ACE2 receptor ectodomain fusion molecules and uses thereof

Abstract

The present invention relates generally to polypeptides capable of neutralizing SARS-CoV-2 and providing ACE2 enzymatic activity, and the use of these polypeptides to treat disorders associated with coronavirus infection (COVID-19) and associated acute respiratory distress syndrome (ARDS) and major organ injury, as well as methods for making such molecules.

Description

[Technical field]

[0001] [Field of the Invention] The present invention relates to human ACE2 receptor ectodomain fusion molecules and uses thereof. More specifically, the present invention relates to protein fusion variants of the human ACE2 receptor catalytic domain containing structural elements of the human IgG1 antibody framework and their use in reducing coronavirus infection (COVID-19) and associated acute respiratory distress syndrome (ARDS).

[0002] [Background of the invention] The COVID-19 pandemic continues as a global healthcare crisis of unprecedented severity. Although clinical trials of potential vaccines are ongoing, uncertainties exist regarding the safety and efficacy of widespread vaccination of the population. There is an absolute lack of biological therapeutic agents for COVID-19 infection, and current strategies have only modest impact on patient outcomes. In this regard, many of the severe complications of COVID-19, including septic shock, acute respiratory distress syndrome (ARDS), and acute kidney injury (AKI), are mediated, at least in part, by host responses, particularly within the renin-angiotensin system (RAS) [1–3]. Importantly, SARS-CoV-2, the virus that causes COVID-19, directly interacts with angiotensin-converting enzyme 2 (ACE2), a plasma membrane protein that mediates cell entry of SARS-CoV-2 [4–6].

[0003] The SARS-CoV-2 virus utilizes its spike protein for attachment and internalization into host cells as a central step required for viral replication. Therefore, the spike protein has become a major molecular target for the development of potential anti-COVID-19 biological therapeutics, vaccines and diagnostics. The spike protein trimer in its prefusion state allows the receptor-binding domain (RBD) of the spike protein to directly interact with the ACE2 receptor on the host cell. Since the outbreak of this pandemic, much has been learned about the structure and function of the spike protein in relation to the human receptor ACE2 of the SARS-CoV-2 virus. Part of this understanding has led to several approaches aimed at developing biological therapeutics against the disease. Among these numerous approaches, the two main directions for blocking viral entry include binding to the spike protein by neutralizing monoclonal antibodies and by ACE2 receptor decoys. Indeed, from the perspective of blocking viral entry, the main disadvantage of antibodies is the risk of reduced efficacy against newly emerging viral strains [7], whereas receptor decoys based on human ACE2 should in principle possess a robust pan-specificity profile.

[0004] Angiotensin (Ang) II is a potent vasoconstrictor with proinflammatory and procoagulant effects. ACE2 is a monocarboxypeptidase that converts Ang-II to Ang-(1-7), a vasodilatory antagonistic peptide to Ang-II. SARS-CoV-2 infection increases pulmonary and coronary microvascular thrombosis and coagulation (increased D-dimer), which is associated with increased COVID-19 mortality [8, 9]. Similar to respiratory viruses H1N1 and H5N1, SARS-CoV-2 binds to and inhibits ACE2 [4-6], and thus ACE2 is a potential biomarker and therapeutic target in patients with COVID-19 infection. ACE2 is downregulated in H1N1, H5N1, H7N9, and SARS, which leads to increased Ang-II levels and exacerbated lung injury [10, 11]. Thus, local activation of the renin-angiotensin system (RAS) may mediate the damaging response of the lung, heart and other organs to SARS-CoV-2 in COVID-19. Strategies to increase local tissue ACE2 activity in SARS-CoV-2 could therefore reduce cytotoxicity by reducing Ang-II (harmful) and enhancing Ang-(1-7) (protective). Therefore, ACE2-based decoys against this virus not only have the potential to neutralize viral entry into host cells, but also, via a dual mechanism of action, result in the enzymatic conversion of Ang-II to Ang-(1-7), thereby restoring the protective RAS pathway and transitioning to alleviate ARDS.

[0005] Several studies have focused on using the human ACE2 ectodomain, either alone or fused to the Fc region of human antibodies, with or without its enzymatic activity [12-15]. However, innovative molecular engineering aimed at multifactorial optimization is necessary to develop ACE2-based biotherapeutics with clinical efficacy and large-scale manufacturability. There remains a need for ACE2-based decoys that provide more effective virus neutralization while providing Ang-II-converting ACE2 enzymatic activity.

[0006] [Summary of the invention] The present invention provides an improved ACE2-based decoy. Different from other approaches in the art, the inventors have focused on a natural variant of the human ACE2 receptor, which carries an isoleucine (Ile) amino acid residue at position 92, where a threonine (Thr) amino acid residue is usually found in the more common ACE2 receptors that are widespread in the human population. The inventors have discovered during the course of the invention that utilizing the structural and functional elements of this naturally occurring human variant, herein referred to as hACE2I92, improves several properties of ACE2-based decoys, the most important of which for the above-mentioned dual mechanism of action are improved catalytic activity and improved virus neutralization. Using ACE2I92 as a starting point in a multi-pronged molecular design endeavor, we now provide a class of polypeptide constructs that possess (a) strong binding avidity and affinity to the spike protein for efficient neutralization of viral infection, (b) high enzymatic activity for reduced ARDS, and (c) improved biological manufacturability, while (d) suitable pharmacokinetics to enable viral clearance while preventing antibody-dependent enhancement (ADE), and (e) structural components for protection against newly emerging viral strains and future pandemics.

[0007] Thus, a polypeptide construct capable of neutralizing SARS-CoV-2 and converting Ang-II into Ang-(1-7) is provided, comprising four regions and having the general formula: R1[hACE2I 92 (18-614), X 27 ,X 261 ,X 330 ]-R2-R3[Hinge S 220 ,X 226 ,X 229 ]-R4[C H 2G 270 -C H 3] having In the formula, hACE2I 92 (18-614), X27 ,X 261 ,X 330 R1, represented as: 27 , X 261 and X 330 The first region at the N-terminus of the human angiotensin-converting enzyme 2 (hACE2) receptor catalytic domain, which contains the naturally occurring variant Ile92 (I92), residues 18 to 614, and X 27 is the amino acid residue at position 27, which is either Thr or Tyr; X 261 is the amino acid residue at position 261 which is either Cys or Ser, and X 330 R1 is the amino acid residue at position 330, which is either Asn or Tyr; R2 is a second region comprising a flexible peptide spacer; R3 is a third region comprising a hinge region of a human IgG1 heavy chain antibody, said hinge region comprising residues S 220 , X 226 and X 229 the amino acid residue at position 220 of the human IgG1 hinge is Ser, and the amino acid residues at positions 226 and 229 of the human IgG1 hinge are either Cys or Ser; and R4 is a fourth region at the C-terminus of the polypeptide, and R4 is C H 2G 270 -C H 3 and includes the second constant domain of the human IgG1 antibody heavy chain (C H 2) and the third constant domain (C H 3) Human IgG1 C H A polypeptide construct is provided, wherein the amino acid residue at position 270 in domain 2 is a glycine.

[0008] In embodiments, the R2 spacer region of the polypeptide construct comprises a flexible peptide spacer comprising Gly and Ser residues.

[0009] In embodiments, the polypeptide construct neutralizes SARS-CoV-2 with an IC50 of at least 500 ng / mL.

[0010] In embodiments, the polypeptide construct enhances the catalytic efficiency (kcat / K M ) and at least 60% of the specific activity.

[0011] In an embodiment, R1 of the polypeptide construct comprises a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and / or any sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7.

[0012] In an embodiment, R2 of the polypeptide construct comprises a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, and / or any sequence that is at least 90% identical to SEQ ID NO:8, SEQ ID NO:9.

[0013] In an embodiment, R3 of the polypeptide construct comprises a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, and / or any sequence that is at least 90% identical to SEQ ID NO:11, SEQ ID NO:12.

[0014] In an embodiment, R4 of the polypeptide construct comprises a sequence having SEQ ID NO:14, or a sequence that is at least 90% identical to SEQ ID NO:14.

[0015] In an embodiment, the polypeptide construct of the invention comprises a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:28, and a sequence which is at least 90% identical to SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:28.

[0016] In an embodiment, the polypeptide construct of the present invention is a dimeric polypeptide. In an embodiment, the dimeric polypeptide may be linked or dimerized via their respective R3 hinge regions by disulfide bridges.

