WO2021202427A2 - Ace2-derived composition and use thereof - Google Patents

Description

ACE2-DERIVED COMPOSITION AND USE THEREOF FIELD OF THE INVENTION Generally, the invention relates to the field of biological pharmaceuticals as well as their use in infectious diseases, e.g. viral infections cause by Coronaviruses 2019-nCoV, (SARS)-CoV, etc., and Hentaviruses. More specifically, the invention relates to an ACE2-derived composition that is capable of inhibiting viral binding to ACE2. BACKGROUND The novel coronavirus 2019-nCoV has recently emerged as a human pathogen, causing fever, severe respiratory illness, and pneumonia—a disease recently named COVID-19 (see J. F. Chan et al., Lancet 395, 514–523 (2020), C. Huang et al., Lancet 395, 497–506 (2020), the entire teachings of which are incorporated by reference herein). The emerging pathogen was rapidly characterized as a new member of the betacoronavirus genus, closely related to several bat coronaviruses and to severe acute respiratory syndrome coronavirus (SARS-CoV) (see R. Lu et al., Lancet S0140 6736(20)30251-8 (2020), F. Wu et al., Nature (2020), the entire teachings of which are incorporated by reference herein). 2019-nCoV makes use of a densely glycosylated spike (S) protein to gain entry into host cells. Recent reports demonstrating that 2019-nCoV S and SARS-CoV S share the same functional host cell receptor, angiotensin-converting enzyme 2 (ACE2) (M. Hoffmann et al., The novel coronavirus 2019(2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells. bioRxiv 929042 [Preprint].31 January 2020. https://doi.org/10.1101/2020.01.31.929042, the entire teachings of which are incorporated by reference herein), albeit, there is evidence to indicate that 2019-nCoV S ectodomain binds to ACE2 with substantially higher affinity (see D. Wrapp et al., Science 367(6483), 1260-1263 (2020), the entire teachings of which are incorporated by reference herein). Further, no binding was observed between several SARS-CoV receptor-binding domain (RBD) specific monoclonal antibodies and 2019-nCoV RBD, indicating that the SARS-CoV specific antibodies are ineffective against 2019-nCoV. It would, therefore, be desirable to have a composition that inhibits viral binding of a broad spectrum of viruses to ACE2 which utilize ACE2 for host cell penetration. SUMMARY OF THE INVENTION

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In certain aspects, the present invention provides for a protein composition capable of binding a Coronavirus or a Hentavirus. The protein composition includes a first polypeptide containing amino acid sequence of SEQ ID NO. 1 and a second polypeptide containing amino acid sequence of SEQ ID NO. 2. The protein composition may comprise a polypeptide of SEQ ID NO.

3 or a polypeptide of SEQ ID NO. 4.

In certain aspects, the present invention provides for a therapeutic composition, the therapeutic composition includes a protein composition, the protein composition includes a first polypeptide containing amino acid sequence of SEQ ID NO. 1, and a second polypeptide containing amino acid sequence of SEQ ID NO. 2. The therapeutic composition may contain a protein composition of SEQ ID NO. 3 or of SEQ ID NO. The therapeutic composition may also include about 1.5% sucrose, about 70 mM NaCl, about 100 mM L-proline, about 7 mM Na-citrate, and about 15 mM Na-Phosphate, at pH 6.0 .

In certain aspects, the present invention provides for an isolated nucleic acid encoding a polypeptide containing amino acid sequence of SEQ ID NO. 3 or of SEQ ID NO. 4. The codon usage of the nucleic acid may be optimized for high expression of the polypeptide in a mammalian cell. The nucleic acid may include the nucleic acid sequence containing the sequence of SEQ ID NO. 5.

In certain aspects, the present invention provides for a composition containing a polypeptide of SEQ ID NO. 3 for use in the treatment of a disease associated with infection with a Coronavirus. The Coronavirus may be a 2019-nCoV or a SARS-CoV.

In certain aspects, the present invention provides for a composition comprising a polypeptide of SEQ ID NO. 3 for use in the treatment of a disease associated with infection with a Hentavirus. In certain aspects, the present invention provides for a method of treating or preventing a disease or condition associated with an infection with a Coronavirus. The method includes administering to a patient in need for treating or preventing a disease associated with a Coronavirus infection a therapeutically effective amount of a pharmaceutical composition containing a protein which includes a polypeptide of SEQ ID NO. 3. The Coronavirus may be a 2019-nCoV or a SARS-CoV.

In certain aspects, the present invention provides for a method of treating or preventing a disease or condition associated with an infection with a Hentavirus. The method includes administering to a patient in need for treating or preventing a disease associated with a Hentavirus infection a therapeutically effective amount of a pharmaceutical composition which includes a protein containing a polypeptide of SEQ ID NO. 3.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings and descriptions are provided to aid in the understanding of the invention:

FIG. 1 illustrates cloning of RPH-137 molecule of the present teachings in to an expressing vector for transfection into CHO cells;

FIG. 2 is a schematic presentation of lentiviral pseudotyping approach: 293T cells were transfected with a plasmid encoding a lentiviral backbone (genome) expressing a GFP IRESLuciferase, plasmids expressing the HIV proteins needed for virion formation, Tat, Gag-Pol, and Rev and a plasmid expressing either Spike-Wt or Spike-D18 protein; the transfected cells produced SARS-CoV-2 S pseudotyped lentiviral particles with Spike on their surface; these viral particles are capable of infection of the cells that express the ACE2;

