WO2022043943A1 - Compositions and methods for treating coronavirus infection at different level of disease severity - Google Patents

Abstract

The present invention is in the technical field of a pharmaceutical composition comprising recombinant polypeptides; wherein the first recombinant polypeptide comprises a genetically modified ACE2 protein to increase binding to SARS-CoV and SARS-Cov2 viruses spike protein and neutralizing activity; and the second recombinant polypeptide comprises a Fc portion common to IgG and IgA; wherein the said composition is characterized in that the recombinant polypeptide are such that they exhibit a synergistic neutralizing activity, improved systemic and the mucosal circulation. Furthermore, invention deals with method of detecting a severe acute lung injury or failure in a subject comprising providing a pharmaceutical composition comprising ACE2 recombinant polypeptide to a subject with a severe acute lung injury or failure. In addition, invention deals with parenteral formulation for critical care and nasal formulation targeted for prophylactic and maintenance therapy.

Description

COMPOSITIONS AND METHODS FOR TREATING CORONAVIRUS INFECTION AT DIFFERENT LEVEL OF DISEASE SEVERITY

[001] PRIORITY PARAGRAPH

[002] This application claims priority to the Indian provisional patent application No. 202041036866, filed on August 27^th, 2020, titled "COMPOSITIONS AND METHODS FOR TREATING CORONAVIRUS INFECTION AT DIFFERENT LEVEL OF DISEASE SEVERITY” and is incorporated herein by reference.

[003] TECHNICAL FIELD OF THE INVENTION

[004] The present invention is in the technical field of compositions and methods comprising ACE2 recombinant polypeptide and Fc portion of IgG and/or IgA, wherein recombinant polypeptides are such that they exhibit a synergistic neutralizing activity, improved systemic and the mucosal circulation. The compositions are useful in the diagnosis of SARS-CoV and the prophylaxis and/or treatment of a condition resulting from SARS-CoV.

[005] BACKGROUND OF THE INVENTION

[006] Global spread of COVID- 19 and the severe morbidity and mortality has effectively brought the world to a grinding halt. Multiple efforts are underway to develop prophylactic protein or mRNA-based vaccines for curbing the spread of disease in the community from future outbreaks. However, the need of the hour is to develop an effective therapeutic intervention to neutralize the virus and reduce the severity of disease in infected patients.

[007] It has been shown that COVID- 19 disease causes acute inflammation thus severely sick patients cannot be treated through any aerosol formulation (dry powder inhalation or nasopharyngeal-route). Thus, only intravenous injections of the drug will be administered for immediate effect. After IV injection, most of the large molecule drugs might accumulate at the sites of inflammation due to high vascular permeability at those sites.

[008] Large molecules such as monoclonal antibodies or therapeutic proteins have demonstrated their superiority in neutralizing the virus compared to small molecule inhibitors.

[009] ACE2( Angiotensin Converting Enzyme 2) has been established as the receptor for SARS- CoV-2 Spike (S) protein for entry into conducive host cells. Therefore, effective inhibition of the binding between the S protein and ACE2 receptor is a powerful strategy to thwart the entry of virus into the cells. [010] Since Ace2 is expressed maximally in the nasal airway followed by oral mucosa, local application of the proposed drug through nasal inhalation would be most efficient. However, since patients in disease severity being unable to inhale independently, there is a need for parenteral formulation demanding high dosage and thus cost.

[011] Thus, there is need in the art to develop an effective formulation for treating coronavirus infection both by intranasal route of administration as well as parenteral.

[012] Enzymatically active Ace2 fusion protein, in addition to trapping the virus, can reverse the conditions following the loss of Ace2 in patients with associated comorbid conditions like renal and cardiac hypertension.

[013] SUMMARY OF THE INVENTION

[014] According to an exemplary aspect, a pharmaceutical composition comprising recombinant polypeptide;

[015] a) wherein the first recombinant polypeptide comprises a genetically modified ACE2 protein comprising the nucleotide sequence of SEQ ID No: 1 to increase binding to SARS-CoV and SARS-Cov2 viruses spike protein and neutralizing activity; and

[016] b) the second recombinant polypeptide comprises a Fc portion common to IgG and IgA;

[017] wherein characterized in that the said recombinant polypeptide are such that they exhibit a synergistic neutralizing activity, improved systemic and the mucosal circulation recombinant polypeptide are such that they exhibit a synergistic neutralizing activity, improved systemic and the mucosal circulation.

[018] According to further exemplary aspect, the present invention discloses mutations those have been introduced to make a soluble protein of human ACE2 coronavirus binding site.

[019] In an embodiment, compositions and methods for treating coronavirus infection and the associated comorbidities like cardiac and renal hypertensin and complications associated with diabetes, with an engineered fusion protein (NOT a product of nature) combining the ectodomain of a receptor, modified for better binding, better expression and lesser immunogenicity, with the Fc region of human antibody for increased dimerization and pharmacokinetics for different level of severity, with parenteral formulation.

[020] In an embodiment, a method of detecting a severe acute lung injury or failure in a subject comprising providing a pharmaceutical composition comprising ACE2 recombinant polypeptide to a subject with a severe acute lung injury or failure, wherein the severe acute lung injury or failure in the subject is detected.

[021] In further embodiment, a method for the production of recombinant ACE2 polypeptides or a preparation of recombinant ACE2, comprising the steps of;

[022] a) introducing an ACE2-encoding polynucleotide, preferably an ACE2 without transmembrane domain-encoding polynucleotide;

[023] b) introducing one or more mutations in ectodomain of ACE2-encoding polynucleotide;

[024] c) creating codon optimized Fc region from hlgG;

[025] d) cloning Ace2 ectodomain and Fc regions into mammalian expression vector;

[026] e) transfecting recombinant HAce2Fc construct into mammalian cells;

[027] f) collecting cell supernatant;

[028] g) purifying recombinant protein on Protein-A column;

[029] h) reconstituting recombinant protein in formulation buffer; and

[030] i) characterizing ACE2 recombinant protein.

[031] In an embodiment, a method for the production of recombinant ACE2 polypeptides, wherein the recombinant ACE2 polypeptides comprises mutations T27Y, N330Y, K26R, E329G or any combination thereof.

[032] In an embodiment, a kit comprising recombinant ACE2 polypeptides for neutralizing SARS-CoV and SARS-Cov2 viruses comprising recombinant polypeptide;

[033] a) wherein the first recombinant polypeptide comprises a genetically modified ACE2 protein comprising the nucleotide sequence of SEQ ID No: 1 to increase binding to SARS-CoV and SARS-Cov2 viruses spike protein and neutralizing activity; and

[034] b) the second recombinant polypeptide comprises a Fc portion common to IgG and IgA;

[035] wherein characterized in that the recombinant polypeptide are such that they exhibit a synergistic neutralizing activity.

[036] Ace2 is an important enzyme in the Renin Angiotensin Pathway. It degrades the hypertensive peptide Angiotensin II and brings down the blood pressure to normalcy. SARS-Cov2 binding to ACE2 receptor not only allows the viral entry by phagocytosis, also diminishes the enzymatic function of the protein. Our construct retains the enzymatic domain, thus will be able to reduce pro-hypertensive Angiotensin II peptide in case of hypertensive patients.

[037] In an embodiment, Ace2 part of the construct has been mutated to increase binding to the spike protein but without losing its catalytic activity.

[038] In an embodiment, the fusion protein can be used for both SARS Cov and SARS-Cov2 viruses as well as in the treatment of Acell induced hypertension.

[039] In an embodiment, the Fc portion common to IgG and IgA has been taken so that the pharmacokinetic profile of the drug improves in systemic as well as the mucosal circulation

[040] In various embodiments, invention deals with cloning, expression, purification, invitro efficacy and toxicity, PK analysis both through Intravenous and nasal route, in vivo toxicity, and efficacy and validation of the constructs for Covid therapy.