[0017] Another embodiment is a nucleic acid molecule encoding any of the polypeptide constructs described herein. The present invention also provides an expression vector for producing a polypeptide, the expression vector comprising a nucleic acid molecule encoding any of the polypeptide constructs described herein. In an embodiment, the nucleic acid sequence encoding a polypeptide of the invention is in a form that is secretable by the selected expression host.

[0018] Another embodiment is a composition comprising a polypeptide construct described herein and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0019] In another embodiment, there is provided a transgenic cell host comprising a nucleic acid molecule encoding any of the polypeptide constructs described herein, or an expression vector for producing any of the polypeptide constructs of the invention.

[0020] In another embodiment, the transgenic cell host further comprises a second nucleic acid molecule or a second vector encoding a second polypeptide construct that is the same as the first polypeptide construct.

[0021] Another embodiment is a method for producing a dimeric polypeptide comprising culturing a provided transgenic cell host and recovering a dimeric polypeptide construct according to the invention from the medium conditioned by growth of said host.

[0022] Another embodiment is the use of a polypeptide construct as described herein for the treatment of a medical condition, disease or disorder, in an embodiment the medical condition, disease or disorder includes coronavirus infection such as COVID-19, acute respiratory distress syndrome (ARDS) and associated major organ failure such as lung, heart, kidney, brain and intestine.

[0023] As described herein, the class of polypeptide constructs of the invention comprises four regions R1, R2, R3 and R4 (Figure 1). These polypeptides are useful for neutralizing SARS-CoV-2 to treat COVID-19, and for converting Ang-II to Ang-(1-7) to treat ARDS. The polypeptides of the invention have the general formula: R1-R2-R3-R4 wherein R1 is hACE2I 92 (18-614), X 27 ,X 261 ,X 330 Contains X 27 is Thr or Tyr, and X 261 is Cys or Ser, and X 330 is Asn or Tyr; R2 comprises a spacer or linker; R3 is Hinge S 220 ,X 226 ,X 229 Contains;X 226 and X 229 is Cys or Ser; and R4 is C H 2G 270 -C H Includes 3.

[0024] In a preferred embodiment, the polypeptide has the general formula: R1[hACE2I 92 (18-614), X 27 ,X 261 ,X 330 ]-R2[Spacer]-R3[Hinge S 220 ,X 226 ,X 229 ]-R4[C H 2G 270 -C H 3] The polypeptide includes a polypeptide having the formula:

[0025] hACE2I 92 (18-614), X 27 ,X 216 ,X 330Region R1, denoted as Ile92, is the first (N-terminal) region containing the naturally occurring variant Ile92 of human angiotensin-converting enzyme 2 (hACE2I92). Since the ACE2 collectrin (neck) domain of the native ACE2 enzyme was incompatible with binding to the SARS-CoV-2 spike trimer, it was decided to lock the ACE2 catalytic domain dimer in a rigid conformation; therefore, the neck (collectrin) domain was removed, thereby generating the R1 region consisting of the receptor catalytic domain composed of residues 18–614, X. 27 is the amino acid residue at position 27, which is either Thr or Tyr; X 261 is the amino acid residue at position 261 which is either Cys or Ser, and X 330 is the amino acid residue at position 330 which is either Asn or Tyr. In preferred non-limiting embodiments, the R1 region of the polypeptide construct is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and sequences substantially identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7.

[0026] Region R2, also referred to herein as spacer or linker, is a second region of the polypeptide construct of the invention comprising a flexible polypeptide linker made of Gly and Ser residues. In a preferred non-limiting embodiment, the R2 region of the polypeptide construct is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, and sequences substantially identical to SEQ ID NO:8, SEQ ID NO:9. As will be understood by a person skilled in the art, the polypeptide construct of the invention is not limited to the R2 regions specifically described herein, but can comprise any suitable spacer or linker (used interchangeably herein), provided that said linker or spacer is of a sequence and length that allows operable function of the polypeptide of the invention.

[0027] Hinge S 220 ,X 226 ,X 229The region R3, represented as follows: 226 ,X 229 are amino acid residues at positions 226 and 229 that are either Cys or Ser. In preferred non-limiting embodiments, the R2 region of the polypeptide construct is selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, and sequences substantially identical to SEQ ID NO:11, SEQ ID NO:12.

[0028] C H 2G 270 -C H Region R4, designated as 3, corresponds to the second constant domain of the human IgG1 antibody heavy chain (C H 2) and the third constant domain (C H 3) a C-terminal region of a polypeptide construct comprising H The R2 domain comprises a Gly amino acid residue at position 270. In a preferred, non-limiting embodiment, the R2 region of the polypeptide construct has the sequence of SEQ ID NO:14 or any sequence substantially identical to SEQ ID NO:14.

[0029] In preferred, non-limiting embodiments, the polypeptide construct of the present invention is selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:28, and sequences substantially identical to SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:28.

[0030] The polypeptide constructs provided in these preferred, non-limiting embodiments can be produced in high yields by transient transfection in CHO cells, can be purified to high purity by Protein A affinity chromatography and preparative size-exclusion chromatography (SEC), possess elevated enzymatic activity, bind with high affinity and avidity to the spike protein of SARS-CoV-2, neutralize pseudotyped SARS-CoV-2 with high potency, and neutralize authentic SARS-CoV-2 virus in cells and animal models. The class of polypeptide constructs described in this invention will be useful for reducing viral load in living organisms, such as mice, hamsters, and monkeys as animal models of disease, and in humans for clinical applications. Thus, these compounds represent useful biological therapeutic agents for treating coronavirus infections, including SARS and COVID-19 and their newly emerging viral strains, as well as ARDS and multiple organ damage, such as lungs, heart, kidneys, brain, and intestine, associated with viral diseases.

[0031] Furthermore, the ACE2 replacement function of these compounds provides additional therapeutic applications in other virus-induced pathologies resulting in acute respiratory distress syndrome (ARDS) or resulting from sepsis-induced ARDS / cytokine storm, such as respiratory syncytial virus, avian H5N1 influenza, etc. Finally, additional therapeutic indications include non-viral indications such as cardiac dysfunction, such as in myocarditis, perivascular and myocardial fibrosis, diabetic nephropathy, renal fibrosis and liver dysfunction resulting in NASH / NAFLD.

[0032] These and other features of the present invention will now be described, by way of example only, with reference to the accompanying drawings. [Brief description of the drawings]