FIG. 3 illustrates assay specificity of SARS-CoV-2 S-protein pseudovirus titration. A: SARS-CoV2-SD18 was added to the parental 293T or ACE2-293T cells at various dilutions, right Y-axis displays luciferase activity on ACE2-293T cells; B: ACE2-293T cells were infected with SARS-CoV-2-S-Wt or pseudotyped lentiviruses at various dilutions;

FIG. 4 shows RPH-137-mediated inhibition of SARS-CoV-2 S-protein pseudovirus infection: A: comparison of inhibitory properties of RPH-137 for SARS-CoV-2-S-Wt and SARS- CoV2-S-D18 pseudoviral particles; both types of particles were incubated with the indicated concentration of RPH-137 and then added to ACE2-293T cells; B: IC50 determination for RPH- 137; SARSCoV2-S-D18 pseudoviral particles were incubated with the indicated concentration of RPH-137 and then added to ACE2-293T cells; two different pseudovirus dilutions, 1:5 and 1:25 were tested; titration curve was analyzed using variable slope 4-parameters curve fit algorithm and IC50 values are shown in the upper right corner of the panel;

FIG. 5 shows IC50 determination for RPH-137: SARS-CoV2-S-D18 pseudoviral particles were preincubated with various concentrations of RPH-137 and then added to ACE2-293T cells, titration curve was analyzed using variable slope 4-parameters curve fit algorithm and IC50 value is shown in the upper right corner;

FIG. 6 shows substrate titration curves in the ACE2 assay;

FIG. 7 shows plots of ACE2 activity in the ACE2 assay in the presence of different concentrations of RPH-137;

FIG. 8 shows RPH-137 ELISA assay standard curve, the data were used to determine RPH- 137 concentration in hamster plasma samples;

FIG. 9 shows pharmacokinetics of RPH-137 in hamster plasma following sc administration at 5 mg/kg;

DETAILED DESCRIPTION OF THE INVENTION

The teachings disclosed herein are based, in part, upon engineering of ACE2-Fc fusion proteins that are capable of binding to 2019-nCoV, SARS-CoV or other viruses which utilize ACE2 for cellular penetration, and thus reducing their virulence. In one example, an ACE2-Fc fusion protein of the present teachings is based on residues 18-615 of human ACE2

(https.//www.uniprot.org/umpfot/09BYF1), having H374N and H378N mutations in the enzyme active center, C-terminally fused via a flexible linker to a Fc fragment of human IgG. A signal peptide may be fused at the N-terminus of the fusion protein to facilitate heterologous protein expression. The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them. The scope or meaning of any use of a term will be apparent from the specific context in which the term is used. "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, preferably within 5- fold and more preferably within 2-fold of a given value.

Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences to each other, including wild-type sequence to one or more mutants (sequence variants). Such comparisons typically comprise alignments of polymer sequences, e.g., using sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few). The skilled artisan can readily appreciate that, in such alignments, where a mutation contains a residue insertion or deletion, the sequence alignment will introduce a "gap" (typically represented by a dash, or "A") in the polymer sequence not containing the inserted or deleted residue.

The methods of the invention may include statistical calculations, e.g. determination of IC50 or EC50 values, etc. The skilled artisan can readily appreciate that such can be performed using a variety of commercially available software, e.g. PRISM (GraphPad Software Inc, La Jolla, CA, USA) or similar.

"Homologous," in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a "common evolutionary origin," including proteins from superfamilies in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions. However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and may or may not relate to a common evolutionary origin.

The term "sequence similarity," in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.

The terms “protein” and “polypeptide” are used interchangeably. The polypeptides described herein may be comprised of more than one contiguous amino acid chain, thus forming dimers or other oligomeric formations. In general, the polypetides of the present teachings for use in mammals are expressed in mammalian cells that allow for proper post-translational modifications, such as CHO or HEK293 cell lines, although other mammalian expression cell lines are expected to be useful as well. It is therefore anticipated that the polypeptides of the present teachings may be post-translationally modified without substantially effecting its biological function.

In certain aspects, functional variants of the fusion protein of the present teachings include fusion proteins having at least an extracellular metalloprotein domain of the human ACE2 or a functional fragment thereof, and one or more fusion domains. Well known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (e.g., an Fc), maltose binding protein (MBP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For example, an ACE2 polypeptide portions may be fused with a domain that stabilizes the ACE2 polypeptides in vivo (a "stabilizer" domain), optionally via a suitable peptide linker. The term "stabilizing" means anything that increases the half life of a polypeptide in systemic circulation, regardless of whether this is because of decreased destruction, decreased clearance, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on certain proteins. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains that confer an additional biological function, e.g. promoting accumulation at the targeted site of action in vivo.

In an exemplary embodiment, a fusion protein of the present teachings includes an extracellular metalloprotein domain of the human ACE2 fragment fused with a Fc portion of human IgGl (h tip s : //w w w uniprot . org/uniprot/P01857). An ACE2 fragment may optionally be mutated as to render it enzymatically inactive. The ACE2 portion of the fusion protein of the present teachings may comprise the amino acid sequence of SEQ ID NO. 1, wherein two His residues in the enzyme active site are replaced with Asn residues.