[041] According to an exemplary aspect, Ace2Fc is designed by fusing the receptor with immunoglobulin Fc fragment, enabling the dimerization of the receptor, increase the half-life of the drug in biological fluids.

[042] In embodiment, parenteral formulation targeted for critical care and maintenance therapy has been made.

[043] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.

[044] BRIEF DESCRIPTION OF THE DRAWINGS

[045] The foregoing and other objects and features of the present invention will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only typical embodiments of the invention and are, therefore, not to be considered limiting of its scope, the invention will be described with additional specificity and detail through use of the accompanying drawings.

[046] FIG 1: illustrates the Flow chart illustrating the experimental steps starting from cloning to efficacy testing in hamster model of the HAce2Fc protein, according to the aspects of present invention.

[047] FIG 2: illustrates the amplification of ACE2 at -2.2 Kbps and immunoglobulin Fc at -697 bps, according to the aspects of present invention. [048] FIG 3: illustrates the Cloning of ACE2 in Mammalian expression vector. Lane 1: pTT5 Ace2-Fc clone 2 undigested, Lane 2: pTT5 Ace2-Fc clone digested with BamHI showing an insert release of an expected size 1.844 Kbps Lane 3: DNA ladder, Lane 4: pTT5 Ace2-Fc clone 2 plasmid amplified with Ace2 Forward and Reverse primer showing the expected band size of ~2.2 Kbps, according to the aspects of present invention.

[049] FIG 4: Illustrates a dot blot with Goat Anti-hlg- HRP of various cell supernatants after transfection of HEK293T cells. Dot Blot of cell supernatant/lysate after transient transfection of Ace2-Fc in HEK293T. 1: Supernatant from control non-transfected cells. 2-5 Supernatant from cells transfected with Lipofectamine 30000. 6-7: purified protein fractions from protein A sepharose column.8: Human IgG (positive control). 9: 1X PBS buffer. 10: Cell lysate of transfected cells. The dot blot was probed with 1 :20X dilution of Goat anti-Human HRP, according to the aspects of present invention.

[050] FIG 5: Table- summarizes the OD450 values of the cell supernatants after appropriate dilution in ELISA. It shows the ELISA result of cell supernatant after transient transfection, according to the aspects of present invention.

[051] FIG 6: depicts the Western Image of cell supernatant and lysate expressing Ace2Fc loaded under non-denaturing. Lane 1 Transfected cell pellet, Lane2 Transfected cell supernatant, Lane 3 un-transfected cell supernatant. The blot was probed with Goat anti-human HRP conjugate at 1 : 10000X dilution, according to the aspects of present invention.

[052] FIG 7A: depicts the Coomassie blue stained image of purified Ace2-Fc loaded under non- reducing condition and Fig 7B depicts the Coomassie blue stained image of purified Ace2-Fc loaded under reducing condition, according to the aspects of present invention.

[053] FIG 8: illustrates the Western image of purified Ace2Fc: Lane 1: SDS-PAGE of Ace2Fc loaded under unreduced condition, lane 2 Ace2Fc loaded under reduced condition. Lane 3 Ace2Fc protein was boiled with 2- ME at 95°C, 5’. The blot was probed with Goat anti-human HRP conjugate at 1 : 10000X dilution, according to the aspects of present invention.

[054] FIG 9A: illustrates the binding of Ace2-hFc to the whole spike lysate of SARS-COV2 protein expressed in HEK293E cells by ELISA and FIG 9 B Binding of Ace2-hFc to Spike 1 protein of SARS-COV2 protein expressed in HEK293E cells by ELISA, according to the aspects of present invention.

[055] FIG 10: illustrates FAR UV CD-SPECTRA of Ace2-hFc. The fusion protein depicting 46% of alpha helical structure of the Ace2 peptide which has a high helical folding propensity in aqueous solution and 22% beta fold mainly from the Fc region, according to the aspects of present invention. [056] FIG 11: illustrates the SARS-COV2 Neutralization by PRNT assay and Cytotoxicity by LDH assay of HAce2FC protein on Vero-E6. A clear Dose response is seen and close to 80% efficacy is achieved at -125 ug/mL concentration with no observed cytotoxicity, according to the aspects of present invention.

[057] FIG 12A: illustrates the SARS-COV2 Neutralization of HAce2Fc protein by qRT-PCR on M08 oral epithelial cells and 12 B on Vero E6 cells, according to the aspects of present invention. [058] FIG 13A: illustrates the pharmacokinetic analysis of the HAce2Fc protein in Phosphate buffer formulation buffer using non-compartmental model; 13B: illustrates the pharmacokinetic analysis of the HAce2Fc protein in Phosphate buffer formulation buffer using compartmental model displaying the 1. Distribution Phase (α-phase), which marks the attenuation of drug in plasma due to distribution to other sites and 2. The Elimination Phase (β-phase), which marks the Attenuation of drug in plasma due to systemic excretion, according to the aspects of present invention.

[059] FIG 14: illustrates histopathology of 1 : Heart, 2:Liver, 3:Kidney and 4:Spleen Post IV administration of the hAce2-hFc for safety studies, according to the aspects of present invention. [060] FIG 15: Lanes 1,2,3 shows: Histopathology of the Lung tissue Post IV administration of the hAce2-hFc for safety studies displaying Moderate congestion with multifocal areas of bronchial epithelium showing mild to moderate hyperplasis, according to the aspects of present invention.

[061] FIG 16: illustrates pharmacokinetic analysis of the HAce2Fc protein in Phosphate buffer formulation buffer using 2-compartmental model after intranasal dosing. The Tmax is observed around 1.5 hours with a peak pulmonary concentration of about 8.3 μg/mL, according to the aspects of present invention.

[062] FIG 17: illustrates pharmacokinetic analysis of the HAce2Fc protein in formulation buffer in Balb/c mice showing a Tmax of 21.08 hrs, according to the aspects of present invention.

[063] FIG 18: illustrates efficacy assay graph. FIG18A: illustrates PFU isolated per lung homogenate between SARS-COV2 infected Hamster groups (control) versus HAce2Fc treated hamsters (Treated), 18B: Graph depicting the qRT-PCR Mean Ct values of RdRp and N gene between the control and Treated group of hamsters, 18C: Graph depicting the correlation between the PFU count and Ct valuesshowing the reduction in viral load in the lung samples isolated from the control and Ace2FC2 treated hamster lung samples, according to the aspects of present invention.

[064] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

[065] DETAILED DESCRIPTION OF THE INVENTION

[066] Embodiments of systems and methods for compositions and methods for treating coronavirus infection at different level of disease severity are described herein.

[067] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way.

[068] In this detailed description of the various embodiments, for purposes of explanation, various details are described to provide a thorough understanding of the embodiments disclosed. One skilled in the art will appreciate, however, that these various embodiments may be practiced with or without these specific details. In other embodiments, structures and devices are shown in block diagram form. Furthermore, one skilled in the art can appreciate that the specific sequences in which methods are presented and performed are illustrative in nature and it is contemplated that the sequences can be different and still remain within the spirit and scope of the various embodiments disclosed herein.

[069] DEFINITIONS:

[070] The following terms are used as defined below throughout this application, unless otherwise indicated.

[071] The phrase "differentially present" refers to differences in the quantity of the marker present in a sample taken from patients as compared to a control subject. A biomarker can be differentially present in terms of frequency, quantity or both.

[072] "Diagnostic" means identifying a pathologic condition.

[073] The terms "detection", "detecting" and the like, may be used in the context of detecting markers or biomarkers.

[074] A "test amount" of a marker refers to an amount of a marker present in a sample being tested. A test amount can be either in absolute amount (e.g., μg/ml) or a relative amount (e.g., relative intensity of signals).

[075] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. "Polypeptide," "peptide" and "protein” can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.

[076] The terms "subject", "patient" or "individual" generally refer to a human or mammals. "Sample" refers to a polynucleotide, antibodies fragments, polypeptides, peptides, genomic DNA, RNA, or cDNA, polypeptides, a cell, a tissue, and derivatives thereof may comprise a bodily fluid or a soluble cell preparation, or culture media, a chromosome, an organelle, or membrane isolated or extracted from a cell.