[0033] [Figure 1]1 is a schematic diagram showing the design of a class of polypeptides of the invention comprising four regions R1, R2, R3 and R4, with reference to SEQ ID NOs in the sequence listing. The numbers of the amino acid positions shown correspond to the conventional numbering of each region and not to the sequential numbering along the full length polypeptide sequence. [Diagram 2] Figure 1 shows the main principle of the structure-based modular design that led to the class of polypeptides of the invention. The ACE2 collectrin (neck) domain locks the ACE2 catalytic domain dimer in a rigid conformation that is incompatible with the CoV-2 spike trimer; therefore, the neck (collectrin) domain was removed. The introduced flexibility provided by the spacer and hinge regions unlocks the ACE2 catalytic domain dimer for strong binding to the CoV-2 spike trimer. [Diagram 3] FIG. 3D rendering of the R1 region (shown as a dark grey ribbon) of a polypeptide of the invention affinity matured for improved binding to the SARS-CoV-2 spike protein receptor binding domain (RBD) (shown as a semi-transparent molecular surface). A natural variant of the human ACE2 catalytic domain carrying the T92I mutation represents the starting point for the structure-based affinity maturation predictions performed in this study using the ADAPT platform. Mutations selected in this invention for improved binding affinity to the SARS-CoV-2 spike RBD are rendered as CPK models at positions 27 and 330 of the ACE2I92 natural variant. See Table 1 for the top 30 binding affinity improving mutations according to the ADAPT calculations. [Figure 4A] Figure 4A depicts the analysis of polypeptide variants produced by transient transfection in CHO cells and purified by Protein A affinity chromatography. Figure 4A provides an SDS-PAGE analysis of denatured proteins under non-reducing and reducing conditions and different elution buffers. [Figure 4B]Figure 4 depicts the analysis of polypeptide variants produced by transient transfection in CHO cells and purified by Protein A affinity chromatography. Figures 4B and 4C provide UPLC-SEC analysis of elution fractions for selected variants eluted from a Protein A column with citrate pH 3.6 and acetate pH 3.7 buffers, respectively. The molecular weights of the major peaks as determined by MALS analysis are shown. [Figure 4C] Figure 4 depicts the analysis of polypeptide variants produced by transient transfection in CHO cells and purified by Protein A affinity chromatography. Figures 4B and 4C provide UPLC-SEC analysis of elution fractions for selected variants eluted from a Protein A column with citrate pH 3.6 and acetate pH 3.7 buffers, respectively. The molecular weights of the major peaks as determined by MALS analysis are shown. [Figure 5A] Figure 5A shows the analysis of polypeptide variants after preparative size exclusion chromatography. Figure 5B provides SDS-PAGE analysis of denatured proteins under non-reducing and reducing conditions. Purity levels for an additional set of polypeptide variants are listed in Table 2. [Figure 5B] Figure 5B shows the analysis of polypeptide variants after preparative size exclusion chromatography. Figure 5B provides the UPLC-SEC analysis of selected purified variants, along with the molecular weight of the main peak determined by MALS analysis. The purity levels for an additional set of polypeptide variants are listed in Table 2. [Figure 5C] Figure 5A depicts the analysis of polypeptide variants after preparative size exclusion chromatography. Figure 5C provides sedimentation velocity analytical ultracentrifugation (AUC) analysis data for selected purified variants. Purity levels for an additional set of polypeptide variants are listed in Table 2. [Figure 6A]Figure 6 depicts enzyme activity data for selected polypeptide variants. Recombinant human ACE2 (rhACE2) ectodomain is used as a control. Figure 6A provides enzyme activity determined using a fluorogenic substrate-based cell-free assay as a function of substrate concentration at a fixed enzyme concentration of 100 ng / mL, which is used to determine catalytic efficiency (kcat / KM) values. [Figure 6B] Figure 6A shows enzyme activity data for selected polypeptide variants. Recombinant human ACE2 (rhACE2) ectodomain is used as a control. Figure 6B provides activity as a function of enzyme concentration, which is used to determine specific activity values ​​determined using a cell-free assay based on fluorogenic substrates. For enzyme activity data for additional tested variants, see Table 4. [Figure 6C] Figure 6A depicts enzyme activity data for selected polypeptide variants. Recombinant human ACE2 (rhACE2) ectodomain is used as a control. Figure 6C depicts the enzyme activity of the polypeptide variants of the invention or control enzymes (rhACE2 and rhACE) at 100 ng / mL upon incubation with 25 nM angiotensin II (Ang II) for 30 min, as assessed by hydrolysis of Ang II to angiotensin-(1-7) assayed by ELISA. Data are presented as mean ± SEM, *P<0.01 vs. rhACE. N=3-4 experiments. [Figure 6D] Figure 6A represents enzyme activity data for selected polypeptide variants. Recombinant human ACE2 (rhACE2) ectodomain is used as a control. Figure 6D presents the fluorescence readouts expressed as RFU for enzyme activity using 11.25 μM Mca-APK (Dnp) fluorogenic substrate at 30 min in the presence of mouse primary proximal tubular epithelial cells (PTEC) treated with 0.6 nM of the polypeptide variant of the invention or control enzyme (rhACE2 and rhACE) for 24 h. Data are presented as mean ± SEM, *P<0.01 vs. rhACE. N=3 experiments. [Figure 6E]Figure 6A represents enzyme activity data for selected polypeptide variants. Recombinant human ACE2 (rhACE2) ectodomain is used as a control. Figure 6E presents the fluorescence readouts expressed as RFU for enzyme activity assessed by hydrolysis of Ang II (5 nM) to angiotensin-(1-7) (ELISA) in the presence of mouse primary proximal tubular epithelial cells (PTEC) treated with 0.6 nM of the polypeptide variants of the invention or control enzymes (rhACE2 and rhACE) for 24 hours. Data are presented as mean ± SEM, *P<0.01 vs. rhACE. N=3 experiments. [Figure 7A] Figure 7A shows the evaluation of the binding ability of the polypeptide variants of the present invention to SARS-CoV-2 spike RBD. Figure 7A provides the normalized SPR sensorgrams used to rank the polypeptide constructs by dissociation rate (koff). The homobivalent variants are run over the immobilized Wuhan strain SARS-CoV-2 spike RBD, which prevents the calculation of the binding dissociation constant due to the avidity effect. [Figure 7B] Figure 7B shows the binding ability of the polypeptide variants of the present invention to SARS-CoV-2 spike RBD. Figure 7B provides surrogate neutralization (sn)-ELISA binding competition data using the immobilized Wuhan strain SARS-CoV-2 spike RBD and bound biotinylated ACE2 detection by streptavidin-polyHRP. IgG antibody isotype is used as a negative control. Table 5 lists the SPR-based koff dissociation rate and snELISA IC90 values ​​of the tested variants. [Figure 7C] Figure 7A shows an evaluation of the binding ability of the polypeptide variants of the present invention to the SARS-CoV-2 spike RBD. Figure 7C shows the dissociation rate of the polypeptides of the present invention from the spike-RBD of SARS-CoV-2 variants Wuhan strain and B.1.351 (beta) and SARS-CoV-1. [Figure 7D]Figure 7 shows the evaluation of the binding ability of the polypeptide variants of the present invention to SARS-CoV-2 spike RBD. Figure 7D and Figure 7E provide SPR sensorgrams from long dissociation kinetics experiments of selected polypeptides of the present invention binding to the spike RBD of SARS-CoV-2 variants Wuhan strain, B.1.351 (beta) and B.1.1.529 (omicron), as well as the corresponding fitted apparent kinetics and equilibrium constants. Data are presented as mean ± SEM from N=3 experiments. [Figure 7E] Figure 7 shows the evaluation of the binding ability of the polypeptide variants of the present invention to SARS-CoV-2 spike RBD. Figure 7D and Figure 7E provide SPR sensorgrams from long dissociation kinetics experiments of selected polypeptides of the present invention binding to the spike RBD of SARS-CoV-2 variants Wuhan strain, B.1.351 (beta) and B.1.1.529 (omicron), as well as the corresponding fitted apparent kinetics and equilibrium constants. Data are presented as mean ± SEM from N=3 experiments. [Figure 8A] Figure 8 shows neutralization data based on pseudotyped lentiviral particles as a surrogate for live SARS-CoV-2 virus. The ability of the polypeptide constructs of the invention to block pseudotyped viral particle entry into a host cell line expressing ACE2 was measured. Pseudotyped lentiviral particles contain the SARS-CoV-2 spike protein, a luciferase reporter and a minimal set of lentiviral proteins required to assemble a virus-like particle. Blockade of viral entry is detected by loss of luciferase reporter signal. Figure 8A provides assay implementation 1 co-expressing human ACE2 and TMPRSS2 on HEK293T cells. [Figure 8B]Figure 8B shows neutralization data based on pseudotyped lentiviral particles as a surrogate for live SARS-CoV-2 virus. The ability of the polypeptide constructs of the invention to block pseudotyped viral particle entry into host cell lines expressing ACE2 was measured. Pseudotyped lentiviral particles contain the SARS-CoV-2 spike protein, a luciferase reporter and a minimal set of lentiviral proteins required to assemble a virus-like particle. Blocking of viral entry is detected by loss of luciferase reporter signal. Figure 8B provides an assay implementation expressing human ACE2 on HEK293T cells. See Table 6 for a listing of IC50 values ​​of the variants tested. [Figure 8C] Figure 8 shows neutralization data based on pseudotyped lentiviral particles as a surrogate for live SARS-CoV-2 virus. The ability of the polypeptide constructs of the invention to block pseudotyped viral particle entry into host cell lines expressing ACE2 was measured. Pseudotyped lentiviral particles contain SARS-CoV-2 spike protein, luciferase reporter and a minimal set of lentiviral proteins required to assemble virus-like particles. Blockage of viral entry is detected by loss of luciferase reporter signal. Figure 8C shows the ability of selected polypeptides of the invention to neutralize cell infection caused by virus-like particles (VLPs) pseudotyped with spike proteins from several SARS-CoV-2 variants of interest, including the Wuhan strain, D614G, B.1.1.7 (alpha), B.1.351 (beta) and B.1.617.2 (delta). The associated IC50 values ​​are listed in Table 6a. [Figure 9] Figure 7 shows the neutralization of live SARS-CoV-2 Wuhan strain virus by selected polypeptides of the present invention for the infection of VERO-E6 cells. A neutralizing monoclonal antibody was used as a positive control. The associated IC50 values ​​are listed in Table 7. [Figure 10A]Figure 10 depicts the in vivo effect of selected polypeptides of the invention following intravenous administration in hypertensive mice: Figure 10A shows the effect of selected polypeptides of the invention on systolic blood pressure measured by the tail cuff method 3, 6, 24, 48, and 72 hours after administration. [Figure 10B] Figure 10A depicts the in vivo effect of selected polypeptides of the invention after intravenous administration in hypertensive mice.Figure 10B depicts ACE2 activity in plasma and various organs 72 hours (endpoint) after administration of selected polypeptides of the invention, as assayed using the fluorogenic substrate Mca-APK(Dnp). [Figure 10C] Figure 10A shows the in vivo effect of selected polypeptides of the present invention after intravenous administration in hypertensive mice. Figure 10C shows ELISA immunoblotting for human ACE2 and IgG-Fc in plasma and various organs 72 hours after administration of selected polypeptides of the present invention (endpoint). [Figure 10D] Figure 10A-D show the in vivo effect of selected polypeptides of the invention after intravenous administration in hypertensive mice. Figure 10D shows the effect of treatment on albuminuria. ACR: urinary albumin / creatinine ratio. Data are presented as mean ± SEM, P<0.05, @ vs. saline, * vs. Ang II, # vs. rhACE2, & vs. Ang II+K, % vs. Ang II+M. n=3-6 / group. [Figure 11A] Figure 11A and 11B show the in vivo therapeutic efficacy of selected polypeptides of the invention following administration to hamsters infected with SARS-CoV-2 (Wuhan strain). Figures 11A and 11B show the change in body weight following intranasal administration as combined prophylactic and therapeutic dosing (Figure 11A) or following therapeutic dosing alone (Figure 11B). Male hamsters were challenged intranasally with 8x103 PFU of SARS-CoV-2 Wuhan isolate. Data plotted as mean + / - SEM. n=6-7 / group. [Figure 11B]Figure 11A and 11B show the in vivo therapeutic efficacy of selected polypeptides of the invention following administration to hamsters infected with SARS-CoV-2 (Wuhan strain). Figures 11A and 11B show the change in body weight following intranasal administration as combined prophylactic and therapeutic dosing (Figure 11A) or following therapeutic dosing alone (Figure 11B). Male hamsters were challenged intranasally with 8x103 PFU of SARS-CoV-2 Wuhan isolate. Data plotted as mean + / - SEM. n=6-7 / group. [Figure 11C] Figure 11C depicts the in vivo therapeutic efficacy of selected polypeptides of the invention after administration to hamsters infected with SARS-CoV-2 (Wuhan strain). Figure 11C shows the viable virus titer in lung tissue at day 3 post-infection after intravenous administration of a combined prophylactic (-4 h) and therapeutic (+24 h) dose of 10 mg / kg of selected polypeptides of the invention. Female hamsters were challenged intranasally with 104 PFU of SARS-CoV-2 Wuhan isolate. Viral titers were determined by plaque assay. Data plotted as median + / - SEM. n=7 / group.

