide linker may also be of type which may easily be modified, e.g. glycosylated.

According to an embodiment of the invention, the linker is (GGSG)3 (SEQ ID NO: 12), where n=l-10 for example (e.g., n=3).

Where a heterologous amino acid sequence is fused to the ectopic (ecto) domain of hACE2 this variant is referred to a fusion protein or a chimeric protein.

As used herein, the term “fused” means that at least a protein or polypeptide is physically associated with another protein or polypeptide, which naturally don’t form a complex. According to a specific embodiment the fused molecule is a “fusion polypeptide” or “fusion protein”, a protein created by joining two or more heterologously related polypeptide sequences together. The fusion polypeptides encompassed in this invention include translation products of a chimeric nucleic acid construct that joins the DNA sequence encoding a hACE2 variant apical domain with the DNA sequence encoding an IgG Fc to form a single open-reading frame. In other words, a "fusion polypeptide" or “fusion protein” is a recombinant protein of two or more proteins which are joined by a polypeptide bond.

The terms “fusion protein”, “chimera”, “chimeric molecule”, or “chimeric protein” are used interchangeably.

According to a specific embodiment, the fusion protein (termed “Coronacept”) is as set forth in SEQ ID NO: 4.

Thus, the molecule of this aspect of the present invention may comprise a heterologous moiety, as described above. Additionally or alternatively, the isolated polypeptide’s amino acid sequence of the present invention may be attached to a non-proteinaceous moiety.

The phrase “non-proteinaceous moiety” as used herein refers to a molecule, not including polypeptide bonded amino acids, that is attached to the above-described isolated polypeptide’s amino acid sequence.

According to one embodiment, the non-proteinaceous moiety is non-toxic.

Exemplary non-proteinaceous moieties which may be used according to the present teachings include, but are not limited to, polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), poly(styrene comaleic anhydride) (SMA), and divinyl ether and maleic anhydride copolymer (DIVEMA).

Such a molecule is highly stable (resistant to in-vivo proteolytic activity probably due to steric hindrance conferred by the non-proteinaceous moiety) and may be produced using common solid phase synthesis methods which are inexpensive and highly efficient, as further described hereinbelow. However, it will be appreciated that recombinant techniques may still be used, whereby the recombinant polypeptide product is subjected to in-vitro modification (e.g., PEGylation as further described hereinbelow).

It will be appreciated that such non-proteinaceous moieties may also be attached to the above mentioned fusion molecules (i.e., which comprise a the apical domain and an amino acid sequence of IgG Fc, the fusion molecules capable of binding an Coronavirus) to promote stability and possibly solubility of the molecules.

Bioconjugation of such a non-proteinaceous moiety (such as PEGylation) can confer the isolated polypeptide’s or fusion protein’s amino acid sequence with stability (e.g., against protease activities) and/or solubility (e.g., within a biological fluid such as blood, digestive fluid) while preserving its biological activity and prolonging its half-life.

Bioconjugation is advantageous particularly in cases of therapeutic proteins which exhibit short half-life and rapid clearance from the blood. The increased half-lives of bioconjugated proteins in the plasma results from increased size of protein conjugates (which limits their glomerular filtration) and decreased proteolysis due to polymer steric hindrance. Generally, the more polymer chains attached per polypeptide, the greater the extension of half-life. However, measures are taken not to reduce the specific activity of the isolated polypeptide or fusion protein of the present invention (e.g. capability of binding a Coronavirus).

Bioconjugation of the isolated polypeptide’s or fusion protein’s amino acid sequence with PEG (z.e., PEGylation) can be effected using PEG derivatives such as N-hydroxy succinimide (NHS) esters of PEG carboxylic acids, monomethoxyPEG2-NHS, succinimidyl ester of carboxymethylated PEG (SCM-PEG), benzotriazole carbonate derivatives of PEG, glycidyl ethers of PEG, PEG p-nitrophenyl carbonates (PEG-NPC, such as methoxy PEG-NPC), PEG aldehydes, PEG-orthopyridyl-disulfide, carbonyldimidazol-activated PEGs, PEG-thiol, PEG- maleimide. Such PEG derivatives are commercially available at various molecular weights [See, e.g., Catalog, Polyethylene Glycol and Derivatives, 2000 (Shearwater Polymers, Inc., Huntsvlle, Ala.)]. If desired, many of the above derivatives are available in a monofunctional monomethoxyPEG (mPEG) form.

In general, the PEG added to the isolated polypeptide’s or fusion protein’s amino acid sequence of the present invention should range from a molecular weight (MW) of several hundred Daltons to about 100 kDa (e.g., between 3-30 kDa). Larger MW PEG may be used, but may result in some loss of yield of PEGylated peptides. The purity of larger PEG molecules should be also watched, as it may be difficult to obtain larger MW PEG of purity as high as that obtainable for lower MW PEG. It is preferable to use PEG of at least 85 % purity, and more preferably of at least 90 % purity, 95 % purity, or higher. PEGylation of molecules is further discussed in, e.g., Hermanson, Bioconjugate Techniques, Academic Press San Diego, Calif. (1996), at Chapter 15 and in Zalipsky et al., "Succinimidyl Carbonates of Polyethylene Glycol," in Dunn and Ottenbrite, eds., Polymeric Drugs and Drug Delivery Systems, American Chemical Society, Washington, D.C. (1991).