[077] "Biological sample” means blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures, or cells derived therefrom and the progeny thereof.

[078] The term "host", as used herein, is intended to refer to an organism or a cell into which a vector such as a cloning vector or an expression vector has been introduced. The organism or cell can be prokaryotic or eukaryotic.

[079] The term "pharmaceutically acceptable excipient" means any inert substance that is combined with an active molecule such as a drug, agent, or binding molecule for preparing an agreeable or convenient dosage form. The excipient is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation comprising the drug, agent or binding molecule.

[080] The term "therapeutically effective amount" refers to an amount of the binding molecule as defined herein that is effective for preventing, ameliorating and/or treating a condition resulting from infection with SARS-CoV.

[081] The term "treatment" means therapeutic treatment as well as prophylactic or preventative measures to cure or halt or at least retard disease progress.

[082] The term "vector" denotes a nucleic acid molecule into which a second nucleic acid molecule can be inserted for introduction into a host where it will be replicated, and in some cases expressed.

[083] The term “Recombinant molecule” includes, but are not limited to, derivatives that are substantially similar in primary structural sequence, that contain in vitro or in vivo modifications, chemical and/or biochemical. Such modifications may include acetylation, acylation, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, giycosylation, methylation, pegylation, proteolytic processing, phosphorylation, and the like.

[084] A “system” refers to a set of components, real or abstract, comprising a whole where each component interacts with or is related to at least one other component within the whole.

[085] A “biomolecule” is any molecule that is produced by biological organisms, including proteins, polysaccharides, lipids, nucleic acids and small molecules such as primary metabolites, secondary metabolites, and other natural products.

[086] “Recombinant polypeptide” means- a manipulated form of protein, encoded by genetically modified DNA. [087] “SARS-COV2” means virus that causes a respiratory disease called coronavirus disease 19 (COVID- 19).

[088] “Neutralizing activity” means protective activity.

[089] “Pharmaceutical composition” means process in which different chemical substances, including the active drug, are combined to produce a final medicinal product.

[090] “Prophylaxis” means treatment given or action taken to prevent disease

[091] “Medicament” means a substance used for medical treatment.

[092] “Renin-Angiotensin-System” is a hormone system that regulates blood pressure and fluid and electrolyte balance as well as systemic vascular resistance.

[093] “Renin inhibitor” are drugs that inhibit the the activity of renin which is responsible for hydrolyzing angiotensinogen to angiotensin 1.

[094] “Fc” refers to the fragment crystallizable region which is the tail region of an antibody that interacts with the cell surface receptors called the Fc receptors.

[095] “hlgG” refers to immunoglobulin G.

[096] “Ectodomain” means the domain of a membrane protein that extends into the extracellular space.

[097] “Transfection” means the process of introducing nucleic acids into mammalian cells.

[098] EMBODIMENTS OF THE INVENTION:

[099] In an embodiment, a pharmaceutical composition comprising recombinant polypeptides;

[0100] wherein the first recombinant polypeptide comprises a genetically modified ACE2 protein comprising the nucleotide sequence of SEQ ID No: 1, to increase binding to SARS- CoV and SARS-Cov2 viruses spike protein and neutralizing activity; and

[0101] the second recombinant polypeptide comprises a Fc portion common to IgG and IgA;

[0102] wherein characterized in that the said recombinant polypeptide are such that they exhibit a synergistic neutralizing activity, improved systemic and the mucosal circulation. [0103] In further embodiment, the pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0104] In an embodiment, pharmaceutical composition is for the diagnosis, prophylaxis, treatment, or combination thereof of a condition resulting from a SARS-CoV or cardiac or renal hypertensin or complications associated with diabetes.

[0105] In further embodiment, the pharmaceutical composition is for neutralizing SARS-CoV, SARS-CoV2 and/or related viruses with similar spike protein structure. [0106] In an embodiment, pharmaceutical composition is for detection and quantification of viral load of SARS-CoV, SARS-CoV2 and/or related viruses with similar spike protein structure.

[0107] In an embodiment, the pharmaceutical composition is a renin-angiotensin-system or a bradykinin receptor inhibitor or a renin inhibitor or an ACE inhibitor.

[0108] In an embodiment, the pharmaceutical composition is not cytotoxic.

[0109] In further embodiment, pharmaceutical composition is for medicament.

[0110] In an embodiment, the pharmaceutical composition is administered as individual or as a combination medicament.

[0111] In an embodiment, the medicament is administered intravenously, intraperitoneally, mucosally, intranasally, orally, intratracheally, and/or as an aerosol composition.

[0112] In an embodiment, a method of detecting a severe acute lung injury or failure in a subject comprising providing a pharmaceutical composition comprising ACE2 recombinant polypeptide to a subject with a severe acute lung injury or failure, wherein the severe acute lung injury or failure in the subject is detected.

[0113] In an embodiment, the severe acute lung injury or failure is further defined as an injury induced by acid aspiration or sepsis, a lung oedema, and/or a lung injury and/or failure connected with infection with severe acute respiratory syndrome (SARS) coronavirus or hypertension or due to diabetes or any other comorbidities.

[0114] In exemplary embodiment, the inhibitor of the renin-angiotensin-system is an ACE inhibitor, ACE2, renin inhibitor or inhibitors of SARS-COV or SARS-CoV2 or similar viral entry, or any combination thereof.

[0115] In an embodiment, method for the production of recombinant ACE2 polypeptides or a preparation of recombinant ACE2, comprising the steps of;

[0116] introducing an ACE2-encoding polynucleotide, preferably an ACE2 without transmembrane domain-encoding polynucleotide;

[0117] introducing one or more mutations in ectodomain of ACE2-encoding polynucleotide;

[0118] creating codon optimized Fc region from hlgG;

[0119] cloning Ace2 ectodomain and Fc regions into mammalian expression vector;

[0120] transfecting recombinant HAce2Fc construct into mammalian cells;

[0121] collecting cell supernatant;

[0122] purifying recombinant protein on Protein-A column;

[0123] reconstituting recombinant protein in formulation buffer; and

[0124] characterizing ACE2 recombinant protein. [0125] In further embodiment, method for the production of recombinant ACE2 polypeptides, wherein the recombinant ACE2 polypeptides comprises mutations K26R, T27Y, L79T, M82I, N90Q, T92Q, E329G, N330Y, R518G or any combination, or any other mutation in the spike protein binding domain thereof.

[0126] In an embodiment, a kit comprising recombinant ACE2 polypeptides for neutralizing SARS-CoV and SARS-Cov2 viruses comprising recombinant polypeptide;

[0127] wherein the first recombinant polypeptide comprises a genetically modified ACE2 protein comprising the nucleotide sequence of SEQ ID No: 1 to increase binding to SARS-CoV and SARS-Cov2 viruses spike protein and neutralizing activity; and

[0128] the second recombinant polypeptide comprises a Fc portion common to IgG and IgA;

[0129] wherein characterized in that the recombinant polypeptide are such that they exhibit a synergistic neutralizing activity.

[0130] In an embodiment, the kit is for qualitative analysis of SARS-CoV or SARS-CoV2 or any other virus with similar spike protein or quantitative analysis of SARS-CoV or SARS-CoV2 or any other virus with similar spike protein.

[0131] In further embodiment, a composition for binding to SARS-CoV and SARS-Cov2 viruses spike protein, wherein said composition comprising of nucleotide sequence: a synthetic, recombinant oligonucleotides sequence of SEQ ID NO 1.