[0034] Additional aspects and advantages of the present invention will become apparent in light of the following description. The detailed description and examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

[0035] [Detailed Description of the Invention] The present invention will be further illustrated in the following examples. However, it should be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.

[0036] Therefore, the following is a detailed description provided to assist those skilled in the art in carrying out the present disclosure.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs.The technical terms used in the description herein are only for describing specific embodiments and are not intended to limit the present disclosure.All publications, patent applications, patents, drawings and other references mentioned herein are expressly incorporated by reference in their entirety.

[0037] definition As used herein, the following terms may have the meanings given to them below unless otherwise specified. However, it should be understood that other meanings known or understood by those skilled in the art are also possible and fall within the scope of this disclosure. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0038] The term "about," as used herein, can be used to account for experimental error, measurement error, and variations that would be expected by a person of ordinary skill in the art. For example, "about" can mean plus or minus 10%, or plus or minus 5% of the referenced indication.

[0039] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0040] The word "and / or" as used herein should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified.

[0041] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" will be interpreted as inclusive, i.e., the inclusion of at least one, but also including two or more of a number or list of elements, and optionally additional unlisted items. The sole term "consisting of" when clearly indicated to the contrary or used in the claims, such as "only one of" or "exactly one of," will mean the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein will only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0042] As used herein, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "consisting of," and the like, shall be understood to be open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0043] As used herein, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that there may optionally be elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the specifically identified elements.

[0044] The term "sequence identity" as used herein means the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions x 100%). In one embodiment, the two sequences are the same length. The determination of the percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm used for the comparison of two sequences is the algorithm incorporated into the NBLAST and XBLAST programs

[16] . BLAST nucleotide searches can be performed using, for example, the NBLAST nucleotide program parameters for score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. BLAST protein searches can be performed using, for example, the XBLAST program parameters for score-50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in

[17] . Alternatively, PSI-BLAST can be used to perform an iterative search that detects distant relationships between molecules. When using BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the NCBI website).Another non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller

[18] . Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0045] A "substantially identical" sequence may include one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence can result in a variant peptide without substantial changes in physiological, chemical, physicochemical or functional properties compared to the reference sequence; in such cases, the reference sequence and the variant sequence will be considered to be "substantially identical" polypeptides. A conservative amino acid substitution is defined herein as the substitution of an amino acid residue with another amino acid residue having similar chemical properties (e.g., size, charge, or polarity).

[0046] In a non-limiting example, a conservative mutation can be an amino acid substitution. Such a conservative amino acid substitution can be a basic, neutral, hydrophobic, or acidic amino acid substitution from another amino acid of the same group. The term "basic amino acid" refers to a hydrophilic amino acid with a side chain pKa value greater than 7, which is typically positively charged at physiological pH. Basic amino acids include arginine (Arg or R) and lysine (Lys or K). The term "neutral amino acid" (also referred to as "polar amino acid") refers to a hydrophilic amino acid with a side chain that is uncharged at physiological pH but has at least one bond in which the pair of electrons shared by the two atoms is held relatively close by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine ​​(Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). The term "hydrophobic amino acid" (also referred to as "non-polar amino acid") is meant to include amino acids that exhibit a hydrophobicity greater than zero according to the standardized consensus hydrophobicity scale of

[19] . Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). "Acidic amino acid" refers to a hydrophilic amino acid with a side chain pKa value less than 7, which is typically negatively charged at physiological pH. Acidic amino acids include glutamic acid (Glu or E) and aspartic acid (Asp or D). Histidine (His or H) is a polar amino acid with a special ionization potential due to its pKa of around 7, more precisely around 6.4 in the case of histidine residues located on the protein surface

[20] . This results in histidine amino acid residues that are "polar" and predominantly uncharged at physiological pH of 7.2-7.4, and predominantly positively charged in acidic environments (pH<7).

[0047] A substantially identical sequence of the present invention may be at least 85% identical at the amino acid or nucleotide level to a sequence described herein; in another example, a substantially identical sequence may be at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical, or any percentage therebetween. Importantly, a substantially identical sequence retains the activity and specificity of the reference sequence. In a non-limiting embodiment, the difference in sequence identity may be due to conservative amino acid mutation(s). In a non-limiting embodiment, the difference in sequence identity may be due to synonymous nucleotide substitutions or nucleotide substitutions that result in conservative amino acid mutation(s). In a non-limiting example, the present invention may be directed to a polypeptide construct comprising an amino acid sequence that is at least 85%, 90%, or 95% identical to the polypeptide construct sequence shown in SEQ ID NO:23.

[0048] As used herein, the terms "peptide" and "polypeptide" refer to a linear chain of two or more amino acids linked by peptide bonds. The term "peptide" is generally used to refer to a short chain of amino acids, including 2-49 amino acids, while the term "polypeptide" is generally used to refer to a relatively long chain of amino acids, including 50 or more amino acids. However, these terms can be used interchangeably. The term "polypeptide construct" is used herein to refer to one or more peptides or polypeptides that have folded and / or assembled to form a three-dimensional structure, although protein and polypeptide construct can also be used interchangeably. Proteins can include post-translational modifications, as would be understood by one of skill in the art. For example, proteins can be glycosylated, lipidated, phosphorylated, ubiquitinated, acetylated, nitrosylated, and / or methylated.

[0049] As used herein, the term "recombinant polypeptide" refers to a polypeptide produced by recombinant techniques, typically in which DNA or RNA encoding the protein to be expressed is inserted into a suitable expression vector, which is introduced into a host cell to allow expression of the recombinant polypeptide. A recombinant polypeptide can contain amino acid sequences from two or more sources, such as different proteins. Such recombinant polypeptides may be referred to as fusion polypeptides. A recombinant polypeptide can also contain one or more synthetic amino acid sequences.