ACE2 polypeptide (SEQ ID NO. 1)

While the Fc portion of the fusion protein of the present teachings may comprise the amino acid sequence of SEQ ID NO. 2.

Fc polypeptide (SEQ ID NO. 2)

In one example, the fusion protein of the present teachings may comprise the amino acid sequence of SEQ ID NO. 3.

ACE2-Fc fusion polypeptide (SEQ ID NO. 3)

QSTIEEQAKT FLDKFNHEAE DLFYQSSLAS WNYNTNITEE NVQNMNNAGD KWSAFLKEQS 60

Using well established molecular biology techniques, synthetic gene corresponding to the protein designs of the fusion proteins of the present teachings can be generated, which may include codon-optimization for expression in mammalian cells, and synthesized. A protein expression vector can then be generated.

In an exemplary embodiment the DNA fragment with an open reading frame corresponding to ACE2-Fc fusion polypeptide may comprise the DNA sequence of SEQ. ID NO. 5.

ACE2-Fc DNA (SEQ ID NO. 5)

EXAMPLES

The following Examples illustrate the forgoing aspects and other aspects of the present teachings. These non-limiting Examples are put forth so as to provide those of ordinary skill in the art with illustrative embodiments as to how the compounds, compositions, articles, devices, and/or methods claimed herein are made and evaluated. The Examples are intended to be purely exemplary of the inventions disclosed herein and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for.

In an exemplary embodiment the DNA sequence of SEQ ID NO. 5 was cloned into pAFF- DHFR and pAFF-NEO expression vectors, as schematically shown in FIG. 1. Both constructs were linearized and co-transfected into DHFR-/- CHO-DG44 cell line. Double selection was carried out using methotrexate for pAFF-DHFR and geneticin for pAFF-NEO vectors, respectively. The overexpressed ACE2-Fc fusion protein (RPH-137) was purified from cell supernatant utilizing Protein A affinity chromatography, followed by anion exchange chromatography utilizing protocols well known in the art.

Example 1: Development and use of SARS-CoV-2 Spike protein pseudotyped lentivirus system for assessing inhibitory properties of RPH-137.

For viral entry into the cells SARS-CoV-2 coronavirus relies on binding of its surface Spike protein to ACE2 expressed on the cell surface. Novel therapeutic agents that prevent Spike protein and ACE2 interaction, as well as other mechanisms of virus entry are currently being developed. Since SARSCoV-2 is biosafety Level 3 virus, it cannot be used in most of research labs that are usually biosafety level 2 or below. To address the biosafety issue, SARS-CoV-2 S-protein can be ‘pseudotyped’ onto replication-defective viral particles replacing their native entry receptors. A lentiviral system based on human HIV was used where SARSCoV-2 S-protein was introduced, thus making pseudovirus entry dependent on S-protein. For qualitative and quantitative evaluation of viral entry, GFP and luciferase, respectively, separated by an IRES, were included into the genome of the pseudovirus. Additionally, a stable cell line expressing full length human ACE2 was generated to increase efficiency of viral infection. The system was tested and further optimized for use with biologies as well as with small molecules that target virus infectivity dependent on ACE2 - SARS-CoV-2 interaction. The S-protein pseudovirus system was successfully used to test inhibition properties of RPH-137 of the viral infection.

Materials and reagents.

The reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein were described elsewhere (Crawford, K.H.D., et al, Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses,

2020. 12(5), hereinafter “Crawford”, the entire teachings of which are incorporated by reference herein). Gene synthesis was carried out by ThermoFisher.

Plasmids

All plasmids used in these studies were generated according to Crawford and the names of the plasmids were kept the same as specified in this publication. Schematically, the approach is illustrated in FIG. 2.

— pHAGE2-EFlaInt-ACE2-WT: Lentiviral backbone plasmid expressing the human ACE2 gene (GenBank ID for human ACE2 is NM 021804) under an EFla promoter with an intron to increase expression. This plasmid was used for generation of stable cell line expressing human ACE2.

— HDM-IDTSpike-fixK-HA-tail: Plasmid expressing under a CMV promoter the Spike from SARSCoV-2 strain Wuhan-Hu-1 (GenbankNC_045512) codon-optimized using IDT, with the Spike cytoplasmic tail replaced by that from the HA protein of A/WSN/1933 (H1N1) influenza, and the Kozak sequence in the plasmid fixed compared to an earlier version of this plasmid.

— pHAGE-CMV-Luc2-IRES-ZsGreen-W: Lentiviral backbone plasmid that uses a CMV promoter to express luciferase followed by an IRES and ZsGreen.

— HDM-Hgpm2: lentiviral helper plasmid expressing HIV Gag-Pol under a CMV promoter.

— HDM-tatlb: Lentiviral helper plasmid expressing HIV Tat under a CMV promoter.

— pRC-CMV-Revlb: Lentiviral helper plasmid expressing HIV Rev under a CMV promoter.