[0132] Ace2 protein can be used in addition to covid infection for angiotensin Il-induced hypertension. Angiotensin-converting enzyme 2 (ACE2) is a carboxy peptidase that potently degrades angiotensin II to angiotensin 1-7. The enzymatic ectodomain of recombinant ACE2 (rACE2) markedly increases circulatory levels of ACE2 activity, and thus effectively lowers blood pressure in angiotensin Il-induced hypertension. However, due to the short plasma half-life of rACE2, its therapeutic potential for chronic use is limited. A chimeric fusion of rACE2 and the immunoglobulin fragment Fc segment is shown to increase its plasma stability and marks its therapeutic potential to ameliorate albuminuria, and reduced kidney and cardiac fibrosis. rACE2- Fc is suitable to develop new renin angiotensin system-based inhibition therapies.

[0133] Also, Dysregulated RAS (Renin Angiotensis system), as seen in diabetes, could lead to an increase in serum levels of Ang-II that could cause a plethora of potentially harmful effects including vasoconstriction, inflammation, and increased oxidative stress.

[0134] Further, loss of ACE2 due to viral binding disrupts the balance of the RAS in a diabetic state and leads to Angll/ATIR-dependent systolic dysfunction and impaired vascular function. [0135] Downregulation of ACE2 following viral intrusion results in decreased degradation of angiotensin-II, increased aldosterone secretion and subsequent increased urinary potassium loss resulting in hypokalemia.

[0136] The present invention is further elaborated with the help of the following examples. However, these examples should not be construed to limit the scope of the present invention.

[0137] EXAMPLE 1: METHODOLOGIES

[0138] Construction and Expression of HACE2-hIg-Fc -2.2 Kb virus binding domain of hACE2 (with SNPs was amplified and cloned in human Fc vector with hemagglutinin (HA) signal peptide (FIG 2 and FIG 3).

[0139] Construct was transiently expressed in HEK293E adherent cultures (FIG 3, FIG 4, FIG 5). [0140] The HAce2-Fc fusion protein was purified using Protein A column from the cleared expression media obtained after removing the cellular debris post spinning (FIG 7A and 7B, FIG 8).

[0141] Formulation of Ace2-Fc.

[0142] The purified protein was provided in formulation buffer (20mM Sodium phosphate containing 100 mM glycine, 150 mM NaCl, and 50 μM ZnC12 at a pH of 7.5) at 2.0 mg/mL or 2.5 mg/mL.

[0143] The binding kinetics of the therapeutic protein was established via a series of immunological assays using SARS-CoV-2 virus.

[0144] A large batch of well characterized fusion protein was produced in mammalian culture system and used for all the experiments.

[0145] In vitro Binding studies with recombinant full-length spike protein of SARS-COV2 expressed in HEK293E cells by ELISA: Microtiter plates were coated at 4 °C overnight with 0.2 μg/mL of anti-SARS-COV2 Spike antibody diluted in 0.1 M carbonate buffer (pH 9.6). The wells were rinsed with 200 μl wash buffer (0.05% Tween 20 in phosphate-buffered saline (PBS), and blocked with 300 μL blocking buffer (5% BSA in washing buffer) by incubating at 37 °C for 1 hour. 100 μL of varying concentration of cell lysates of HEK293E cells expressing Spike protein (S1+S2) of SARS-COV or null cell lysate of HEK293 E cells diluted in PBST was added and incubated at 37 °C for 1 hour.

[0146] The wells were washed and the absorbed protein in each well was challenged with 1 μg/mL of ACE2-hIg-Fc protein diluted in PBST and incubated at 37 °C for 1 h. The wells were washed and incubated with peroxidase-conjugated goat anti-Human IgG and developed with 1XTMB/H₂O₂. The absorbance was read at 450 nm in an ELISA plate reader. The ACE2-hIg-Fc protein showed a dose dependent binding to the spike protein by ELISA (FIG 9A) [0147] Invitro Binding studies with recombinant Spike 1 fragment of SARS-COV2 expressed in HEK293E cells by western blot: 10 μgs of total cell lysate of HEK293E cells expressing Spike 1 of SARS-COV2 was probed with ACE2-hIg-Fc (lug/mL) over night at 4°C. The blot was probed with Goat anti-Human-HRP conjugate and developed with super signal west Pico Chemiluminescent substrate. The ACE2-hIg-Fc protein bound to the spike 1 fragment (~70 kDa; Fig 9B) of the viral protein by western blot.

[0148] Biophysical characterization of The ACE2-hIg-Fc protein by CD spectroscopy to predict the secondary structure: CD data were collected on a Jasco J-815 spectrometer using purified ACE2-hIg-Fc protein solution in 2-mm quartz cuvettes. HAce2FC protein was diluted in 600 μL of 20 mM Phosphate buffer, pH 7.4 to a concentration of 0.2 mg/mL. The spectra were collected from 200 to 250 nM. The spectrum showed that the HAce2FC protein consisted largely the Apha Helix structure, 46% corresponding to the Ace2 region followed by 22% beta sheet mainly the Fc region and 32% random coil (FIG 10).

[0149] EXAMPLE 2: IN VITRO NEUTRALIZATION ASSAY

[0150] Determination of viral titer

[0151] The plaque assay is the gold standard test for quantifying infectious virus in a sample. The plaque assay measures “plaques,” which describe the zone of cellular death that occurs after one infectious unit has entered a cell and spread to adjacent cells over the time of incubation. The plaque reduction neutralization test (PRNT) is used to quantify the titer of neutralizing antibody/protein for a virus. As this is a cell-based assay it may not be reliable when the samples themselves are cytotoxic (e.g., homogenate from certain tissues) or when the virus is poorly cytopathic in each cell type. Thus, it is important to choose a highly permissive cell type (e.g., Vero E6 cells) for which SARS-CoV-2 causes substantive cell death.

[0152] Neutralization assay by Plaque Reduction Neutralization Test (PRNT) assay

Vero E6 cells were seeded at a density of 3 x 10⁴ /well of 96 well plate and incubated overnight (12-18 h) at 37 °C. On the following day, 100 PFU of infectious clone-derived wild-type SARS- CoV-2 was incubated with different concentrations (500 μg/mL and 5 serial dilutions) of HAce2FC-2 (total volume of 60 μL) at 37°C for 1.5 h. After the completion of 1.5 h incubation, the existing cell culture media was removed from 96-well plates and virus-HAce2FC-2 mixture (60 μL) of each dilution was added in duplicate. The plate was incubated at 37° C in 5% CO₂ for 1 h, and were rocked (manually) every 15 min to prevent cells from drying out. 2X MEM + 4% FBS with 2% methylcellulose in a 1:1 ratio was mixed and placed in 37° C incubator to decrease viscosity of the solution. After 1 h incubation the infection medium was removed and 200 μL of MEM: methylcellulose mixture was added to each well of the 96-well plates. The plates were incubated at 37° C in 5% CO₂ for 3 days. On day 3, the methylcellulose overlays were gently removed with a pipette and the cells by adding 200 μL of paraformaldehyde (PFA) to each well. [0153] The plate was Incubated at room temperature for 20 min. Removed PFA from the wells and dispensed into an appropriate hazardous waste container. 200 μL of 0.05% (w/v) crystal violet in 20% methanol was added to each well and incubated for 20-30 min. The crystal violet was removed with a pipette and washed twice with distilled water (dH₂O) or until excess crystal violet was removed, and plaques were easily visualized. The plaques were counted at the dilution in which there are 10-100 plaques. The viral titer was calculated in PFU/mL using the following formula:

[0155] Average number of plaques given by t _{[0156] x} Diluti [0154] PFU/mL= identified virus dilution factor (counted)

[0157] 0.1 or volume of virus used for infectioi

[0158] Cytotoxicity assay was performed by assaying for LDH assay: The cytotoxicity of HAce2FC protein on VeroE6 cells was analysed by LDH (Lactate Dehydrogenase) assay. VeroE6 cells were treated with different concentrations (500, 250,125 & 62.5, 31.25 and 15.625 μg/ml) ofAce2Fc protein for 90 minutes and the cell viability was assessed by LDH assay. As seen in Figure 11, HAce2FC protein did not show any cytotoxicity on VeroE6 cells in any of the measured concentrations.

[0159] Neutralization assay by real time quantitative PCR (qRT-PCR) by following the below mentioned steps.