[0050] As used herein, the term "spacer" or "linker" refers to a peptide that directly and covalently links two polypeptides. The linker can be an amino acid or a peptide containing two or more amino acids. When the linker is an amino acid or a peptide, the N-terminus of the linker can be covalently linked to the C-terminus of the first polypeptide by a peptide bond, and the C-terminus of the linker can be covalently linked to the N-terminus of the second polypeptide by a peptide bond. Typically, the two polypeptides covalently linked by the linker are polypeptides that are not naturally linked, for example, the two polypeptides can be encoded by different genes and / or by different organisms, or can be different portions or domains of a single polypeptide or protein. In a non-limiting example, the linker or spacer can be less than 20 amino acids.

[0051] As used herein, the term "antigen" refers to any molecule, moiety or entity capable of eliciting an immune response. The immune response includes a cellular and / or humoral immune response. An antigen is generally a biological molecule, usually a protein, peptide, polysaccharide, lipid or conjugate, that contains at least one epitope to which a cognate antibody can selectively bind.

[0052] A "viral surface antigen" is an antigen, such as a polypeptide, that can be found on the surface of a virus. The viral surface antigen can be a trimeric viral surface antigen. Examples of trimeric viral surface antigens include, but are not limited to, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2) spike, SARS-CoV-1 spike, Middle East Respiratory Syndrome (MERS)-CoV spike, influenza hemagglutinin (HA), human immunodeficiency virus (HIV) gp120, respiratory syncytial virus (RSV) RSV F protein, rabies virus glycoprotein (RABVG), and human metapneumovirus (hMPV) glycoprotein.

[0053] As used herein, the term "pharmaceutical acceptable carrier" refers to a carrier that is non-toxic.Suitable pharmaceutical acceptable carriers include, for example, one or more of water, physiological saline, phosphate buffered saline, dextrose, glycerol, ethanol, and combinations thereof.Pharmaceutical acceptable carriers may further contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance shelf life or effectiveness.

[0054] As used herein, with respect to a molecule such as a nucleic acid molecule or a polypeptide, the term "fragment" means a portion of the molecule that is less than the full length of the molecule.

[0055] As used herein, the term "subject" means a human or non-human animal, such as a mammal, bird, reptile, fish, or amphibian.

[0056] As used herein, the term hinge fragment, C H 2 domain and C H The three domains refer to the corresponding regions of an IgG antibody heavy chain, having their nucleotide and protein sequences as defined and numbered according to the ImMunoGeneTics (IMGT) database (http: / / www.imgt.org / ) [21-23]. Hinge fragment, C H2 domain and C H A preferred, non-limiting embodiment of the three domains is derived from a human antibody, and preferably the human IgG1 isotype.

[0057] It should also be understood that, for certain methods described herein that include two or more steps or actions, the order of the method steps and actions is not necessarily limited to the order in which the method steps or actions are recited, unless the context dictates otherwise.

[0058] To address the problems in the art, the present invention provides an improved ACE2-based decoy variant of the human ACE2 receptor that carries an isoleucine (Ile) amino acid residue at position 92, where a threonine (Thr) amino acid residue is normally found in the more common ACE2 receptors that are widespread in the human population.The present invention uses ACE2I92 as a starting point in a multi-faceted molecular design effort, which now provides a class of polypeptide constructs that carry (a) strong binding avidity and affinity to spike proteins for efficient neutralization of viral infection, (b) high enzymatic activity for reduced ARDS, and (c) improved biological manufacturability; meanwhile, (d) suitable pharmacokinetics to allow viral clearance while preventing antibody-dependent enhancement (ADE), and (e) structural components for protection against newly emerging viral strains and future pandemics.

[0059] The present invention relates to a compound having the general formula: R1-R2-R3-R4 providing a polypeptide construct having Wherein R1 is hACE2I 92 (18-614), X 27 ,X 261 ,X 330 Contains X 27 is Thr or Tyr, and X 261 is Cys or Ser, and X 330 is Asn or Tyr; R2 comprises a spacer or linker; R3 is Hinge S 220 ,X 226 ,X 229 Contains;X 226 and X 229 is Cys or Ser; and R4 is C H 2G 270 -C H 3, including The structural features and designed advantages of each of the four regions are presented and described in detail in the following section entitled "Molecular Engineering of Polypeptide Constructs" in Example 1. Experimental data demonstrating the advantages of the designed polypeptide constructs of the present invention are presented in the following section under Examples 2-7. Non-limiting illustrative examples of the present disclosure are provided in SEQ ID NOs: 15-29, more specifically SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28.

[0060] Example 1. Molecular Engineering of Polypeptide Constructs Structure-based modular design for optimizing avidity against the SARS-CoV-2 spike homotrimer FIG. 2 illustrates the structural domains utilized in the modular design of the polypeptides of the invention. The molecular model of the SARS-CoV-2 spike homotrimer was taken from the cryo-EM structure with PDB ID 6VSB. The human ACE2 ectodomain (18-740) homodimer, including the catalytic and neck (collectrin) domains in complex with the SARS-CoV-2 receptor binding domain (RBD), was taken from the cryo-EM structure with PDB ID 6M17. Hinge, C H 2 and C H The human IgG1 Fc fragment homodimer containing three domains was obtained from the crystal structure of human IgG1 mAb with PDB ID 1HZH. Structure manipulation, visualization and rendering were performed using the PyMOL Molecular Graphics System (Schroedinger, LLC).

[0061] As a first step in this modular design, we determined that the neck (collectrin) domain functions as a non-covalent dimerization domain and locks the ACE2 catalytic domain (R1 region) in a rigid mutual orientation that we determined in this study to be incompatible with simultaneous binding to the two RBD domains of the spike protein homotrimer, and therefore the neck domain was removed, as shown in Figure 2. The next step was the construction of a human IgG1 C domain that non-covalently assembles the remaining human ACE2 catalytic domain (R1 region) as a homodimer via a flexible polypeptide spacer (R2 region) followed by a flexible human IgG1 hinge (R3 region). H 2-C H The aim of the present invention was to link the two R1 regions of the homodimeric polypeptide constructs of the present invention to the two RBD domains of the same viral spike protein homotrimeric molecule, as well as to the two RBD domains of different spike protein homotrimers that are adjacent to each other on the virion surface. Therefore, using the unique design platform of the polypeptide constructs of the present invention, residues that confer high binding avidity and affinity to the viral spike protein, as determined by ADAPT affinity maturation (see below), were selected. It is understood that the deletion of the collectrin (neck) domain is non-limiting, and utilizing the full-length hACE2(18-740) ectodomain in the design scheme described in Figures 1 and 2 is an obvious modification that one skilled in the art would understand, and can retain significant levels of enzymatic activity and virus neutralization for the polypeptide constructs of the present invention.

[0062] Additional sequence optimization in each of the four regions of the polypeptide construct The sequence variations described in this paragraph are shown diagrammatically in Figure 1. First, in the ACE2 catalytic domain (R1 region), a naturally occurring human mutation T92I [24-26] was selected. This natural mutation is N 90 XT 92 It is predicted that the ACE2I92 partially deglycosylated natural variant will eliminate the N-glycosylation sequon and thus the N-glycosylation of Asn90. The first advantage of utilizing this partially deglycosylated natural variant in our design is that it results in a relatively low molecular weight of the polypeptide construct of the present invention, which may improve tissue penetration. Furthermore, as will be shown for the first time herein, the ACE2I92 partially deglycosylated natural variant confers an improved activity profile to the polypeptide construct of the present invention, both in terms of increased enzymatic activity and increased virus neutralization potency. Importantly, the natural occurrence of the Ile amino acid at position 92 of human ACE2 eliminates immunogenicity concerns that may arise due to the protein surface exposure resulting from the removal of the carbohydrate structure at Asn90.

[0063] To evaluate the effect on the flexibility and relative freedom of movement of the two independent ACE2 catalytic domains (R1 regions) in the homodimer polypeptide construct, two different lengths of spacers (R2 regions) were designed; a 5-residue spacer SGGGG, and a 15-residue spacer SGGGGSGGGGSGGGG. Geometric measurements were performed in PyMOL on the molecular model to ensure that the relatively short spacers allow the two independent ACE2 catalytic domains (R1 regions) to fit into the homodimeric polypeptide construct without steric hindrance.