— SARS-CoV-2 S -D18: SARS-CoV-2, the spike gene of Wuhan-Hu-1 strain (GenBank: MN908947) was codon-optimized for expression in human cells. The plasmid was generated as described in (Xiong, H.L., et al, Robust neutralization assay based on SARS- CoV-2 S-proteinbearing vesicular stomatitis virus (VSV) pseudovirus and ACE2- overexpressing BHK21 cells. Emerg Microbes Infect, 2020. 9(1): p. 2105-2113, hereinafter “Xiong”, the entire teachings of which are incorporated by reference herein). A stop codon was introduced after K1255 position thus removing 18 C-terminal amino acids. This plasmid was used to replace full-length S-protein (plasmid HDMIDTSpike-fixK-HA-tail) in most of the experiments since the concentration pseudovirus particles produced with S- D18 was about 100-fold higher than for S Wt.

Stable cell line generation.

Human embryo cell line 293 T was used as a parental cell line for generation a cell line expressing human ACE2. The cells were transfected with pHAGE2-EFlaInt-ACE2-WT plasmid and plated on 5 different P-10 plates at various dilutions. After propagation in culture for 10-14 days, visible colonies were harvested using cloning cylinders, expanded in 24-well plates and expression of ACE2 was analyzed by Western blot analysis.

Protocol for generation of pseudotyped lentiviral particles.

1. Seed 293T cells in DMEM supplemented with 10% FBS (DMEM-10%) so that they will be 50%-70% confluent the next day. For a six-well plate, this is 5.0E+05 cells per well (2.5E+05 cell/ml).

2. At 16-24 h after seeding, transfect the cells with the plasmids required for lentiviral production. Transfection was carried out using BioT (Bioland Scientific, Paramount, CA, USA) following the manufacturer's instructions and using the following plasmid mix per well of a six-well plate (plasmid amounts should be adjusted for larger plates):

• 1 μg of lentiviral Luciferase-IRES-ZsGreen backbone

• 0.22 μg each of plasmids HDM-Hgpm2, pRC-CMV-Revlb, and HDM-tatlb

• 0.34 μg viral entry protein — either SARS-CoV-2 Spike-Wt SARS-CoV-2 Spike-D18.

3. At 18 to 24 h post-transfection, culture medium was replaced with fresh, pre-warmed DMEM-10%.

4. At 60 h post transfection, virus was collected by harvesting the supernatant from the cells and filtering it through a 0.45 μ SFCA low protein-binding filter. Virus can be stored at 4° C for immediate use or frozen at -80° C. For long-term storage, it is recommended to freeze virus in small aliquots to avoid multiple freeze-thaw cycles.

Protocol for titration pseudotyped lentiviral particles.

1. Seed 96-well plate with ACE2-293T cells in DMEM-10% at 1.25E+04 cells per well (6.25E+04 cell/ml). Plate 0.2 ml per well of ACE2-293T cell suspension at 6.25E+04 cell/ml. After 16-24 h after seeding, the cells are ready for viral infection. 2. In sterile 2.0 ml 96-well plate prepare virus dilutions. To 1.5 ml of undiluted virus preparation add 3.0 μl of 10.0 mg/ml polybrene to a final concentration 5.0 μg/ml. For assay controls and dilutions use DMEM-10% supplemented with 5.0 μg/ml polybrene (DMEM- 10%-PBR) .

3. For 8-point calibration curve, to the wells A - G add 700 μl DMEM-10%-PBR. Add 700 pi of undiluted virus supplemented with polybrene to the well H. Add 700 pi of undiluted virus supplemented with polybrene to the well G, mix well by pipetting up and down several times, transfer 700 μl of the well content to the well F. Repeat serial 2-fold dilutions for the remaining wells E to B. Use well A as a negative control, place 700 μl of DMEM- 10%-PBR

4. From the plate with attached ACE2-293T cells, remove culture medium using multichannel aspirator, aspirate columns 1, 2 and 3. Using multichannel pipette, from wells A through H with virus dilutions, transfer 3 x 200 μl of the virus dilutions to ACE2-293T cells into columns 1, 2 and 3, respectively.

5. At 48 h post-infection, cells could be observed under fluorescent microscope for GFP expression. After 60-72 hours, harvest the cells for luciferase assay.

Protocol for titration of inhibitors.

1. Seed 96-well plate with ACE2-293T cells in DMEM-10% at 1.25E+04 cells per well (6.25E+04 cell/ml). Plate 0.2 ml per well of ACE2-293T cell suspension at 6.25E+04 cell/ml. After 16-24 h after seeding, the cells are ready for viral infection.

2. Identify appropriate virus dilution from virus titration experiment that provides >1,000 difference between background (no virus added wells) and a given virus dilution.

3. In sterile 0.5 ml 96-well plate prepare virus solution, controls and inhibitor dilutions. Use columns 1 to 4 for virus preparations and controls. Use columns 6 to 20 for controls and inhibitor dilutions. Note that all dilutions in the plate should be 2-fold higher since virus preparations and inhibitor preparation will be mixed at 1 : 1 ratio prior to adding to cells.

4. To columns 1 through 4 add 350 μl of virus dilutions (prepared in DMEM-10%-PBR) and controls. As a negative no virus control use DMEM- 10%-PBR.