[0160] VeroE6 and MO8 oral epithelial cells were plated in a 96-well plate at 3.0x10⁴cells per well and incubated overnight. Different concentration of the HAce2FC protein (500, 250,125 & 62.5, 31.25 and 15.625 μg/ml) was preincubated with SARS-CoV-2 virus at an MOI of 0.01 at 37°C for 1.5 hours and then added to the seeded cells and incubated for 1 h at 37 °C with 5% CO₂. The infection media was removed and fresh growth media was added onto the cells and incubated for 48 hours. After the completion of 48 hours the supernatant was collected into viral transport media. The virus was inactivated and the viral load was checked by q-RT-PCR assay with Nucleocapsid-N-gene, Envelop-E- gene and RdRp (RNA dependent RNA polymerase) gene primers. As observed in Figure 12 A and B, there was a dose dependent decrease in viral load with an increasing concentration of HAce2FC2 protein. [0161] EXAMPLE 3: PHARMACOKINETICS ASSAYS AND TOXICITY STUDY

[0162] Pharmacokinetics assays and toxicity study in hamsters in 20mM Phosphate buffer with 0.9% NaCl formulation buffer.

[0163] Pharmacokinetics assays in hamsters: PK studies of HAce2FC were performed via two routes of administration.

[0164] In single dose IV PK study, HAce2FC2 was administered via Juglar vein at a concentration of 5 mg/Kg body weight of the hamster and serum samples collected at indicated time points; Imin, 2 h, 48 h, 15 mins, 8 h and 24 h and analysed for the HAce2FC-2 levels by ELISA As observed in FIG13 A, the half life of the protein was 29.56 hours (non-compartmental model) and FIG 13 B, the half life explained in terms of distribution half-life of 3.2 minutes and elimination half-life of 23.4 hours (compartmental model) the protein . At 48 hours post serum collection lung, liver, spleen, kidney and heart were also collected for toxicity analysis (FIG 14).

[0165] For intranasal PK study, 0.25 mg of HAce2FC-2 at a concentration of 2.5 mg/mL was dosed per animal through nasal route after anesthetizing the animals. For intranasal route of administration, both serum (1 and 15min, 2, 8, 24 and 48 hours) and half lung homogenates (1,2,4, 8, 24 and 48 hours) were collected post administration and analysed for the HAce2FC-2 levels by ELISA. As observed in FIG 16, the Cmax (maximum concentration) reached 8 ug/mL in the lung tissue.

[0166] Toxicity analysis: From each of the animal’s lung, liver, spleen, kidney and heart were collected from PK studies: Slices from all the organs collected at various timepoints were made for H&E staining for toxicity studies.

[0167] Composition of the formulation buffer

[0168] The purified protein was provided in formulation buffer (20mM Sodium phosphate containing 100 mM glycine, 150 mM NaCl, and 50 μM ZnC12 at a pH of 7.5) at 2.0 mg/mL or 2.5 mg/mL for the PK assay in balb/c mice.

[0169] Pharmacokinetics assays in balb/c mice in Formulation buffer

[0170] For PK studies in balb/c mice, 5mg/Kg of HAce2Fc-2 was dosed per animal through intravenous route and serum samples collected at 1 h,2 h ,8 h, 24 h, 48 h, 96 h and 192 hours and analyzed for the HAce2Fc-2 levels by ELISA (FIG 17).

[0171] EXAMPLE 4: IN VIVO EFFICACY OF ACE2FC IN REVERSING THE SEVERITY OF SARS-COV2 INFECTION

[0172] Animal models of SARS-COV2 are relatively limited and difficult to perform due to highly infectious nature of the virus. Thus hinders the testing of vaccines and therapeutics. Moreover, animal model must mimic clinical disease and pathology in humans (1-3). [0173] Though, Transgenic BALB/C mice expressing human ACE-2 receptors in mice are preferred models for vaccine and therapeutic development. However, these are costly and difficult to procure .

[0174] Many laboratory animal species have been recently evaluated and the Golden Syrian Hamster has been well characterized and validated for SARS-CoV2 infection with typical organ pathology and cytokines produced and are thus suitable for testing efficacy of vaccine/antibody therapeutics (4).

[0175] Disease model was developed by intranasal inoculation of ~10⁵ PFU. The hamsters developed severe infection in 2-3 days associated with laboured respiration and succumbed due to severe pathology by day 7. This hamsters model represents infection/disease and pathology similar to human SARS infection with viremia, typical lung lesions, haemorrhages, lymphocytic infiltration, elevated pro-inflamatory cytokine and chemokine levels, and severe organ pathology demonstrable by immuno-histochemistry^6-8. Viral loads in organs were estimated by detection of viral mRNA in various organs by Q-RTPCR to estimate viral gene copy numbers or following titration in cell culture assay by plaque forming assays.

[0176] Efficacy: Hamsters were infected with 10⁵pfu/Hamster on Day 0. On Day 1, 2.5 mg/kg of the Ace2Fc protein was administered per hamster by intravenous route and the blood and Lung samples were collected at Ohr, 1,6 12, 24, 48 and 96 hrs from both control and Ace2Fc treated Hamsters, to check for the distribution of Ace2Fc protein into the serum and Lung samples as well as to check for the efficacy of the HAce2Fc protein by plaque reduction assay from the lung homogenates of Ace2Fc treated versus non-treated animals. The viral load reduction was also further assessed by qRT-PCR from the lung homogenates of control versus treated animals (FIG 18 A, B, and C).

[0177] EXAMPLE 5: IN VITRO EFFICACY TESTING OF ACE2FC FOR MANAGING HYPERTENSION

[0178] In vitro Ace2Fc peptidase activity will be measured using Mca-APK(Dnp) substrate, which is a surrogate Anorogenic substrate for Ace2 and the Auorescence intensity were measured at 320nm excitation and 420 nm emission wavelength. ACE2-Fc (different concentrations were used) peptidase activity assay using surrogate Anorogenic substrate Mca-APK(Dnp) were performed in black microtiter plates. The reaction buffer contains 50 mM 4- morpholineethanesulfonic acid, pH = 6.5, 300 mM NaCl, 10 μM ZnC12, 0.01% Triton X-100 and 20 μMof Mca-APK(Dnp). The total reaction volume will be 100 μL at room temperature and the duration of the reactions were set for 20 min. Peptidase activities are calculated as Auorescence intensity at 320 nm excitation and 420 nm emission wavelength. [0179] Efficacy testing of HAce2-Fc2 in Renal hypertension rodent model: The renal hypertension was induced by two kidney s/one clip model with slight modification. Male rats (190-220gm) are adapted to systolic blood pressure (SBP) measurement protocol by measuring BP once in three days for 1 week prior to 2K/1C surgical procedure. The readings recorded are considered as baseline systolic blood pressure (SBP). A U-shapes silver clip with a gauge of 0.25mm will be placed around the renal artery and secured in place. In sham operated group, the left renal artery exposed without disturbing the renal blood flow. In the sham operated rats, the surgery procedure is implicated without the artery clipping. Postoperative care is performed for the animals for 14days. Two weeks subsequent to postoperative care, SBP are measured for one week. Animals with SBP ≥160mm Hg are selected and randomized to different groups (Group 1, Sham operated; Group 2, positive control; Group 3, Reference drug (Ace Inhibitor); Group 4 HAce2-Fc2 (low and high dose). At the end, animals will be anaesthetized for blood collection and will be euthanized lung, heart and kidney will be collected.

[0180] EXAMPLE 6: IMPORTANT ATTRIBUTES OF THE INVENTION

[0181] Global spread of COVID-19 and the severe morbidity and mortality has effectively brought the world to a grinding halt. Multiple efforts are underway to develop prophylactic protein or mRNA-based vaccines for curbing the spread of disease in the community from future outbreaks (9,10). However, the need of the hour is to develop an effective therapeutic intervention to neutralize the virus and reduce the severity of disease in infected patients.