[0064] The human IgG1 hinge (R3 region) was also modified. First, we mutated the unpaired Cys residue at hinge position 220, which is naturally involved in disulfide bridges to the antibody light chain, aiming to reduce the possibility of forming undesired covalent multimeric species via Cys220, which was absent in our construct and was therefore replaced by a Ser amino acid. Furthermore, we designed a variant in which the remaining two Cys residues in the native human IgG1 hinge region at hinge positions 226 and 229 were additionally replaced by Ser residues. These Cys residues may also give rise to undesired covalent multimeric species that affect the manufacturability of Fc fusion proteins, since these Cys residues normally form disulfide bridges between the two heavy chains in the Fc fragment homodimer [27, 28]. Structural features were explored to reduce sample heterogeneity during large-scale manufacturability. It was recalled that the hinge mutant R3 region lacking Cys residues results in a homodimeric variant, which is mainly composed of C H It is stabilized only by non-covalent inter-polypeptide chain interactions through three-domain homodimerization, and therefore not by inter-polypeptide chain covalent disulfide bonds. In the same spirit, certain embodiments of the present invention also include a surface-exposed unpaired Cys residue at position 261 of the ACE2 catalytic domain (R1 region) that is mutated to a Ser residue.

[0065] Finally, human C from the R4 region H The 2 domain was selected to contain the mutation D270G, which can attenuate immune effector functions through reduced binding to FcγR receptors and the Cq complement complex. This modification aims to reduce certain side effects of antibody treatment of viral infections, namely: (i) the antibody-dependent enhancement (ADE) effect, which has been shown to exacerbate pathology in certain viral infections

[29] , and (ii) increased inflammation in organs and tissues already affected by acute respiratory distress syndrome (ARDS) due to COVID-19 infection [30-32].

[0066] ADAPT affinity maturation against SARS-CoV spike RBD In addition to the sequence improvements and modifications described above, the human ACE2 catalytic domain (R1 region) was further engineered by incorporation of residues identified by affinity maturation against the SARS-CoV-2 spike protein. The starting point for affinity maturation was the atomic coordinates of hACE2 bound to the SARS-CoV-2 spike protein receptor-binding domain (RBD), taken from the cryo-EM structure with PDB ID 6M17 [4]. Only one copy of human ACE2 catalytic domain residues 21–615 and SARS-CoV-2 spike RBD residues 336–518 were retained, and all other atoms were removed. Hydrogen atoms were added to the complex and adjusted to maximize H-bond interactions. Structural refinement of the complex was subsequently performed by energy minimization using the AMBER force field with distance-dependent electrostatic and infinity-point cutoffs for non-bonded interactions [33, 34]. Non-hydrogen atoms were added at 40 and 10 kcal / (mol A for backbone and side chain atoms, respectively. 2 ) were constrained to their crystallographic positions. The ADAPT platform was then used for affinity maturation [35, 36]. Single-point scanning mutagenesis simulations were performed at 57 positions in the ACE2 catalytic domain (R1 region) that may affect binding affinity to the spike RBD upon mutation. We used the ADAPT protocol to build structures and evaluate the energies of single-point mutations to 17 other possible natural amino acids (Cys and Pro were excluded) at these positions of the parent hACE2 structure. A consensus approach across specific versions of these three protocols

[37] was applied to build and score the hACE2 variants. Scoring of binding affinity was primarily based on the average Z-score for scores calculated using the three component energy functions. Prior to binding affinity prediction, mutations predicted to destabilize the correct folding of the ACE2 catalytic domain were discarded from further evaluation.

[0067] Selection of the most likely single point mutants with improved spike RBD binding affinity was primarily guided by the top 30 consensus Z-scores from ADAPT (Table 1). Visual inspection of molecular interactions predicted using the three sampling protocols of ADAPT was used to detect suboptimal complementarity at the antibody interface (e.g., burial of polar groups in a non-polar environment), steric crowding and distortion in the covalent geometry (e.g., deviation of aromatic rings from planarity), and replacement of native Gly residues with their main-chain torsion angles in the rejected region of the Ramachandran plot. Two positions, Thr27 and Asn330, were finally selected where aromatic substitutions were predicted to improve binding affinity to the parent ACE2 structure with high probability. Among those possible substitutions, we chose Tyr as the replacement residue given its increased solubility relative to the other predicted substitutions Phe and Trp (Figure 3). In the polypeptide constructs of the invention, the T27Y and N330Y mutations are used either alone or in combination, preferably also in combination with the sequence variations described in the preceding sections.

[0068] [Table 1]

[0069] Example 2. Production of designed polypeptide constructs in CHO cells The various polypeptide constructs of the present invention contain the native signal sequence of human ACE2 at their N-terminus. DNA coding regions for the constructs were prepared synthetically (GenScript) and cloned into the HindIII (5' end) and BamH1 (3' end) sites of the pTT5 mammalian expression plasmid vector

[38] . Fusion proteins were produced by transient transfection of Chinese Hamster Ovary (CHO) cells. Briefly, plasmid DNA was transfected into 0.5 L or 1 L cultures of CHO-55E1 cells. Transfections were performed at 1.8 x 10 with viability of >98%. 6 ~2.0×10 6The transfection was performed at a cell density of 100000 cells / mL. Cells were distributed into 1.0 L-2.8 L shake flasks and transfected with 1 μg total DNA per mL of product using PEI MAX™ (Polysciences, Inc., Warrington, PA). The final DNA:PEI MAX™ ratio was 1:4 (w / w). Cell cultures were incubated for 24 hours on an orbital shaking platform at 37°C with an agitation speed of 110 rpm in a humidified 5% CO2 atmosphere. After 24 hours, cultures were fed with tryptone N1 at a final concentration of 1% w / v and valproic acid sodium salt at a final concentration of 0.5 mM and transferred to 32°C for 6 days. Cell density and cell viability were determined by direct counting of cell samples with a Vi-CELL automated cell counting system (Beckman Coulter Life Sciences, Indianapolis, IN) using the trypan blue dye exclusion method.

[0070] The polypeptide constructs were produced in yields ranging from 104 to 328 mg / L, with a typical yield of approximately 300 mg / L for the majority of variants. This demonstrates that the polypeptide constructs of the invention can be efficiently produced by transient transfection in CHO cells. Further yield increases are expected by selection and propagation of pools of CHO cells with the best expression of specific variants.

[0071] Example 3. Purification of polypeptide constructs by affinity chromatography Purification from cell culture supernatant was performed by Protein A affinity chromatography. Cell culture supernatant was loaded onto a 5 mL HiTrap MabSelect™ SuRe™ column (GE Healthcare Life Sciences) equilibrated in DPBS. The supernatant was loaded using a linear flow rate for the binding set of approximately 45 cm / h (1.7 mL / min), resulting in a residence time of approximately 2.9 minutes. The column was washed with DPBS and the protein was eluted with 0.1 M citrate buffer pH 3.6 or 0.1 M acetate buffer pH 3.7. Neutralization was performed with 10% (v / v) 1 M HEPES. The neutralized elution pool was buffer exchanged using a Zeba spin column and sterile filtered. Using a citrate pH 3.6 elution buffer, the tested samples of variants carrying cysteines at positions 226 and 229 in the hinge (R3 region) and at position 261 in the ACE2 catalytic domain (R1 region) contained approximately 50% homodimers according to SDS-PAGE studies under denaturing non-reducing conditions, with the remainder being aggregates of high molecular weight (HMW) species, the majority of which disappeared under reducing conditions (Figure 4A, left). This level of heterogeneity was confirmed by analytical UPLC-SEC on a high-resolution BEH-450 column (Figure 4B). However, the homogeneity of the preparation was improved by using acetate pH 3.7 as the elution buffer. For variants with mutated cysteines either at both hinge positions 226 and 229 (ACE2m4-hinge2CS-SG4-Fc; SEQ ID NO: 23) or at position 261 in the ACE2 catalytic domain (ACE2m4-C261S-SG4-Fc; SEQ ID NO: 22), there are no HMW species, as can be seen from the denaturing SDS-PAGE data under non-reducing conditions in Figure 4A (right side). As expected, the ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) variant appears as a monomer under denaturing non-reducing conditions due to the absence of a covalent disulfide between the hinge regions of the native homodimer.Furthermore, analytical UPLC-SEC data on the BEH-450 column shown in FIG. 4C suggests that the elution buffer is the main driver of improved homogeneity of the purified sample (approximately 50% homodimers using citrate pH 3.6 elution buffer vs. approximately 70% homodimers using acetate pH 3.7 elution buffer) and not mutation of cysteine ​​residues. This is evident from variant ACE2-SG4-Fc (SEQ ID NO: 15), which was tested with both elution buffers and shows that the size and amount of HMW species is reduced with acetate pH 3.7 elution buffer (FIG. 4C) compared to citrate pH 3.6 elution buffer (FIG. 4B). We also observed that mutating C261 to Ser in the ACE2 catalytic domain (R1 region) did not further improve homogeneity (using acetate pH 3.7 elution buffer), containing 72% homodimers. However, a novel finding was that when the two Cys at positions 226 and 229 of the hinge (R3 region) were both mutated to Ser, the homogeneity improved significantly, resulting in 88% homodimers with only 12% HMW species of relatively small size (Figure 4C). Furthermore, the MW determined by MALS-RI analysis for the homodimeric species of this purified sample of ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) variant was 198 kDa, which is in good agreement with the theoretical homodimer MW of 190 kDa, with the difference likely being due to glycosylation. All these results indicate that using acetate pH 3.7 elution buffer and mutating the cysteines at positions 226 and 229 of the human IgG1 hinge are important to achieve a reasonably high homogeneity of about 90% by Protein A purification for this class of polypeptides.