5. Prepare 1.5 ml of Inhibitor with 2x highest concentration using DMEM- 10%-PBR. Leave the wells for the highest concentration of Inhibitor empty. To the remaining wells add 400 μl per well of DMEM-10%-PBR. Add 400 μl of 2x the highest concentration of Inhibitor to the empty well(s). Add 400 μl of 2x the highest concentration of Inhibitor to the well(s) where next 2-fold dilution should be. Mix well by pipetting up and down, transfer 400 μl of the well content to the well with next dilution. Repeat for other dilutions as needed.

6. Using multichannel pipette, sequentially add 350 μl content from wells 6 through 10 to wells with virus dilutions, 1 through 4, respectively. Mix well by pipetting up and down.

7. Incubate in the hood for 25-30 min.

8. From the plate with attached ACE2-293T cells, remove culture medium using multichannel aspirator, aspirate columns 1, 2 and 3. Using multichannel pipette, from column 1 with virus/inhibitor mixture, transfer 3 x 200 μl of virus dilutions to ACE2-293T cells into columns 1, 2 and 3. Repeat procedure for the remaining columns 2 through 4.

Lucifer ase assay.

1. After 60-72 hours post-infection, harvest cells for luciferase assay. Shake off 96-well plate with infected ACE2-293T cells to remove culture medium and add 55 μl per well of lysis buffer. (Lysis buffer: 25mM Tris-phosphate (pH 7.8), 2mM DTT, 2mM 1,2- diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, 1% Triton® X-100). Incubate at 37°C for 10 min, then place the plate-on-plate shaker and shake at 825 rpm for 10 min. Take 30 μl and transfer into white 96-well plate.

2. To the 30 μl of cell lysate in white 96-well plate add 100 μl of luciferase reagent, put the plate into a plate reader, mix and measure luminescence. (Luciferase reagent: 25 mM Tris- HC1, pH 8.0, 15 mM MgS04, 0.1 mM ethylene diamine tetraacetic acid (EDTA), 4 μM EGTA, 250 mM Coenzyme A, (0.233 mg/ml, M. w. = 767.5), 500 μM luciferin, (0.159 mg/ml, M.w. = 318.4), 1.0 mM ATP, 33.3 mM DTT).

Upon optimization of transfection, virus production and infection and luciferase assay, series of experiments were carried out to establish reliable protocol for analysis of inhibitory properties of drug candidates. Assay validation.

To evaluate specificity of SARS-CoV-2-S pseudovirus infection, parental 293T and ACE2- expressing cell line ACE2-293T were infected with SARS-CoV2-S-D18. It was found that the virus is capable of infection of both cell lines, however infection of parental 293T cells was about 1,000-fold less efficient (FIG. 3, A). This finding demonstrates that SARS-CoV2-S-D18 requires ACE2 for efficient infection. However, low but detectable level of infection in the parental 293 T cells suggests that there is an ACE2-independent mechanism of viral entry. For further optimization of optimization of experimental conditions, comparison of virus titers was carried out. Stable cell line ACE2-293T was infected with virus preparations generated by co-transfection of plasmids encoding non-surface proteins for lentivirus production and either SARS-CoV-2 S- protein Wt or its truncated version, SARS-CoV-2-D18. Both transfections were prepared in parallel and the only difference was the presence of Wt or D18 S-protein-encoding plasmid. Consistent with previous reports, titer of SARS-CoV-2-S-Wt was apparently lower than that of SARS-CoV2-S-D18, most probably due to less efficient packaging into pseudoviral particles (Xiong) apparent titer of Wt pseudovirus was about 10- fold lower than of the D18, therefore all further experiments were carried out using SARS-CoV2-S-D18 pseudotyped virus (FIG. 3, B).

Infection inhibition assay for RPH-137.

RPH-137 is a novel SARS-CoV2 targeting biologies, a chimeric protein comprised of an extracellular fragment of human 2 (ACE2) genetically linked to Fc potion of human IgGl. It was hypothesized that soluble ACE2-Fc fusion protein will bind S-protein of the surface of viral particles thus preventing their clinically relevant interaction with ACE2 receptor expressed on the surface of susceptible cells. To test this hypothesis, inhibition of infection of ACE2-293T cells by SARS-CoV2-S pseudovirus was carried out. Virus titer was optimized in preliminary experiments to provide at least 3 logs difference between signals from uninfected vs. infected cells. First, in a pilot experiment several concentrations of RPH-137 were tested. Both SARSCoV2-S-D18 and SARS-CoV2-S-Wt were tested in this experiment. Both versions of pseudovirus were mixed with a control medium or at the indicated concentrations of RPH-137, incubated for 30 min and then added ACE2-293T cells. It was found that at RPH-137 concentration of 20 μg/ml, inhibition of viral entry for SARS-CoV2-S-Wt was about 20-fold and for SARS-CoV2-S-D18 this number was about 50-fold (FIG. 4, A). Since SARS-CoV2-S-D18 pseudovirus preparation has higher titer and thus generates higher signal, only this version of pseudovirus was further used.