[0182] ACE-2 is believed to be the receptor for SARS-CoV 2 to gain entry into human cells and establish infection.

[0183] The ectodomain of ACE-2 has two distinct sites for the virus binding and the catalytic domain for converting pro-angiotensin to angiotensin by cleavage

[0184] Since ACE2 already contains complementary region for SARS-CoV-2 S protein, a soluble version of the protein can specifically bind to the virus and attenuate or effectively neutralize the virus.

[0185] Since ACE-2 functions as a dimeric-receptor and in order to exhibit desirable pharmacokinetic properties, the fusion protein will be fused to the Fc portion of a human IgG molecule

[0186] The ACE2-Fc fusion protein has already been shown to neutralize the effect of S protein in a cell-based assay (5).

[0187] SARS-CoV-2 enters the human body as droplet mainly through the respiratory tract and ACE-2 is highly expressed in the lungs. Therefore, the viral load in the respiratory tract is expected to be high. [0188] Interestingly, this transgenic mouse model represents infection/disease and pathology similar to human SARS infection with viremia, typical lung lesions, hemorrhages, lymphocytic infiltration, elevated pro-inflammatory cytokine and chemokine levels, and severe organ pathology demonstrable by immuno-histochemistry (6-8). In this model, blocking of Spike protein (S) by injecting ACE2 have been shown to protect transgenic mice from severe SAR CoV infection and severe pathology with 100% survival till day 14 suggesting dominating role of ACE2 receptors in virus uptake.

[0189] Alternatively, the Golden Syrian Hamster infection model can also be used as this model also develops SARS-COV2 infection similar to that in humans showing elevated cytokine levels. In this model also, blocking of Spike proteins by injecting Ace2Fc have been shown to protect it from disease severity.

Further, the catalytically active HAce2Fc can also be used for treatment of complications associated with comorbid conditions like diabetes, renal and cardiac hypertension (9-11).

[0190] EXPERIMENTAL EMBODIMENTS

[0191] EXAMPLE 7: INVENTION IS DESCRIBED IN DIFFERENT EMBODIMENTS THROUGH FOLLOWING FIGURES

[0192] FIG. 1 shows flow chart illustrating the experimental steps starting from cloning to efficacy testing in hamster model of the HAce2Fc protein. FIG. 1 Flow chart illustrating the experimental steps starting from cloning to efficacy testing in hamster model of the HAce2Fc protein, stepl: Mutations were introduced to the ectodomain of the hAce2-Fc protein. Step 2: selection of the Fc region from hlgG. Step 3: The selected sequences were codon optimized for optimum expression in HEK293 cell system. Step 4: the codon optimized DNA was synthesized. Step5: The synthetic Ace2Fc ectodomain and Fc regions were cloned into mammalian expression vector as a fusion protein. Step6: The recombinant HAce2Fc construct was transfected into HEK293 E cells transiently. Step7: The secreted protein from the cell supernatant was purified on Protein-A column. Step 8: The purified protein was reconstituted into different formulation buffer. Step9: The biophysical characterization of the purified protein to predict the secondary structure was performed by CD spectroscopy. Step 10: The Ace2Fc protein used either as a trap protein for SARS-COV2 infections or can be used for Step 11 : treating hypertension. Step 12: Invitro efficacy and toxicity testing of the protein on Vero E6 cells Step 14: Pharmacokinetic analysis in Hamster and Balb/c animal models. Stepl5: Efficacy testing in SARS-COV2 hamster infected model. Step 16: Invitro efficacy testing of the protein for its catalytic activity. Stepl7: In vivo efficacy testing to be used for hypertension in rodent model.

[0193] FIG. 2 illustrates the amplification of ACE2 and immunoglobulin Fc. FIGG illustrates the Cloning of ACE2.in Mammalian expression vector.

[0194] FIG. 4. illustrates the dot blot with Goat Anti-hlg- HRP of various cell supernatants after transfection of HEK293T cells. FIG 5. Summarizes the OD450 values of the cell supernatants after appropriate dilution in ELISA. FIG. 6 Western Image of cell supernatant and lysate expressing Ace2Fc protein. FIG. 7 A depicts the Coomassie blue stained image of purified Ace2- Fc under non-reducting condition. Fig 7B Coomassie blue stained image of purified Ace2-Fc reducing condition.

[0195] Fig. 8 depicts the Western image of purified Ace2Fc protein.

[0196] FIG9A. Binding of Ace2-hFc to SARS-COV2 spike protein by ELISA. FIG 9 B Binding of Ace2-hFc to SARS-COV2 Spike 1 protein by western blot.

[0197] FIG10: FAR UV CD-SPECTRA of Ace2-hFc.

[0198] FIG11 SARS-COV2 Neutralization by PRNT assay and Cytotoxicity by LDH assay of HAce2FC protein on Vero-E6.

[0199] FIG12A: SARS-COV2 Neutralization by qRT-PCR assay of HAce2FC protein on MO8 cells. And 12 B: Vero cells.

[0200] FIG 13A: illustrates the pharmacokinetic analysis of the HAce2Fc protein in Phosphate buffer formulation buffer using non-compartmental model; 13B: illustrates the pharmacokinetic analysis of the HAce2Fc protein in Phosphate buffer formulation buffer using compartmental model displaying the 1. Distribution Phase (α-phase), which marks the attenuation of drug in plasma due to distribution to other sites and 2. The Elimination Phase (β-phase), which marks the Attenuation of drug in plasma due to systemic excretion, according to the aspects of present invention.

[0201] Fig 14: Histopathology of hamster Lung, Liver, Kidney and spleen tissues post IV administration of the Ace2-Fc for safety studies.

[0202] Fig 15 Histopathology of the Lung tissue Post IV administration of the Ace2-hFc for safety studies displaying Moderate congestion with multifocal areas of bronchial epithelium showing mild to moderate hyperplasis.

[0203] FIG 16: Pharmacokinetic analysis post intranasal administration of the HAce2Fc protein in hamsters.

[0204] Fig 17: pharmacokinetic analysis of the HAce2Fc protein in Balb/c. [0205] FIG18 A and B: Efficacy assay graphs by PFU and qRT-PCR. Fig18 C: Correlation graph. [0206] EXAMPLE 8: MUTATIONS IN MAKE A SOLUBLE PROTEIN OF HUMAN ACE2 CORONAVIRUS BINDING SITE

[0207] Aim is to make a soluble protein of human ACE2 coronavirus binding site as well as the protease activity site and improving it by (i) introducing mutation for better binding to SARS- COV2 and similar corona viruses, (ii) Replacing the leader peptide with HA leader peptide (MKTIIALSYIFCLVFA) for efficient extracellular secretion, (iii) Introducing mutation for increased productivity in HEK293T. (iv) optimizing the codon usage for increased productivity in HEK293 cells, (v) fusing with human Ig Fc region common to IgG and IgA for dimerization as well as increased half life in systemic and mucosal circulation.

[0208] Backbone: AA 19-740 [QI 8 is removed, 1-17 - original leader sequence)

[0209] TABLE 1: SUMMARY OF MUTATIONS

[0210] Later t0 be tried out: K26D, D30E, K31Y.

[0211] ACE2 uniprot accession number: Q9BYF1

[0212] Human Ace2

[0213] PDB ID 6vwl ACE2-spike complex

[0214] PDB ID: 6M17: cryo-EM structures

[0215] Computational prediction for Ace2 peptides. After a detailed analysis of interface residues, a small stretch of the ACE2 PD (peptidase domain) N-terminal region (23-amino acids: Glu23 to Leu45) was found to be interacting majorly with the SARS-CoV-2 spike protein (12).

[0216] The highlighted residues in orange should not be changed (F28 to L45) which is involved in binding to COV2-RBD.