Conveniently, PEG can be attached to a chosen position in the isolated polypeptide’s or fusion protein’s amino acid sequence by site-specific mutagenesis as long as the activity of the conjugate is retained (e.g. capability of binding a Coronavirus). A target for PEGylation could be any Cysteine residue at the N-terminus or the C-terminus of the isolated polypeptide’s or fusion protein’s amino acid sequence. Additionally or alternatively, other Cysteine residues can be added to the isolated polypeptide’s or fusion protein’s amino acid sequence (e.g., at the N- terminus or the C-terminus) to thereby serve as a target for PEGylation. Computational analysis may be effected to select a preferred position for mutagenesis without compromising the activity.

Various conjugation chemistries of activated PEG such as PEG-maleimide, PEG- vinylsulfone (VS), PEG-acrylate (AC), PEG-orthopyridyl disulfide can be employed. Methods of preparing activated PEG molecules are known in the arts. For example, PEG- VS can be prepared under argon by reacting a dichloromethane (DCM) solution of the PEG-OH with NaH and then with di-vinylsulfone (molar ratios: OH 1: NaH 5: divinyl sulfone 50, at 0.2 gram PEG/mL DCM). PEG- AC is made under argon by reacting a DCM solution of the PEG-OH with acryloyl chloride and triethylamine (molar ratios: OH 1: acryloyl chloride 1.5: triethylamine 2, at 0.2 gram PEG/mL DCM). Such chemical groups can be attached to linearized, 2-arm, 4-arm, or 8-arm PEG molecules.

While conjugation to cysteine residues is one convenient method by which the isolated polypeptide’s or fusion protein’s amino acid of the present invention can be PEGylated, other residues can also be used if desired. For example, acetic anhydride can be used to react with NH2 and SH groups, but not COOH, S— S, or — SCH3 groups, while hydrogen peroxide can be used to react with — SH and — SCH3 groups, but not NH2. Reactions can be conducted under conditions appropriate for conjugation to a desired residue in the polypeptide employing chemistries exploiting well-established reactivities.

For bioconjugation of the isolated polypeptide’s or fusion protein’s amino acid sequence of the present invention with PVP, the terminal COOH-bearing PVP is synthesized from N-vinyl- 2-pyrrolidone by radical polymerization in dimethyl formamide with the aid of 4,4'-azobis-(4- cyanovaleric acid) as a radical initiator, and 3 -mercaptopropionic acid as a chain transfer agent. Resultant PVPs with an average molecular weight of Mr 6,000 can be separated and purified by high-performance liquid chromatography and the terminal COOH group of synthetic PVP is activated by the N-hydroxysuccinimide/dicyclohexyl carbodiimide method. The isolated polypeptide’s or fusion protein’s amino acid sequence is reacted with a 60-fold molar excess of activated PVP and the reaction is stopped with amino caploic acid (5-fold molar excess against activated PVP), essentially as described in Haruhiko Kamada, et al., 2000, Cancer Research 60: 6416-6420, which is fully incorporated herein by reference.

Resultant conjugated isolated polypeptide or fusion protein molecules (e.g., PEGylated or PVP-conjugated isolated polypeptide or fusion protein) are separated, purified and qualified using e.g., high-performance liquid chromatography (HPLC). In addition, purified conjugated molecules of this aspect of the present invention may be further qualified using e.g., in vitro assays in which the binding specificity of isolated polypeptide or fusion protein to its ligand (e.g., SPIKE protein of a Coronavirus) is tested in the presence or absence of the isolated polypeptide or fusion protein conjugates of the present invention, essentially as described for other polypeptides e.g. by surface plasmon resonance assay.

Molecules of this aspect of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation and classical solution synthesis. These methods are preferably used when the polypeptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involve different chemistry.

Thus, the polypeptides of some embodiments of the invention may be synthesized by any techniques that are known to those skilled in the art of polypeptide synthesis. For solid phase polypeptide synthesis, a summary of the many techniques may be found in J. M. Stewart and J. D. Young, Solid Phase Polypeptide Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973. For classical solution synthesis see G. Schroder and K. Eupke, The Peptides, vol. 1, Academic Press (New York), 1965.

In general, these methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing polypeptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then either be attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions suitable for forming the amide linkage.

The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or concurrently, to afford the final polypeptide compound. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide and so forth. Further description of polypeptide synthesis is disclosed in U.S. Pat. No. 6,472,505. A preferred method of preparing the polypeptide compounds of some embodiments of the invention involves solid phase polypeptide synthesis.

Large scale polypeptide synthesis is described by Andersson Biopolymers 2000; 55(3):227-50.

Synthetic polypeptides can be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.] and the composition of which can be confirmed via amino acid sequencing.

In cases where large amounts of the polypeptides of the present invention are desired, the polypeptides of the present invention can be generated using recombinant techniques such as described by Bitter et al. (1987) Methods in Enzymol. 153:516-544; Studier et al. (1990) Methods in Enzymol. 185:60-89; Brisson et al. (1984) Nature 310:511-514; Takamatsu et al. (1987) EMBO J. 6:307-311; Coruzzi et al. (1984) EMBO J. 3:1671-1680; Brogli et al. (1984) Science 224:838-843; Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

According to another aspect of the invention, there is provided a method of producing the polypeptide of some embodiments of the invention. The method comprises:

(a) expressing the polypeptide in a host cell culture system;

(b) recovering the polypeptide from the culture.

For example, a nucleic acid sequence encoding an isolated polypeptide of the present invention (e.g., the amino acid sequences set forth in SEQ ID NO: 2) is ligated to a nucleic acid sequence which may include an inframe sequence encoding a proteinaceous moiety such as immunoglobulin (e.g., SEQ ID NO: 5).

Also provided is an expression vector, comprising the isolated polynucleotide of some embodiments of the invention. According to one embodiment, the polynucleotide sequence is operably linked to a cis- acting regulatory element.