[0072] All Protein A purified samples were then further purified by preparative SEC on a Superose 6 Increase column (GE Healthcare Life Sciences). Selected peak fractions were concentrated by ultrafiltration using Vivaspin® 6 centrifugal concentrators with a membrane molecular weight cutoff of 10 kDa (GE Healthcare Life Sciences) at 15° C. according to the manufacturer's instructions. During the process, protein concentration was monitored with a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA) using the absorbance at 280 nm and the calculated specific extinction coefficient of each variant. SDS-PAGE analysis of denatured purified variants shows highly pure homodimers for all polypeptide constructs under both non-reducing and reducing conditions ( FIG. 5A ). The ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) variant appears as a monomer under denaturing non-reducing conditions due to the absence of a covalent disulfide between the hinge regions of the native homodimer. The high purity was subsequently confirmed by analytical UPLC-SEC on a high-resolution BEH-450 column, with representative chromatograms shown in FIG. 5B and the homodimer fractions for all variants listed in Table 2. It is important to note that both ACE2m4-SG4-Fc (SEQ ID NO: 21) and ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) have nearly identical UPLC-SEC chromatograms, both indicative of homodimers, despite the differences between these variants in the presence (in the former variant) or absence (in the latter variant) of interpolypeptide chain disulfide bridges at the level of the hinge (R3 region). As shown in FIG. 5B and Table 2, the determined MW for the homodimers of these molecular species closely matches the glycosylated protein with a calculated protein MW of 190 kDa.Of note, as shown in Table 2, the determined MW for variants based on the natural variant of human ACE2 Ile92 lacking carbohydrate at position Asn90 (SEQ ID NOs: 19, 20, 21, 22 and 23) is smaller than those with glycosylation at Asn90 (SEQ ID NOs: 15, 17 and 18).

[0073] Samples purified by preparative SEC were further analyzed by sedimentation velocity analytical ultracentrifugation (SV-AUC) performed on a Beckman Proteomelab XL-I using an AN-50 8-hole rotor while monitoring absorbance at 280 nm at a protein concentration of 1 mg / mL in PBS. A cell containing a two-sector charcoal epon centerpiece with 3 mm path length and sapphire windows was used. Proteins were centrifuged at 45000 rpm and scans were taken every 4 min. c(s) distributions were obtained using SEDFIT software and integrated using GUSSI software. The SV-AUC data tabulated in Table 2 show that purified samples of the tested polypeptide constructs have one major peak at approximately 8S, which accounts for 88-97% of the protein peak area and is attributed to homodimeric species. The content of HMW species is very low. The friction coefficients for these samples ranged from 1.8 to 2.1, implying an asymmetric conformation for these homodimeric assemblies, which is consistent with the engineered conformational flexibility described above. Figure 5C shows similar sedimentation coefficient distributions for a pair of variants that differ only in the presence or absence of a covalent disulfide bond between the polypeptide chains in the hinge (R3 region): ACE2m4-SG4-Fc (SEQ ID NO: 21) versus ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23).

[0074] [Table 2]

[0075] Example 4. Stability characterization of polypeptide constructs Freeze-thaw cycle Purified samples of the polypeptide constructs of the present invention were subjected to a freeze-thaw stability stress test consisting of three cycles of freezing at -80°C and thawing for 30 minutes. Sample heterogeneity was determined before and after each freeze-thaw cycle using analytical UPLC-SEC on a BEH-200 column. All samples were highly robust to freeze-thaw stress. The homodimer fraction determined after the third freeze-thaw cycle is listed in Table 3. Samples before stress and after the third freeze-thaw cycle were also tested for SARS-CoV-2 spike RBD binding using SPR (see below). Similarity scores comparing the SPR sensorgrams before and after freeze-thaw stress are recorded in Table 3 and show very similar binding levels. Taken together, these data indicate that the polypeptide constructs of the present invention are highly robust to freeze-thaw stress and can be stored at -80°C.

[0076] [Table 3]

[0077] Folding stability The inventors were also interested in finding out whether there are differences in the stability of the various structured domains of this class of polypeptide constructs. To this end, differential scanning calorimetry (DSC) was used to measure the temperature transition midpoints (T m) was determined. DSC experiments were performed using a VP-capillary DSC system (Malvern Instruments Ltd, Malvern, UK). Samples were diluted in HyClone™ Dulbecco's Phosphate Buffered Saline (DPBS; GE Healthcare Life Sciences) to a final concentration of 0.4 mg / mL. Thermal denaturation was performed under 70 psi nitrogen pressure by increasing the temperature from 20°C to 100°C at a rate of 60°C / h, with feedback mode / gain set to "low", a filtering time of 8 seconds, and a prescan time of 3 minutes. All data were analyzed using Origin 7.0 software (OriginLab Corporation, Northampton, MA). Thermograms were corrected by subtraction of the corresponding buffer blank scan and normalized to protein molar concentration. T m Let the value be T m The peaks were determined using automated data processing with a rectangle peak finder algorithm for . The data listed in Table 3 show that all variants have transitions at both .50°C and .82°C of roughly equal enthalpy that confer two common features. The two common structural features of these compounds are the ACE2 catalytic domain (R1 region) and the C H 2-C H The C3 domain (R4 region) of the antibody IgG1 Fc fragment H The ACE2 catalytic domain is known to have a melting temperature of approximately 82 °C. The ACE2 catalytic domain corresponds to a transition at around 50 °C

[39] . m Data-based structure-stability relationship analysis showed that the N330Y mutation present in some variants reduces the T of the ACE2 catalytic domain by approximately 2°C. m This suggests that this is the cause of the decline in

[0078] Example 5. Enzymatic activity of polypeptide constructs Catalytic efficiency The enzymatic activity of the polypeptide constructs of the invention was assayed using the fluorogenic substrate Mca-APK(Dnp) (AnaSpec, San Jose, CA, Cat#: AS-60757) as previously described

[40] . Recombinant human ACE2 (rhACE2) was used as a positive control (R&D Systems Inc., Minneapolis, MN, Cat#: 933-ZN) and a blank was used as a negative control. The assay buffer contained 50 mM 2-(N-morpholino)ethanesulfonic acid (MES), 300 mM NaCl, 10 μM ZnCl2, pH 6.8. The variants and rhACE2 control were tested at a concentration of 100 ng / mL, and the substrate concentrations were 1, 2, 4, 6, 8 and 16 μM. Samples were assayed using 10 -5 M ACE2 inhibitor MLN-4760 (Calbiochem, Cat#:530616) was used with or without the assay in triplicate. Reactions were followed kinetically on a FLUOstar Galaxy fluorometer (BMG Labtechnologies, Durham, NC, USA) using excitation at 320 nm to detect emission at 405 nm, with readouts every 62 seconds for up to 32 minutes. Data were fitted and plotted using Grafit (Sigma-Aldrich). Relative fluorescence units (RFU) were subtracted for samples with MLN-4760 and converted to product concentration units (μM) based on a standard curve of product formation. Catalytic efficiency k cat / K M For the calculation of k, the initial velocity (V), determined from the linear phase of product formation over time, was plotted against the substrate concentration. The linear phase of the V versus substrate concentration plot was used to calculate k by dividing V by the substrate concentration at substrate concentrations below 6 μM. cat / K M [The inventors gratefully acknowledge that some of the technical data regarding the catalytic efficiency assay was kindly provided by the laboratory of Dr. Kevin Burns (OHRI, University of Ottawa, Ottawa, Canada)].