To confirm RPH-137 inhibitory properties further, a titration experiment was carried out for IC50 determination. RPH-137 was titrated down from 80 μg/ml using 2-fold serial dilutions. Two dilutions of SARS-CoV2-S-D18 preparation was used, 1:5 and 1:25 dilutions. Each pseudovirus dilution was preincubated with the indicated concentrations of RPH-137, and then were added to the cells. Titration curve was plotted and analyzed using variable slope 4-parameters curve fit algorithm and the corresponding IC50 values for 1:5 and 1:25 dilutions were 0.454 μg/ml and 0.741 μg/ml, respectively (FIG. 4, B). Previously reported values for ACE2-Fc fusion proteins in assays of viral entry inhibition, were within the range of 0.48 μg/ml and 2.5 μg/ml (Crawford), therefore IC50 determined in our hands are within reasonable range indicating suitability of the pseudovius assay for evaluation of RPH-137 inhibitory proteins.

To verify experimental reproducibility and data reliability, another RPH-137 titration experiment was carried out. In this experiment, RPH-137 concentration was increased, and it was titrated down from 200 μg/ml using 2-fold serial dilutions (FIG. 5). A single dilution of SAR.S- CoV2-S-D18 preparation was used for preincubation with the indicated concentrations of RPH- 137, and then were added to the cells. Titration curve was plotted and analyzed using variable slope 4-parameters curve fit algorithm and IC50 value was 0.612 μg/ml. This value is in agreement with our previous data as well as with IC50 values previously reported elsewhere (Crawford). It was concluded that the assay based on infection of ACE2-293T by pseudotyped SAR.SCoV-2-S- protein virus is reliable and robust method for evaluation of inhibitory properties of RPH-137 and other similar biomolecules.

Thus, this example demonstrates establishing and optimizing a virus system based on pseudotyping human HIV lentivirus by SARS-CoV-2 S-proteins. It was demonstrated that pseudoviral particles that express SARS-CoV-2 S-protein on their surface are able to effectively infect cells expressing human ACE2. Cell line expressing ACE2 on the surface of parental 293 T cells was generated and used. Although parental 293T cells were also infected with SARS-CoV-2 S pseudovirus particle, the infection was about 1,000-fold less efficient than that of ACE2-293T cells, suggesting that the pseudovirus can infect cells in ACE2-independen mechanism.

The pseudovirus system was used to test inhibitory properties of RPH-137, a novel biologies based on a fusion protein of extracellular domain of human ACE2 to human IgGl Fc fragment. It was demonstrated that RPH-137 inhibited pseudoviral infection in a concentration dependent manner. IC50 value for infection inhibition was 0.5-0.7 μg/ml and this value was consistent with IC50 values for similar molecules reported before. The SARS-CoV-2 S pseudovirus infection assay is reliable and reproducible approach for testing inhibitory properties of molecules that target the SARS-CoV-2 S-protein - ACE2 interactions.

Example 2: Assessment of binding angiotensin II by RPH-137 in ACE2 assay.

ACE2 is an ubiquitously expressed transmembrane metalloprotease. In vivo it converts the peptide angiotensin II into the vasodilator angiotensin. In addition, some viral spike proteins (S- proteins) target host cells by selectively binding to ACE2. ACE2 was identified as the receptor for entry SARS-CoV-2 into host cells in the human body. This role in the viral pathogenesis has made ACE2 a protein of interest in the field of anti-SARS-CoV-2 biotherapeutic development. RPH-137 comprises a homodimer of ACE2 catalytic domain fused to a human Fc fragment of immunoglobulin 1 (IgG-1). RPH-137 employs its ACE2 domain to bind viral particles, thereby functioning as a decoy receptor that prevents anchoring viral particles to ACE2 bearing host cells. Surface Plasmon Resonance-based approach confirmed strong binding between RPH-137 and recombinant S-protein trimer of SARS-CoV-2 (KD «10-20 nM). In this S-protein binding decoy receptor role, RPH-137 is not intended to have catalytic activity of ACE2. Therefore, selective mutations were made to RPH-137's ACE2 active site in order to abolish its catalytic activity. Previous studies confirmed the absence of ACE2 catalytic activity in RPH-137. Nevertheless, because RPH-137 still has a portion of the ACE2 active site, it potentially may bind ACE2 substrate, angiotensin II, and, as a consequence, cause shifting equilibrium in the renin-angiotensin system important for control of blood pressure. The study described in this example was designed to assess RPH-137 binding activity toward angiotensin II using ACE2 enzymatic assay as a test system. Since RPH-137 still has a catalytically inactive binding site that may accommodate angiotensin II, its presence in blood and tissues can hypothetically cause unintended side effects associated with reduction of angiotensin II concentration. Therefore, it was important to demonstrate that RPH-137 does not interfere with ACE2 activity in vitro and, therefore, does not bind angiotensin II. RPH-137 was purified and concentrated essentially as described above to a final concentration of 2 mg/mL, in 25 mM sodium citrate, 250 mM Tris-HCl, pH 7.0. For the purposes of this study ACE2 Activity Assay Kit (Fluorometric) from BioVision, catalog # K897-100 was utilized. Two 96-well polystyrene assay plates, black, flat bottom (Costar, catalog # 3915) and a Spectramax iD3 plate reader (Molecular Devices) were used to conduct the experiments.

Principle of the Assay.