The nucleic acid construct (also referred to herein as an "expression vector") of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. The nucleic acid construct of some embodiments of the invention typically includes a signal sequence for secretion or presentation of antibody from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence.

Preferably, the promoter utilized by the nucleic acid construct of some embodiments of the invention is active in the specific cell population transformed.

In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of TCRL mRNA translation.

In addition to the elements already described, the expression vector of some embodiments of the invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.

Improvements in recombinant polypeptide expression in mammalian cells can be achieved by effectively increasing the gene dosage in a transfected host cell. Increases in gene copy number are most commonly achieved by gene amplification using cell lines deficient in an enzyme such as dihydrofolate reductase (DHFR) or glutamine synthetase (GS) in conjunction with expression vectors containing genes encoding these enzymes and agents such as methotrexate (MTX), which inhibits DHFR, and methionine sulfoxamine (MSX), which inhibits GS.

Exemplary systems for expression are described in EP2861741, US20120178126, and US20080145895, each of which is incorporated herein by reference in its entirety. Also provided are cells which comprise the polynucleotides/expression vectors as described herein.

Suitable host cells for cloning or expression include prokaryotic or eukaryotic cells. See e.g. Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N. J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli; see Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006) for suitable fungi and yeast strains; and see e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 for suitable plant cell cultures which can also be utilized as hosts.

After expression, the isolated polypeptide or fusion protein may be isolated from the cells in a soluble fraction and can be further purified.

Recovery of the isolated polypeptide or fusion protein may be effected following an appropriate time in culture. The phrase "recovering the recombinant polypeptide or fusion protein” refers to collecting the whole fermentation medium containing the polypeptide or fusion protein and need not imply additional steps of separation or purification.

Notwithstanding the above, proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

Molecules of the present invention are preferably retrieved in "substantially pure" form. As used herein, "substantially pure" refers to a purity that allows for the effective use of the protein in the applications, described herein.

It will be appreciated that the composition of matter comprising the isolated polypeptide or fusion protein of the present invention may comprise a single isolated polypeptide or fusion protein or alternatively may comprise two or more isolated polypeptides or fusion proteins fused together according to any of the methods described hereinabove.

Once polypeptides are obtained, they may be tested for binding affinity as discussed in detail above.

According to one embodiment, the composition of matter comprising the isolated polypeptides or fusion proteins of some embodiments of the invention is also selected capable of neutralizing the Coronaviruses for maximizing therapeutic efficacy.

The term "neutralizing" refers the ability of the composition of matter comprising the isolated polypeptides or fusion proteins to block the site(s) on viruses that they use to enter their target cell. According to one embodiment, the composition of matter comprising the isolated polypeptides or fusion proteins of some embodiments of the invention are capable of neutralizing the virus infectivity by at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, or by 100 % as compared to infectivity in the absence of the composition of matter comprising the isolated polypeptides or fusion proteins of the invention.

Determination of neutralizing of Crononaviruses can be carried out using any method known in the art, such as, by in vitro neutralization assays (such as the one described in the ‘general materials and experimental procedures section’ below).

According to one embodiment, the composition of matter comprising the isolated polypeptides or fusion proteins of some embodiments of the invention is also selected capable of initiating antibody-dependent cellular cytotoxicity (ADCC), i.e. the killing of an antibody-coated target cell by a cytotoxic effector cell (e.g. NK cells, monocytes, macrophages, neutrophils eosinophils and dendritic cells) through a non-phagocytic process (e.g. by the release of the content of cytotoxic granules or by the expression of cell death-inducing molecules).

Determination that the isolated peptides or fusion proteins initiate ADCC can be carried out using any method known in the art such as by measuring lactate dehydrogenase (LDH) release using LDH Cytotoxicity Detection kit (available e.g. from Roche Applied Science).

According to one embodiment, the composition of matter comprising the isolated polypeptides or fusion proteins of some embodiments of the invention is typically capable of promoting eradication of infected cells as well as directly neutralizing Coronaviruses.

According to one embodiment, the composition of matter comprising the isolated polypeptides or fusion proteins of some embodiments of the invention is also selected thermostable (e.g. stable up to 45 °C, up to 50 °C, up to 55 °C, up to 60 °C, or even up to 65 °C). Such determinations can be carried out using any method known in the art, such as by circular dichroism measurements (such as described in the ‘general materials and experimental procedures section’ below).

The polypeptide described herein is endowed with an exceptionally high binding affinity towards the SPIKE protein.

As used herein “binding” or “binds” refers to receptor-ligand mode of binding and in this case hACE2 to the Spike protein.

As used herein the term “KD” refers to the equilibrium dissociation constant between the RBD of Spike and the polypeptide variant of hACE2. According to a specific embodiment, the KD is below 0.2 nM (e.g., 0.01-0.1 nM, 0.01- 0.09 nM, 0.01-0.08 nM, 0.01-0.07 nM, 0.01-0.06 nM, 0.01-0.05 nM, 0.01-0.04 nM, 0.01-0.03 nM, 0.01-0.03 nM, 0.03 nM), as determined by Surface Plasmon Resonance assay (SPR) e.g., where SPIKE RBD is the SOLUBLE analyte.

The affinity of the pair is determined by Surface Plasmon Resonance (SPR) using a captured or immobilized hACE2 polypeptide variant format to minimize contribution of avidity.