[0217] Three SNVs, E329G (rsl43936283), M82I (rs267606406) and K26R (rs4646116), had a significant reduction in binding free energy, which indicated higher binding affinity than wild- type ACE2 and greater susceptibility to SARS-CoV-2 infection for people with them (13), and hence these mutations are included in our design. This ref is correct

[0218] one of the variants of sACE2 (sACE2.v2.4 with mutations T27Y, L79T and N330Y) was expressed and purified with higher yields than wild type (20% and 80% higher for 8his and IgGl- Fc tagged proteins, respectively), although some protein remained aggregated after storage at 37 °C for 60 h (Figure 10D and 10E) (14). These three mutations have also been included. This ref is correct

[0219] Interestingly the T27Y and N33OY mutations for increased production yield is just besides the residues K26R and E329G mutations which are seen to increase the binding to SARS-COV2 RBD.

[0220] Following is genetically modified ACE2 nucleotide sequence of SEQ ID No: 1

[ 0221 ] ATGAAAACCATCATCGCCCTTTCTTACATCTTCTGCCTGGTGTTCGCCTCTACAATCGAGGAA CAGGCAAGATACTTTCTGGACAAGTTCAACCACGAGGCCGAGGATCTGTTTTATCAGAGCAGCCTGGCC AGCT GGAACTACAATACCAACAT CACAGAGGAGAAT GT GCAGAACAT GAACAAT GCCGGCGACAAGT GG TCCGCCTTCCTGAAGGAGCAGTCTACAACCGCCCAGATCTACCCCCTCCAGGAGATCCAGCAGCTGCAG GTGAAGCTCCAGCTCCAGGCCCTCCAGCAGAATGGCTCTAGCGTGCTGAGCGAGGATAAGTCCAAGCGG CTGAATACAATCCTGAACACCATGTCTACAATCTATAGCACCGGCAAGGTGTGCAATCCAGACAACCCA CAGGAGTGTCTGCTGCTGGAGCCCGGCCTGAATGAGATCATGGCCAACTCCCTGGATTACAATGAGAGG

CTGTGGGCATGGGAGAGCTGGCGCTCCGAAGTGGGCAAGCAGCTGCGGCCTCTGTACGAGGAGTATGTG

GTGCTGAAGAACGAGATGGCCAGAGCCAATCACTACGAGGACTATGGCGATTACTGGAGGGGCGACTAC

GAGGTGAACGGCGTGGACGGCTACGATTATAGCCGCGGCCAGCTGATCGAGGATGTGGAGCACACCTTC

GAGGAGATCAAGCCTCTGTATGAGCACCTGCACGCCTACGTGCGGGCCAAGCTGATGAATGCCTATCCT

TCTTACATCAGCCCAATCGGATGCCTGCCAGCACACCTGCTGGGCGACATGTGGGGCAGATTCTGGACA

AACCTGTACTCCCTGACCGTGCCTTTTGGCCAGAAGCCAAATATCGACGTGACCGATGCTATGGTGGAC

CAGGCCTGGGATGCCCAGAGGATCTTCAAGGAGGCCGAGAAGTTCTTCGTGAGCGTGGGCCTGCCTAAC

ATGACACAGGGCTTTTGGGGCTACTCCATGCTGACCGACCCTGGCAACGTGCAGAAGGCCGTGTGCCAC

CCAACAGCCTGGGACCTGGGCAAGGGCGATTTCCGCATCCTGATGTGCACCAAGGTGACAATGGACGAT

TTTCTGACCGCCCACCACGAGATGGGCCACATCCAGTATGATATGGCCTACGCCGCACAGCCATTCCTG

CTGCGGAATGGCGCCAACGAGGGCTTTCACGAGGCCGTGGGCGAGATCATGAGCCTGTCCGCCGCCACA

CCCAAGCACCTGAAGTCTATCGGCCTGCTGAGCCCTGACTTCCAGGAGGATAACGAGACAGAGATCAAT

TTTCTGCTGAAGCAGGCCCTGACCATCGTGGGCACACTGCCATTCACCTATATGCTGGAGAAGTGGCGC

TGGATGGTGTTTAAGGGCGAGATCCCCAAGGACCAGTGGATGAAGAAGTGGTGGGAGATGAAGAGGGAG

ATCGTGGGAGTGGTGGAGCCCGTGCCTCACGACGAGACATACTGTGATCCCGCCAGCCTGTTCCACGTG

TCCAACGACTATTCTTTTATCAGGTACTATACCGGCACACTGTACCAGTTCCAGTTTCAGGAGGCCCTG

TGCCAGGCAGCAAAGCACGAGGGACCCCTGCACAAGTGTGACATCTCTAACAGCACAGAGGCCGGCCAG

AAGCTGTTCAATATGCTGAGACTGGGCAAGTCCGAGCCCTGGACCCTGGCCCTGGAGAACGTGGTGGGA

GCCAAGAATATGAACGTGAGGCCCCTGCTGAATTATTTCGAGCCTCTGTTTACATGGCTGAAGGATCAG

AATAAGAACTCCTTCGTGGGCTGGAGCACCGACTGGTCCCCATACGCCGATCAGTCTATCAAGGTGAGA

ATCTCCCTGAAGTCTGCCCTGGGCGACAAGGCCTATGAGTGGAATGATAACGAGATGTACCTGTTTAGA

TCCTCTGTGGCCTATGCCATGAGGCAGTACTTCCTGAAGGTGAAGAACCAGATGATCCTGTTTGGCGAG

GAGGACGTGCGGGTGGCCAATCTGAAGCCCAGAATCAGCTTCAACTTCTTTGTGACAGCCCCAAAGAAC

GTGAGCGACATCATCCCAAGGACCGAGGTGGAGAAGGCAATCCGGATGAGCCGGAGCCGGATCAACGAC

GCCTTTAGGCTGAATGATAACTCTCTGGAGTTTCTGGGCATTCAGCCTACCCTGGGACCACCTAATCAG CCACCCGTGAGTGCTAGC

[0222] Note that change in T27A is increasing the free energy, thereby may reduce the binding strength.

[0223] UNIPROT

[0224] Family & Domains¹

[0225] TABLE 2: TOPOLOGICAL REGION

[0226] Following are amino acid sequence of ACE2

[0227] Following is genetically modified ACE2 protein sequence SEQ ID No: 2

Recombinant Protein sequence:

MKTIILALSYIFCLVFASTIEEQARYFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQ NMNNAGDKWSAFLKEQSTTAQIYPLQEIQQLQVKLQLQALQQNGSSVLSEDKSKRLNTIL NTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLY EEYW LKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHL HAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQ AWDAQRIFKEAEKFFVSVGLPNMTQGFWGYSMLTDPGNVQKAVCHPTAWDLGKGDFRILM CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKS IGLLSPDFQEDNETEINFLLKQALT IVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEM

KREIVGW EPVPHDETYCDPASLFHVSNDYSFIRYYTGTLYQFQFQEALCQAAKHEGPLH KCDISNSTEAGQKLFNMLRLGKSEPWTLALENW GAKNMNVRPLLNYFEPLFTWLKDQNK NSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKN QMILFGEEDVRVANLKPRISFNFFVTAPKNVSDI IPRTEVEKAIRMSRSRINDAFRLNDN SLEFLGIQPTLGPPNQPPVS

[0228] Following is the wild type ACE2 protein sequence SEQ ID No: 3

Wild Type Protein sequence:

MSSSSWLLLSLVAVTAAQSTIEEQAMTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQ

NMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTIL

NTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLY

EEYW LKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHL

HAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQ

AWDAQRIFKEAEKFFVSVGLPNMTQGFWEN'SMLTDPGNVQKAVCHPTAWDLGKGDFRILM

CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKS

IGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEM

KREIVGW EPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLH

KCDISNSTEAGQKLFNMLRLGKSEPWTLALENW GAKNMNVRPLLNYFEPLFTWLKDQNK NSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKN

QMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDN

SLEFLGIQPTLGPPNQPPVS

[001] EXAMPLE 9: VALIDATION OF ACE2-FC (AROGYACE) PROTEIN

[002] Validation for the Ace2-Fc protein would be carried out both by invitro model system and in-vivo model system

[003] In vitro validation

[004] Cloning of SARS-COV2 SI region and full-length Viral Spike protein into mammalian expression vector.