This assay employs an ACE2 substrate peptide labeled with the fluorophore 7- methoxycoumarin-4-acetic acid (MCA). MCA fluoresces with an excitation wavelength of 320 nm and an emission wavelength of 420 nm. In vitro, catalytically active ACE2 hydrolyzes this peptide to release free MCA, which results in a gain of total MCA fluorescence. Therefore, an increase in the fluorescence of the experimental system corresponds to ACE2 activity. Fluorescence is measured using a plate reader. The assay was run in accordance with the BioVision Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric) supplied protocol, 1 modified here first to determine optimum substrate concentration and then to assess enzymatic inhibition.

Reagent Preparation and assay setup.

1. Prepare the recombinant ACE2 Positive Control by diluting it with 95 microliters of ACE2 Dilution Buffer.

2. Prepare the ACE2 Substrate by diluting it 25-fold with ACE2 Assay Buffer, to a final volume of >1.5 mL. This is the lx ACE2 substrate solution.

3. Add 100 μL of ACE2 Assay Buffer to 2 other wells to use as blank wells.

4. Dilute ACE2 substrate solution 3-fold and add 50 μL of this diluted ACE2 Substrate Solution to each well.

5. Set the plate in the plate reader and perform fluorescence readings with an excitation wavelength of 320 nm and a detection wavelength of 420 nm. 6. Set the plate reader to shake the sample (to mix) before the first reading. Measure fluorescence every 5 minutes for 35 minutes. Fluorometric ACE2 Activity Assay.

Because the concentration of the fluorescent substrate in BioVision's kit is unknown, we could not initially rule out the possibility that it is high enough to saturate the recombinant ACE2. In the latter case, binding of a relatively small portion of the substrate by RPH-137 present in the reaction mixture would not result in the loss of ACE2 activity because the enzyme would remain saturated by the substrate. We performed this initial assay to determine a substrate dilution factor at which the recombinant ACE2 is definitively not saturated.

Assay Procedure.

1. In each of 8 wells on the 96-well plate, 2 μL of the diluted ACE2 Positive Control solution. To 2 other wells, add 1 μL of diluted ACE2 Positive Control solution. To 2 other wells, add 0.4 μL of diluted ACE2 Positive Control solution. Use ACE2 Assay Buffer to bring each of these wells up to a total volume of 50 μL.

2. Use some of the lx ACE2 Substrate Solution to prepare the following dilutions (in ACE2 Assay Buffer) of ACE2 substrate: l/3x, l/9x, and l/27x.

3. Add 100 μL of ACE2 Assay Buffer to 2 wells to use as blank wells.

4. Add 50 μL of ACE2 Substrate Solution to each well, using the diluted ACE2 Substrate Solutions in 2 wells each.

5. Set the plate in the plate reader and perform fluorescence readings with an excitation wavelength of 320 nm and a detection wavelength of 420 nm. Set the plate reader to shake the sample (to mix) before the first reading. Measure fluorescence every 5 minutes for 30 minutes.

ACE2 Activity in the Presence of RPH-137.

1. In each of 10 wells on the 96-well plate, 2 μL of the diluted ACE2 Positive Control solution.

2. Add RPH-137 to 8 of the wells and then add ACE2 Assay Buffer to bring each well up to a total volume of 50 μL. At this point the ACE2 will have a concentration of 180 nM, and the RPH-137 will have concentrations of 900, 1800, 3600, and 9000 nM.

3. Add 100 μL of ACE2 Assay Buffer to 2 other wells to use as blank wells. 4. Dilute ACE2 substrate solution 3-fold and add 50 μL of this diluted ACE2 Substrate Solution to each well.

5. Set the plate in the plate reader and perform fluorescence readings with an excitation wavelength of 320 nm and a detection wavelength of 420 nm. Set the plate reader to shake the sample (to mix) before the first reading. Measure fluorescence every 5 minutes for 35 minutes.

Effect of substrate concentration on ACE2 activity.

ACE2 assay was ran with different substrate concentration. Raw data of the assay are shown in the Table 1 and graphically presented in FIG. 6. Fluorescence for each duplicate set of wells was averaged and tabulated.

Table 1. Substrate Titration Results.

* Data for 90 nM ACE2 and lx substrate was an outlier.

ACE2 activity in the presence ofRPH-137.

As shown in the FIG. 7 (raw data are presented in the Table 2), the rate of substrate hydrolysis was not significantly affected (within one S.D.) by RPH-137 at RPH-137:ACE2 molar ratios as high as 50:1. As is apparent from the forgoing, substrate titration results (FIG. 6) show that the BioVision ACE2 Activity Assay kit can be used to detect competitive inhibition of ACE2 activity. As shown in FIG. 6, ACE2 activity shows a decrease in Vmax at the tested reduced substrate concentrations (1/3x, 1/9x, and 1/27x). Because the Vmax is concentration-dependent within this concentration range, the recombinant ACE2 is not saturated with the substrate. Therefore, in this system a competitor that binds the substrate can be detected by a reduction in Vmax.

The competition assay model shows that in vitro hydrolysis of the peptide substrate by ACE2 is not substantially affected by even a large excess of RPH-137. This result indicates that RPH-137 does not bind angiotensin II, ACE2 substrate, therefore RPH-137 does not possess angiotensin II binding activity.

Table 2. Fluorescence values (RFU) for each duplicated set of ACE2 assay in the presence of different concentrations of RPH-137 (averaged and tabulated).