According to a specific embodiment, conditions for SPR are provided as follows: the polypeptide variants are first immobilized at a coupling density of -1000 response units (RU) on a series S sensor chip protein A (GE Healthcare) in PBS and 0.02% (w/v) sodium azide buffer. RBD was then injected at 0.16, 0.8, 4, 20, and 100 nM concentrations, at a flow rate of 60 pL/min. Single-cycle kinetics was performed for the binding assay. The sensor chip was regenerated using 10 mM glycine-HCl pH 1.5 buffer.

By virtue of their high affinity the polypeptides of the invention, can be used in various clinical applications including diagnostics.

It is contemplated that the polypeptides described herein can be used as a broad tool to identify infection with various Coronaviruses, such as listed hereinbelow.

Thus, according to an aspect of the invention, there is provided a method of diagnosing a Coronavirus infection in a subject in need thereof, the method comprising:

(a) contacting a biological sample which may comprise a SPIKE protein of a Coronavirus with the composition of matter of any one of claims 1-32 under conditions which allow complex formation between the composition and the SPIKE;

(b) analyzing presence or level of said complex, wherein said presence and/or level is indicative of the Coronavirus infection.

As used herein the term "diagnosing" refers to classifying a disease, determining a severity of a disease (grade or stage), monitoring progression, forecasting an outcome of the disease and/or prospects of recovery.

The subject may be a healthy subject (e.g., human) undergoing a routine well-being check-up. Alternatively, the subject may be at risk of the disease or infection. Yet alternatively, the method may be used to monitor treatment efficacy.

As used herein “biological sample” refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, sputum, and also samples of in vivo cell culture constituents. It should be noted that a “biological sample obtained from the subject” may also optionally comprise a sample that has not been physically removed from the subject, as described in greater detail below.

Numerous well known tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of Coronaviruses or infected cells in the sample. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made.

As mentioned, the method of the present invention is effected under conditions sufficient to form protein-protein interactions i.e., complex (e.g. a complex between the composition of matter comprising the isolated polypeptide or fusion protein of the present invention and the Coronavirus). Such conditions (e.g., appropriate concentrations, buffers, temperatures, reaction times) as well as methods to optimize such conditions are known to those skilled in the art, and examples are disclosed herein below.

The composition of matter comprising the isolated polypeptide or fusion protein of the present invention may comprise e.g., be attached, to an identifiable moiety. Alternatively or additionally, the composition of matter comprising the isolated polypeptide or fusion protein may be identified indirectly such as by using a secondary antibody.

According to one embodiment, diagnosis is corroborated using any diagnostic method known in the art, such as by measuring the viral load or titer, by antigen level measurement, antibody level measurement, virus isolation and/or genomic detection by reverse transcriptase- polymerase chain reaction (RT-PCR), etc. For example, a higher viral load or titre often correlates with the severity of an active viral infection. The quantity of virus per mL can be calculated for example by estimating the live amount of virus in an involved body fluid (e.g. serum sample or whole blood).

The ability to bind the SPIKE protein of a Coronavirus renders the polypeptides suitable as a preventive and therapeutic tool for Coronavirus infection.

Thus, according to an aspect of the invention there is a method of treating a Coronavirus infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of matter comprising the polypeptide described herein, thereby treating the infection.

Alternatively or additionally, there is provided the composition of matter comprising the polypeptide as described herein for use in treating a Coronavirus infection in a subject in need thereof. As used herein, “Coronavirus” refers to enveloped positive- stranded RNA viruses that belong to the family Coronaviridae and the order Nidovirales.

Examples of Corona viruses which are contemplated herein include, but are not limited to, 229E, NL63, OC43, and HKU1 with the first two classified as antigenic group 1 and the latter two belonging to group 2, typically leading to an upper respiratory tract infection manifested by common cold symptoms.

However, Coronaviruses, which are zoonotic in origin, can evolve into a strain that can infect human beings leading to fatal illness. Thus particular examples of Coronaviruses contemplated herein are SARS-CoV, Middle East respiratory syndrome Coronavirus (MERS- CoV), and the recently identified SARS-CoV-2 [causing 2019-nCoV (also referred to as “COVID-19”)].

It would be appreciated that any Corona virus strain is contemplated herein even though SARS-CoV-2 is emphasized in a detailed manner.

According to specific embodiments, the Corona virus is SARS-CoV-2.

The term “treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

As used herein, the term “preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.

As used herein, the term “subject” includes mammals, preferably human beings, male or female, at any age or gender, which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology (e.g., above 65 of age).

The composition of matter comprising the isolated polypeptides or fusion proteins of the present invention can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. Herein the term "active ingredient" refers to the composition of matter comprising the isolated polypeptides or fusion proteins accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intrapulmonary or intraocular injections.

Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebro ventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport polypeptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin polypeptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (composition of matter comprising the isolated polypeptides or fusion proteins) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., Coronaviral infection) or prolong the survival of the subject being treated.

According to an embodiment of the present invention, an effective amount of the composition of matter comprising the isolated polypeptides or fusion proteins of some embodiments of the present invention is an amount selected to neutralize Coronaviruses and/or eliminate infected cells e.g. by initiating ADCC.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For example, any in vivo or in vitro method of evaluating Coronavirus viral load may be employed.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1

P-l).

Dosage amount and interval may be adjusted individually to provide the active ingredient at a sufficient amount to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

The present teachings further envisage treating with other anti-viral drugs or antiinflammatory drugs or anti-coagulants as separate treatments or in a co -formulation.

Without being limited to COVID19 but for the sake of example, according to a specific embodiment, the antiviral drug is selected from the group consisting of remdesivir, an interferon, ribavirin, adefovir, tenofovir, acyclovir, brivudin, cidofovir, fomivirsen, foscamet, ganciclovir, penciclovir, amantadine, rimantadine and zanamivir. Also contemplated are plasma treatments from infected persons who survived and/or anti-HIV drugs such as lopinavir and ritonavir, as well as chloroquine.