[005] Expression of Spike 1 of SARS-COV2 in mammalian host.

[006] Purification of the expressed protein.

[007] Expression of Full-length spike of SARS-COV2 in mammalian host.

[008] The binding of Ace2-Fc to the Spike protein is being determined by Enzyme Linked Immunosorbent Assay (ELISA) and western blotting.

[009] Neutralization Assay by SARS-COV2 virus

[010] Neutralization assay: The neutralization potential of Arogyace is being determined by its ability to neutralize the SARS-COV2 virus by preventing its binding to the Ace2 expressed by the VERO-E6 cells.

[Oi l] In-vivo validation (Efficacy testing of Arogyace in Rhodent model: Golden Syrian Hamster)

[012] Disease model is developed by inoculating live viruses by intranasal inoculation.

[013] Efficacy of Arogyace is determined by administering the drug molecule 24 hours after the lethal viral challenge.

[014] The protective effect of Arogyace in combating the infection is tested by Plaque reduction Neutralization Test from the lung homogenates, qRT PCR and 'assaying the levels of IL6, IL10 and IFN-gamma levels in the disease model.

[015] Pharmacokinetics of Arogyace is measured.

[016] The toxicity of Arogyace is being studied.

[017] According to the aspects of embodiments, the specification may have presented a method and/or process as a particular sequence of steps. However, that the method or process does not depend on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps as described. As person with ordinary skill in the art would appreciate, other sequences of steps may be possible. It is therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and/or process should not be limited to the performance of their steps in the order written, and person skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.

[018] The embodiments described herein, can be practiced with different computer system configurations including hand-held devices, microprocessor-based, microprocessor systems, or programmable consumer electronics, minicomputers, mainframe computers and the like. The embodiments can also be practiced in distributing computing environments where steps are performed by remote processing devices that are linked over a network.

[019] According to a non-limiting exemplary aspect of the present invention, it should be understood that the embodiments described herein can employ various computer-implemented operations involving data stored in computer systems. These operations can be those requiring physical manipulation of computer systems. These quantities can be in form of electrical or magnetic signals capable of being stored, compared, transferred, combined, and otherwise manipulated. Further, the manipulations performed are often referred to in terms, such as producing, identifying, determining, or comparing.

[020] Any of the operations that form part of the embodiments described herein are useful machine operations. The embodiments, described herein, also relate to a device or an apparatus for performing these operations. The systems and methods described herein can be specially constructed for the required purposes or it may be a general-purpose computer selectively activated or configured by a computer program stored in the computer. In particular, various general-purpose machines may be used with computer programs written in accordance with the teachings herein, or it may be more convenient to construct a more specialized apparatus to perform the required operations.

[021] Merely for illustration, only representative number/type of graph, chart, block and sub- block diagrams were shown. Many environments often contain many more block and sub- block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.

[022] According to a non-limiting exemplary aspect of the present invention, the method(s) can be used for the development of technologies that enable pathogen detection in point-of-care settings. These diagnostic technologies developed can then be utilized by hospitals/private clinic s/dental doctors or the public as such to screen/diagnose different pathogens.

[023] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[024] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.

[000] References:

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Claims

1/ We Claim.

1) A pharmaceutical composition comprising recombinant polypeptides; a) wherein the first recombinant polypeptide comprises a genetically modified ACE2 protein comprising the nucleotide sequence of SEQ ID No: 1, to increase binding to SARS-CoV and SARS-Cov2 viruses spike protein and neutralizing activity; and b) the second recombinant polypeptide comprises a Fc portion common to IgG and IgA; wherein characterized in that the said recombinant polypeptide are such that they exhibit a synergistic neutralizing activity, improved systemic and the mucosal circulation.

2) The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

3) The pharmaceutical composition as claimed in claim 1, wherein pharmaceutical composition is for the diagnosis, prophylaxis, treatment, or combination thereof of a condition resulting from a SARS-CoV or cardiac or renal hypertensin or complications associated with diabetes.

4) The pharmaceutical composition as claimed in claim 1, wherein pharmaceutical composition is for neutralizing SARS-CoV, SARS-CoV2 and/or related viruses with similar spike protein structure.

5) The pharmaceutical composition as claimed in claim 1, wherein pharmaceutical composition is for detection and quantification of viral load of SARS-CoV, SARS-CoV2 and/or related viruses with similar spike protein structure.

6) The pharmaceutical composition as claimed in claim 1, wherein pharmaceutical composition is a renin-angiotensin-system or a bradykinin receptor inhibitor or a renin inhibitor or an ACE inhibitor.

7) The pharmaceutical composition as claimed in claim 1, wherein pharmaceutical composition is not cytotoxic.

8) The pharmaceutical composition as claimed in claim 1, wherein pharmaceutical composition is for medicament.

9) The pharmaceutical composition as claimed in claim 8, wherein pharmaceutical composition is administered as individual or as a combination medicament.

10) The pharmaceutical composition as claimed in claim 9, wherein the medicament is administered intravenously, intraperitoneally, mucosally, intranasally, orally, intratracheally, and/or as an aerosol composition. 11) A method of detecting a severe acute lung injury or failure in a subject comprising providing a pharmaceutical composition comprising ACE2 recombinant polypeptide to a subject with a severe acute lung injury or failure, wherein the severe acute lung injury or failure in the subject is detected.

12) The method as claimed in claim 11, wherein the severe acute lung injury or failure is further defined as an injury induced by acid aspiration or sepsis, a lung oedema, and/or a lung injury and/or failure connected with infection with severe acute respiratory syndrome (SARS) coronavirus or hypertension or due to diabetes or any other comorbidities.

13) The method as claimed in claim 10, wherein the inhibitor of the renin-angiotensin-system is an ACE inhibitor, ACE2, renin inhibitor or inhibitors of SARS-COV or SARS-CoV2 or similar viral entry, or any combination thereof.

14) A method for the production of recombinant ACE2 polypeptides or a preparation of recombinant ACE2, comprising the steps of; a) introducing an ACE2-encoding polynucleotide, preferably an ACE2 without transmembrane domain-encoding polynucleotide; b) introducing one or more mutations in ectodomain of ACE2-encoding polynucleotide; c) creating codon optimized Fc region from hlgG; d) cloning Ace2 ectodomain and Fc regions into mammalian expression vector; e) transfecting recombinant HAce2Fc construct into mammalian cells; f) collecting cell supernatant; g) purifying recombinant protein on Protein-A column; h) reconstituting recombinant protein in formulation buffer; and i) characterizing ACE2 recombinant protein.

15) A method for the production of recombinant ACE2 polypeptides, wherein the recombinant ACE2 polypeptides comprises mutations K26R, T27Y, L79T, M82I, N90Q, T92Q, E329G, N330Y, R518G or any combination, or any other mutation in the spike protein binding domain thereof.

16) A kit comprising recombinant ACE2 polypeptides for neutralizing SARS-CoV and SARS- Cov2 viruses comprising recombinant polypeptide; a) wherein the first recombinant polypeptide comprises a genetically modified ACE2 protein comprising the nucleotide sequence of SEQ ID No: 1 to increase binding to SARS-CoV and SARS-Cov2 viruses spike protein and neutralizing activity; and b) the second recombinant polypeptide comprises a Fc portion common to IgG and IgA; wherein characterized in that the recombinant polypeptide are such that they exhibit a synergistic neutralizing activity.

17) The kit as claimed in claim 16, wherein the kit is for qualitative analysis of SARS-CoV or SARS-CoV2 or any other virus with similar spike protein or quantitative analysis of SARS- CoV or SARS-CoV2 or any other virus with similar spike protein.

18) A composition for binding to SARS-CoV and SARS-Cov2 viruses spike protein, wherein said composition comprising of nucleotide sequence: a synthetic, recombinant oligonucleotides sequence of SEQ ID NO 1.