Example 3. Development and use of SARS-CoV-2 Spike protein pseudotyped lentivirus system for assessing inhibitory properties of RPH-137.

The purpose of this study was to characterize pharmacokinetics of RPH-137 in the Syrian hamster animal model. Plasma samples were taken from Syrian hamsters dosed subcutaneously with RPH-137 at 5 mg/kg.

Six female CrkLVG(SYR) Golden Syrian hamsters were assigned to one study group. Hamsters weighed between 110.7 g and 124.5 g at the initiation of dosing. Animals were individually housed. All hamsters had ad libitum access to certified pelleted commercial laboratory diet (PMI Certified Rodent 5002; PMI® Nutrition International Inc.). Water was available ad libitum. The hamsters were individually identified using a subcutaneously implanted electronic identification chip. The RPH-137 formulation was administered once by the subcutaneous injection. Study parameters included: viability, clinical observations, body weights, body weight changes, and bioanalysis. Food consumption was not measured; however, food was monitored and replenished as necessary to monitor the health and well-being of the animals.

RPH-137 at concentration of 12.5 mg/ml was formulated in a buffer containing 1.5% sucrose, 70 mM NaCl, 100 mM L-proline, 7 mM Na-citrate, 15 mM Na-Phosphate, pH 6.0. Blood samples for bioanalysis evaluations were collected as shown in the Table 3.

Table 3. Bioanalytical Sample Collection.

X = Sample collected; - = Not applicable. a Samples collected before administration. b Samples collected ± 5 minutes of the time of administration. c Samples collected ± 10 minutes of the time of administration.

No mortality occurred on study. There were no clinical observations related to administration of RPH-137. Body weights and body weight changes were unaffected by administration of RPH- 137. All hamsters gained weight thorough out the study period. RPH-137 was quantified in 96-well microplates using human IgGFc ELISA kit (GenWay, Cat. #GWB-EKA001) following manufacture's protocol and standard procedures well know in the art. Absorbance at 450nm was measured utilizing Spectramax iD3 plate reader (Molecular Devices). The standard curve for the assay is shown in FIG. 8. A450 values for the IgG Fc standards with and without supplemental hamster plasma were compared. No significant difference resulted from the presence of plasma in the system. Pharmacokinetic curve of RPH-137 is shown in FIG. 9. The data show detectable RPH-137 concentrations in the plasma of Syrian hamsters. RPH-137 pharmacokinetics is characterized by the following major parameters: Cmax = 1.6 μg/mL, Tmax = 8-30 hours, T1/2 = 90 hours. All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

What is claimed is:

1. A protein composition, said protein composition comprising: a first polypeptide comprising amino acid sequence of SEQ ID NO. 1; and a second polypeptide comprising amino acid sequence of SEQ ID NO. 2.

2. The protein composition of claim 1, said protein composition comprising a polypeptide of SEQ ID NO. 3.

3. The protein composition of claim 1, said protein composition comprising a polypeptide of SEQ ID NO. 4.

4. A therapeutic composition, the therapeutic composition comprising: a protein composition, the protein composition comprising a first polypeptide comprising amino acid sequence of SEQ ID NO. 1, and a second polypeptide comprising amino acid sequence of SEQ ID NO. 2.

5. The therapeutic composition of claim 5, comprising a protein composition of SEQ ID NO.

3.

6. The therapeutic composition of claim 5, comprising a protein composition of SEQ ID NO.

4.

7. The therapeutic composition of claim 3, further comprising about 1.5% sucrose, about 70 mM NaCl, about 100 mM L-proline, about 7 mM Na-citrate, and 15 mM Na-Phosphate, at pH 6.0

8. An isolated nucleic acid encoding a polypeptide comprising amino acid sequence of SEQ ID NO. 3.

9. An isolated nucleic acid encoding a polypeptide comprising amino acid sequence of SEQ

ID NO. 4.

10. The nucleic acid of claims 7 or 8, wherein the codon usage is optimized for high expression of said polypeptide in a mammalian cell.

11. The nucleic acid of claim 9, wherein the nucleic acid sequence comprises the sequence of SEQ ID NO. 5.

12. A composition comprising a polypeptide of SEQ ID NO. 3 for use in the treatment of a disease associated with an infection with a Coronavirus.

13. The composition of claim 12, wherein said Coronavirus is a 2019-nCoV.

14. The composition of claim 12, wherein said Coronavirus is a SARS-CoV.

15. A composition comprising a polypeptide of SEQ ID NO. 3 for use in the treatment of a disease associated with an infection with a Hentavirus.

16. A method of treating or preventing a disease or condition associated with an infection with a Coronavirus, the method comprising administering to a patient in need for treating or preventing a disease associated with a Coronavirus infection a therapeutically effective amount of a pharmaceutical composition comprising a protein comprising a polypeptide of SEQ ID NO. 3.

17. The method according to claim 17, wherein said Coronavirus is a 2019-nCoV.

18. The method according to claim 17, wherein said Coronavirus is a SARS-CoV.

19. A method of treating or preventing a disease or condition associated with an infection with a Hentavirus, the method comprising administering to a patient in need for treating or preventing a disease associated with a Hentavirus infection a therapeutically effective amount of a pharmaceutical composition comprising a protein comprising a polypeptide of SEQ ID NO. 3.