Specific examples for drugs that are routinely used for the treatment of COVID-19 include, but are not limited to, Lopinavir /Ritonavir, Nucleoside analogues, Neuraminidase inhibitors, Remdesivir, polypeptide (EK1), abidol, RNA synthesis inhibitors (such as TDF, 3TC), anti-inflammatory drugs (such as hormones and other molecules), Chinese traditional medicine, such ShuFengJieDu Capsules and Lianhuaqingwen Capsule, could be the drug treatment options for 2019-nCoV.

As used herein the term “about” refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

The term “consisting of’ means “including and limited to”.

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1- 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

METHODS

Modeling the interaction of ACE2-orthologs with SARS-CoV-2 by the Rosetta suite - Orthologous sequences of ACE2 were collected by using a protein blast ³³ search of the human- ACE2 sequence at GenBank and filtering the results to mammalian origin, and to sequences with greater than 80% identity to human-ACE2. Sequences were aligned using MUSCLE ³⁴. Each sequence was threaded on the human- ACE2/Spike structure (PDB entry: 6VW1) and relaxed using sidechain packing and backbone, sidechain and rigid-body minimization subject to harmonic constraints on the Ca coordinates observed in the experimental structure. The ref2015 energy function was used in all calculations (xml and command line are NOT SHOWN). 100 models were generated for each sequence and the best scoring one was used for comparisons and analyses.

Construction of expression vectors - Codon-optimized forms of human ACE2 binding region (amino-acids 19-615) and modified ACE2 genes were chemically synthesized (Genscript), and were subcloned upstream of a human Fc region (derived from IgGl) as previously described ³⁵. Additionally, modified ACE2 was subcloned upstream of same Fc region, which was pre-cloned to contain three Gly-Gly-Ser-Gly (GGSG) linker on its N'-terminus. Plasmid (pCMV3) encoding the Full-length Clone DNA of SARS-CoV-2 spike was purchased from Sino Biological, and subcloned to the same plasmid after removing 19AA from the C’-terminus (A19 S_covid-pCMV3, done by Fab of Yossi Shaul). Luciferase-pLenti6 and AR89 vectors for lentivirus production were used. The plasmid encoding the His-tagged SARS-CoV-2 Receptor Binding Domain (RBD)³⁶. Full-length human ACE2 is described elsewhere ²² (Addgene plasmid #1786). His-tagged SARS-CoVl RBD was generated by subcloning the DNA-coding RBD region (AA ) from pLVX-EFlalpha-SARSl-Spike-2xStrep-IRES-Puro plasmid.

Protein expression and purification -

ACE-Fc fusion proteins and the His-tagged SARS-CoV-1 or 2 RBDs were expressed in suspension-HEK293F cells grown in FreeStyle media (Gibco). Transfections were done using 40 kDa polyethyleneimine (PEI-MAX ; Polysciences) at 1 mg of plasmid DNA per 1 L of culture at a cell density of 10⁶/ml. Media were collected six days post-transfection and supplemented with 0.02% (w/v) sodium azide and PMSF. SARS RBDs were buffer exchanged to Phosphate Buffered Saline (PBS) using a tangential flow filtration system (Millipore), and captured using a HiTrap IMAC FF Ni⁺² (GE Healthcare) affinity column followed by size exclusion chromatography purification with a Superdex 200 10/300 increased column (GE Healthcare). Fc- Fusion proteins were isolated using HiTrap protein- A (GE Healthcare) affinity columns.

Surface Plasmon Resonance (SPR) measurements - Binding of SARS-CoV-2 RBD or SARS- CoV-1 RBD to ACE2 -Fc and ACE2^mod-Fc fusion proteins were measured using a Biacore T200 instrument (GE Healthcare). Fusion proteins were first immobilized at a coupling density of -1000 response units (RU) on a series S sensor chip protein A (GE Healthcare) in PBS and 0.02% (w/v) sodium azide buffer. RBD was then injected at 0.16, 0.8, 4, 20, and 100 nM concentrations, at a flow rate of 60 pL/min. Single-cycle kinetics was performed for the binding assay. The sensor chip was regenerated using 10 mM glycine-HCl pH 1.5 buffer.

Estimation of antibody pharmacokinetic profiles in vivo - Male 8 weeks C57BL/6J01aHsd mice (Harlan) were injected intravenously with 0.5mg ACE2-Fc or modified ACE2^mod-Fc in lOOul PBS on day 0. Blood was extracted on days 1,3,6,10,14 and 21, and the plasma was frozen after centrifugation at 3000 rpm for 10 min at 4C. Total human IgG serum concentrations were determined by ELISA. Briefly, high binding ELISA plates (Corning) were coated overnight with goat anti-human IgG (Jackson ImmunoResearch) and blocked with 2% BSA, 1 pM EDTA and 0.1% Tween-20 in PBS for 2 h. Serum and standard samples were incubated for 90 min, followed by HRP-conjugated goat anti-human IgG (Jackson ImmunoResearch) for 90 min. Absorbance at 370 nm was determined (Tecan) after the addition of TMB (Sigma). Plates were washed with 0.05% Tween-20 in PBS between each step. The absence of detectable human IgG was confirmed in all mice using pre-infusion serum samples. Mice experiments were approved by the Weizmann Institute Animal Care and Use Committee.

Lentiviral particles production and Neutralization - Lentiviruses expressing S-Covidl9 spikes were produced by transfecting HEK293T cells with Luciferase-pLenti6, A19 S_covid- pCMV3 and AR89 vectors at 1 : 1 : 1 ratio, using Lipofectamine 2000 (Thermo Fisher). Media containing Lentiviruses was collected at 48h post-transfection, centrifuged at 600g for 5min for clarifying from cells, and aliquots were frozen at -80°C.

For neutralization assays, HEK293T were transiently transfected with hACE2-pCDNA using Lipofectamine 2000. Following 18h post-transfection, cells were re-seeded on a poly-L-lysine pre-coated white, chimney 96-well plates (Greiner Bio-One). Cells were left to adhere for 8 h, followed by the addition of S-covidl9 lentivirions, which were pre-incubated with 4-fold descending concentration series of either ACE2-Fc or ACE2^mod-Fc. Luminescence from the activity of luciferase was measured 48 h post-infection using a TECAN infinite M200 pro plate reader after applying Bright-Glo reagent (Promega) on cells.

ACE2 activity assay - ACE2 activity was evaluated using SensoLyte® 390 ACE2 Activity Assay Kit (ANASPEC; cat# 72086) according to the manufacturer's protocol. 10 ng or 100 ng of ACE2-Fc and ACE2^mod-Fc samples were compared blank control. Measurement of product formation (fluoro genic polypeptide cleavage) as a function of time was taken every 10 seconds. EXAMPLE 1

Diversity of the SARS-CoV-2/ACE2 interface

The binding of SARS-CoV-2 to its ACE2 receptors is mediated by the receptor-binding domain (RBD), which is part of its spike complex ^23,28’³⁰. ACE2 has a long helical segment at its N’ terminus, which forms most of the RBD-recognition site on ACE2 (Figure 1A). Multiple sequence alignment of over 200 ACE2 sequences derived from mammals indicates that many of the ACE2 residues that make part of the SARS-CoV-2 recognition site are not conserved (Figure IB). This notion indicates an enormous putative sequence space that ACE2 can assume.

To identify advantageous alterations of ACE2 that may enhance the binding to SARS- CoV-2, 70 orthologous of ACE2 genes were selected having sequence identity to the human- ACE2 greater than 80% and modeled by Rosetta to identify their interaction with SARS-CoV-2 based on a crystal structure of SARS-CoV-2 RBD in complex with human- ACE2 (PDB: 6VW1) ³⁰. These models were analyzed using five different metrics that measure the interfaces' energy and structure properties (Figure 1C). For in-depth analysis the top-20 models in terms of computed binding energy (AAG of binding) were selected. These models showed broad distributions of their total Rosetta scores, buried surface areas, packing statistics, and shape complementarities (Figure 1C). All top-20 models were visually inspected and specific residues predicted to make better contacts with the SARS-CoV-2 RBD compared with the human-ACE2 counterparts were identified.

EXAMPLE 2

Optimized ACE2 interface for improved binding of SARS-CoV-2

The manual inspection of the top-20 models provided multiple potential alterations that may enhance the binding of ACE2 to the SARS-CoV-2 RBD. To select the favorable ones, alterations were selected with a predicted significant impact that avoid tryptophan residues, which may promote undesired promiscuous interactions. In addition, library screening data produced by Procko E. ²⁷ was used, to make sure that each alteration that is made is either enriched or at least not depleted in his dataset of improved ACE2 binders to SARS-CoV-2 RBD. Three of the mutations that were incorporated are located at the first N'-terminal helix of human- ACE2. These three mutations include a T27L mutation that allows better packing with hydrophobic residues of SARS-CoV-2 RBD (Figure 2A), a D30E mutation that forms a new saltbridge with Lys417 of SARS-CoV-2 RBD (Figure 2b), and a Q42R mutation that could have a dual effect. An arginine in position 42 can make a salt-bridge with Asp38 of ACE2 and stabilize it in a configuration that favors the formation of a hydrogen bond with Tyr449 of SARS-CoV-2 RBD (Figure 2C). Also, an arginine in this position could assume a different rotamer that will allow it to make advantageous electrostatic interactions with main-chain carboxylic oxygen of Gly447 from the SARS-CoV-2 RBD (Figure 2C). Besides these three mutations at the N-terminal helix of ACE2, two additional sites in the surrounding regions of ACE2 were identified. In the first site, we identified a putative change of Glu75 and Leu79 to arginine and tyrosine, respectively, that can form a stabilized motif that should interact better with Phe486 of SARS- CoV-2 RBD (Figure 2D). In the second site outside the first helix of ACE2, a phenylalanine instead of asparagine in position 330 would pack better against the aliphatic portion of Thr500 from SARS-CoV-2 RBD (Figure 2E). These six mutations were combined in-silico and the binding of this ACE2 variant with SARS-CoV-2 RBD modeled. The present design showed a remarkable improvement in AAG of binding as well as in the buried surface area (Figure 1C).

Additional modification at other sites on top of modifying ACE2 residues that directly interact with SARS-CoV-2 RBD were incorporated. Human-ACE2 has a putative glycosylation site at Asn90 that was shown to bear a glycan according to the SARS-CoV-2 RBD/human-ACE2 cryo-EM structure ²¹. This N-linked glycan projects toward the SARS-CoV-2 RBD, and presumably impose steric constraints for the binding of SARS-CoV-2 RBD. The library of Procko E. ²⁷ is highly enriched with mutations in this N-linked glycosylation site, further supporting this notion. To eliminate this glycosylation site, Thr92, from the N-X-T glycosylation motif was mutated to an arginine that can make polar interactions with nearby glutamine (Figure 2F). Besides serving as a cellular receptor for SARS-CoV-2, ACE2 is an enzyme that has a critical biological function in regulating blood pressure by hydrolyzing angiotensin II ³¹. Considering the potential use of ACE2 for anti-SARS-CoV-2 therapy, the enzymatic activity of ACE2 may complicate its use by having an undesired effect on blood pressure. Therefore the catalytic activity was deleted by mutating Glu375, which is a catalytic residue that participates in coordinating a metal ion at the active site to leucine.

EXAMPLE 3

An Fc-fusion of ACE2 design exhibits improved binding to viral SPIKE and anti viral activity

To test the design, two chimeric proteins were produced that include amino acids 19-615 of the human-ACE2 ectodomain (omitting the original signal polypeptide) fused to an Fc portion of human IgGl, with or without the eight above-mentioned mutations (i.e., T27L, D30E, Q42R, E75R, L79Y, N33OF, T92R, & E375L). Both the WT construct (ACE2-Fc SEQ ID NO: 3) and the designed ACE2 construct (ACE2^mod-Fc, SEQ ID NO: 4) readily expressed as secreted proteins using HEK293F cells in suspension and were easily purified to near homogeneity using protein-A affinity chromatography (Figure 3A). Testing the enzymatic activity of both ACE2-Fc and ACE2^mod-Fc verified that the latter is indeed catalytic dead (Figure 5). The two immunoadhesins were immobilized on a surface plasmon resonance sensor chip and either purified SARS-CoV-2 RBD (Figure 3B) or SARS-CoV-1 RBD (Figure 3C) were used as an analyte to determine their binding affinities, which is a configuration that does not allow avidity. A simple 1:1 binding model gave a good description for the binding of ACE2^mod-Fc to SARS- CoV-2 RBD, but the binding of ACE2-Fc to SARS-CoV-2 RBD could not be fitted using this model, and we, therefore, used a more complex heterogeneous-ligand model that assumes some heterogeneity of the ACE2-Fc (Figure 3B). Such heterogeneity could presumably originate from partial glycosylation at Asn90 of ACE2. Remarkably, the binding affinity of ACE2^mod-Fc to SARS-CoV-2 RBD is more than two orders of magnitude stronger compared with the binding affinity of ACE2-Fc (Figure 3B). While the association rate (k_a) of ACE2^mod-Fc to SARS-CoV-2 RBD is a bit faster compared to ACE2-Fc, the major factor that improves affinity is a dramatic two-orders of magnitude slower dissociation rate (kd). Hence, an improved ACE2 variant that binds stronger to SARS-CoV-2 RBD was successfully designed.

In both SARS2 (Figure 3B) and SARS1 (Figure 3C) same results were obtained, i.e. enhancement in binding of their RBDs to ACEmod.

To test if the enhanced affinity of ACE2^mod-Fc could translate to improved biological functions, the present inventors conducted a pseudovirus neutralization assay. The neutralization profile of ACE2^mod-Fc is apparently better compared to the profile of ACE2-Fc (Figure 4A). There is more than a 10-fold improvement in both IC50 and ICso values, comparing the two reagents. Anti SARS-CoV-2 immunoadhesin that binds to cell-surface displayed spike complexes might recruit beneficial immune factions via its Fc portion. The present inventors used flow cytometry to monitor the ability of ACE2-Fc and of ACE2^mod-Fc to stain HEK293 cells that were transiently transfected express the SARS-CoV-2 spike complex (Figure 4B). ACE2^mod-Fc has an apparent higher capacity to recognize the spike complex compared to ACE2- Fc. Achieving improved recognition of the SARS-CoV-2 spike complex suggested testing the ability of ACE2^mod-Fc to directly neutralize live authentic viruses. For that, the present inventors performed plaque reduction neutralization test in a BSE-3 facility (Figure 4C). Both ACE2-Fc and ACE2^mod-Fc displayed better neutralization capacity of the live viruses compared with the pseudoviral system (Figures 4A and C), and the potency of ACE2^mod-Fc was significantly higher compared to ACE2-Fc, achieving sterilizing effect well below 1 p.g/ml. Altogether, a superior ACE2-based immunoadhesin was created that displays an improved capacity to target SARS- CoV-2.

Figure 4E shows the neutralization of pseudotyped viruses of indicated strains [mutations in RBD only, meaning that mutation in viruses strains outside the RBD region -AA 319-541 of the SARS CoV2 spike- were excluded (were not generated)]. ACE2^mod-Fc neutralizes all strains with comparable rates of IC50. Supposedly, it could bind and block newly arising strains as well and be unsusceptible to escape mutations by the virus.

It is envisaged that recombinant ACE2^mod-Fc blocks SARS-CoV-2 entry. It is suggested hypothesized that addition of a linker between ACE2^mod and the Fc portion would create a more flexible hinge, thus allowing binding to additional spikes on the same virus or even adjacent viruses in the virus surroundings. A linker composed of 3 Gly-Gly-Ser-Gly (GGSG) (SEQ ID NO: 11) repeats was added between the ACE2^mod and the Fc. It was expressed (ACE2^mod GS3- Fc; Figure 6A) and used for neutralization of pseudotyped SARS-CoV2 viruses, side by side with ACE2^mod-Fc. ACE2^mod GS3-Fc was 5-10 times more potent in this assay than ACE2^mod-Fc (Figure 6B).

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety. References

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Claims

49 WHAT IS CLAIMED IS:

1. A composition of matter comprising a soluble polypeptide comprising an amino acid sequence of an ecto domain of human ACE2, said amino acid sequence comprising a plurality of mutations as compared to said ecto domain of wild type human ACE2 set forth in SEQ ID NO: 1, wherein said plurality of mutations increase binding of said polypeptide to a SPIKE protein of SARS-CoV-2 to a KD below 0.2 nM, as measured by surface plasmon resonance (SPR).

2. The composition of matter of claim 1, wherein said decreased KD is in the range of 0.1-0.01 nM.

3. The composition of matter of any one of claims 1-2, wherein the polypeptide inhibits binding of said SPIKE protein to human ACE2 expressing cells.

4. The composition of matter of claim 1, wherein said plurality of mutations comprise at least one mutation in an N-terminal helix of human ACE2.

5. The composition of matter of claim 1, wherein said plurality of mutations comprise at least one mutation which improves packing with hydrophobic residues in SARS- CoV-2 receptor binding domain (RBD).

6. The composition of matter of claim 5, wherein said plurality of mutations comprise at least one mutation at position T27 corresponding to SEQ ID NO: 1.

7. The composition of matter of claim 6, wherein said at least one mutation is T27L.

8. The composition of matter of any one of claims 1-7, wherein said plurality of mutations comprise at least one mutation which generates a salt bridge with SARS-CoV-2 RBD.

9. The composition of matter of claim 8, wherein said at least one mutation is at position D30 and/or Q42 corresponding to SEQ ID NO: 1. 50

10. The composition of matter of claim 9, wherein said at least one mutation is D30E and/or Q42R.

11. The composition of matter of any one of claims 1 and 5-10, wherein said plurality of mutations are at position Glu75 and/or Leu79 which impart improved interaction with Phe486 of SARS-CoV-2 RBD.

12. The composition of matter of claim 11, wherein said plurality of mutations comprise Gly75R and/or Leu79Y.

13. The composition of matter of any one of claims 1 and 5-12, wherein said plurality of mutations comprise at least one mutation in N33O for improved packing against Thr500 of SARS-CoV-2 RBD.

14. The composition of matter of any one of claims 1-13, wherein said plurality of mutations comprise at least one mutation that abolishes a glycosylation site of said human ACE2.

15. The composition of matter of claim 10, wherein said glycosylation site comprises an N-X-T glycosylation motif.

16. The composition of matter of claim 15, wherein said at least one mutation is at positions 89-92 corresponding to SEQ ID NO: 1.

17. The composition of matter of any one of claims 1-14, wherein said plurality of mutations comprise at least one mutation that renders said human ACE2 catalytically inactive.

18. The composition of matter of claim 17, wherein said at least one mutation is at position Glu375 corresponding to SEQ ID NO: 1.

19. The composition of matter of any one of claims 1-18, wherein said polypeptide is of a length not exceeding 600 amino acid residues. 51

20. The composition of matter of any one of claims 1-19, wherein said plurality of mutations comprise T27L, D30E, Q42R, E75R, L79Y and N33OF.

21. The composition of matter of any one of claims 1-20, wherein said amino acid sequence is as set forth in SEQ ID NO: 2.

22. The composition of matter of any one of claims 1-21 wherein said polypeptide is attached to a heterologous moiety.

23. The composition of matter of claim 22 further comprising a linker between said polypeptide and said heterologous moiety.

24. The composition of matter of claim 23, wherein said linker comprises at least one GGSG.

25. The composition of matter of claim 22, wherein said heterologous moiety is capable of inducing an antibody dependent cellular-mediated cytotoxicity (ADCC) response.

26. The composition of matter of claim 22, wherein said heterologous moiety is for increasing avidity of the polypeptide.

27. The composition of matter of claim 22, wherein said heterologous moiety is for multimerization .

28. The composition of matter of any one of claims 22-27, wherein said heterologous moiety is a proteinaceous moiety.

29. The composition of matter of claim 28, wherein said proteinaceous moiety is selected from the group consisting of an immunoglobulin, a galactosidase, a glucuronidase, a glutathione-S -transferase (GST), a carboxy terminal polypeptide (CTP) from chorionic gonadotrophin (CG0), and a chloramphenicol acetyltransferase (CAT).

30. The composition of matter of claim 28, wherein said proteinaceous moiety is an immunoglobulin . 52

31. The composition of matter of claim 30, wherein said immunoglobulin is an IgG Fc.

32. The composition of matter of claim 31, as set forth in SEQ ID NO: 4 or 8.

33. The composition of matter of any one of claims 22-27, wherein said heterologous moiety is a non-proteinaceous moiety.

34. A pharmaceutical composition comprising the composition of matter of any one of claims 1-33, and a pharmaceutically acceptable carrier.

35. A method of treating a Coronavirus infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of matter of any one of claims 1-33, thereby treating the infection.

36. The composition of matter of any one of claims 1-33 for use in treating a Coronavirus infection in a subject in need thereof.

37. A method of diagnosing a Coronavirus infection in a subject in need thereof, the method comprising:

(a) contacting a biological sample which may comprise a SPIKE protein of a Coronavirus with the composition of matter of any one of claims 1-33 under conditions which allow complex formation between the composition and the SPIKE;

(b) analyzing presence or level of said complex, wherein said presence and/or level is indicative of the Coronavirus infection.

38. The method of claim 37 being performed ex vivo.

39. The method of claim 37 or 38, wherein said composition of matter is attached to a detectable moiety.

40. The method or composition for use of any one of claims 35-39, wherein said coronavirus is SAR-CoV-2, Middle East respiratory syndrome Coronavirus (MERS-CoV) or severe acute respiratory syndrome Coronavirus (SARS-CoV).