[0079] Catalytic efficiency k cat / K M are listed for all tested polypeptide constructs in Table 4, and V versus substrate concentration plots are shown for the rhACE2 positive control and selected variants in Figure 6A. As can be seen from these data, all polypeptide constructs of the present invention exhibited a 2.67 x 10 5 M -1 s -1 k cat / K M The same order of magnitude (10 5 M -1 s -1 ) with high catalytic efficiency. This indicates that the structural changes introduced during the molecular design and optimization as described in Example 1 do not have a major impact on the catalytic activity. Furthermore, structure-activity relationship analysis of the enzyme activity data reveals the important and unexpected role of the T92I spontaneous mutation in increasing the ACE2 enzyme activity in our novel construct. This effect is best highlighted by comparing the variant ACE2m1-SG4-Fc (SEQ ID NO: 18) with Thr at position 92 with ACE2m4-SG4-Fc (SEQ ID NO: 21) with Ile at position 92, the only difference between these variants being this spontaneous mutation and the resulting loss of the carbohydrate structure linked at Asn90. Further support for this finding is provided by the variant ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) with exactly the same ACE2 catalytic domain (R1 region) as ACE2m4-SG4-Fc (SEQ ID NO: 21). The higher catalytic efficiency of ACE2m4-SG4-Fc (SEQ ID NO: 21) and ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) compared to ACE2m1-SG4-Fc (SEQ ID NO: 18) is immediately apparent from FIG. 6A and Table 4.

[0080] Specific activity We also characterized the specific activity of the polypeptide constructs and compared them to that of the rhACE2 positive control. Specific activity was determined using an Angiotensin-Converting Enzyme (ACE2) Fluorimetric Activity Assay Kit (BioVision, Milpitas, CA, Cat#: K897-100). We replaced the enzyme provided in the kit (concentration not disclosed) with the commercially available rhACE2 used in the catalytic efficiency study above. The manufacturer's protocol was followed. The assay was performed in 96-well format in duplicate. The concentrations of all polypeptide constructs ranged from 1 nM to 0.03125 nM with 2-fold serial dilutions. The reactions were followed kinetically on a Cytation 5 instrument (BioTek) with excitation at 320 nm and emission at 420 nm. For the calculation of specific activity, two time points in the linear range of the activity time course plot were selected with enzyme concentrations of 0.25 nM (time points 20 and 25 min) and 1 nM (time points 5 and 10 min), which correspond to the linear phase of the plot (Figure 4B). The duplicate values ​​for each time point were averaged and normalized to zero based on the blank data. Specific activity (pmol / min / mg) was then calculated by dividing the change in RFU between the time points by the corresponding time range and amount of enzyme used. The final specific activity data reported in Table 4 are the average between the data at the two enzyme concentrations (0.25 nM and 1 nM) normalized to the specific activity of the rhACE2 control on each plate.

[0081] The polypeptide construct of the present invention is 6It is readily apparent that the ACE2-SG4-Fc variants have high specific activities, on the order of pmol product / min / mg enzyme (FIG. 6B and Table 4), and are in the range of 68-89% compared to the rhACE2 control. In agreement with the catalytic efficiency data presented in the preceding section, mutations at positions 27 and 330 of the ACE2 catalytic domain (R1 region) slightly reduce enzymatic activity, as can be seen for variant ACE2m1-SG4-Fc (SEQ ID NO: 18). However, specific activity is restored by the addition of the naturally occurring mutation to Ile at position 92, accompanied by removal of the N-linked carbohydrate at position 90 of the ACE2 catalytic domain: compare ACE2m4-SG4-Fc (SEQ ID NO: 21) and ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) variants to ACE2m1-SG4-Fc (SEQ ID NO: 18).

[0082] Taken together, the catalytic efficiency and specific activity data of the polypeptide constructs of the present invention suggest that these compounds will be effective in restoring the protective effects of the alternative renin-angiotensin system by efficiently converting Ang-II to Ang(1-7), and therefore will function as therapeutic agents to reduce the severity of ARDS.

[0083] [Table 4]

[0084] Angiotensin II hydrolysis activity ACE2 enzyme activity was also determined directly by incubation with angiotensin II (Ang II) and subsequent measurement of angiotensin-(1-7) (BMA biomedicals, Cat#: S-1330). Selected polypeptides of the invention or control enzymes (rhACE2, rhACE) at 0.5 μg / mL were added to 10 -5The Ang II hydrolysis activity of all tested polypeptides of the present invention was significantly higher than that of the negative control human recombinant ACE (rhACE) and comparable to that of the positive control commercial recombinant human ACE2 (rhACE2). [The inventors gratefully acknowledge the gracious provision of some of the technical data on angiotensin II hydrolysis activity by the laboratory of Dr. Kevin Burns (OHRI, University of Ottawa, Ottawa, Canada)].

[0085] Enzyme activity and stability in primary cultures of mouse proximal tubular epithelial cells. Proximal tubular epithelial cells (PTECs) were isolated from 12-16 week old C57BL / 6 male and female mice obtained from Charles Rivers (Saint-Constant, Quebec, Canada). Isolations were performed as previously described

[41] . For each isolation, kidneys from one male and one female mouse were harvested in chilled perfusion solution [containing (in mM) 1.5 CaCl2, 5.0 D-glucose, 1.0 MgSO4, 24 NaHCO3, 105 NaCl, 4.0 sodium lactate, 2.0 Na2HPO4, 5.0 KCl, 1.0 L-alanine, 10 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), and 0.2% bovine serum albumin (BSA)]. Kidney cortices were minced and digested in perfusion solution supplemented with 0.1% collagenase (Sigma-Aldrich, Cat#: C9262) and 0.05% soybean trypsin inhibitor (Sigma-Aldrich, Cat#: T6522), pH 7.2. The cortical digest was passed through a 250 μm sieve, pelleted, and resuspended in 40% Percoll (Sigma-Aldrich, Cat#: P1644) solution containing (mM): 5.0 D-glucose, 10 HEPES, 1.0 MgCl2, 120 NaCl, 4.8 KCl, 25 NaHCO3, 1.0 NaH2PO4, 1.0 L-alanine, 1.4 CaCl2, 60 U / mL penicillin, and 60 μg / mL streptomycin. The digested products were centrifuged at 18,500×g for 30 min. After centrifugation, the fourth layer containing PT cells was aspirated, pelleted, and resuspended in culture medium (DMEM / F12-Gibco, Cat#: 31600034 and 21700075). Cells were seeded on 24-well plates and cultured for 24 hours in DMEM / F12 (1:1) medium containing 10% fetal bovine serum (FBS) and chemically defined medium (5 μg / mL insulin transferrin sodium selenate, 50 nM hydrocortisone, 2 nM 3,3',5-triiodo-L-thyronine (Sigma Aldrich Cat#: I1884, H0888, T5516), 100 U / mL penicillin, and 100 mg / mL streptomycin).After 24 hours, cells were grown in DMEM / F12 and chemically defined media. On day 6 (approximately 70% confluence), PTECs were treated with 0.6 nM rhACE2, polypeptides of the invention, rhACE (negative control), or phosphate-buffered saline (PBS, control) for 24 hours. After treatment with the variants, angiotensin II or Mca-APK (Dnp) was added to each well to a final concentration of 5 nM Ang II or 11.25 μM Mca-APK (Dnp). For PTECs treated with Ang II, media was collected and assayed for angiotensin-(1-7) by ELISA (BMA Biomedicals, Cat#: S-1330). PTECs incubated with fluorescent substrate were removed from each well in 100 μL and transferred to a black 96-well plate, and fluorescence was measured as described for ACE2 enzyme activity.

[0086] To test the activity and stability of ACE2-Fc variants in vitro as described above, cultured primary mouse proximal tubule cells were treated with selected polypeptides of the invention, enzyme controls (rhACE2, rhACE) or PBS for 24 hours, followed by incubation with either fluorogenic substrates (Figure 6D) or Ang II (Figure 6E). All tested polypeptides of the invention remained active after 24 hours at 37°C, and their activity with either fluorogenic substrates (Figure 6D) or Ang II (Figure 6E) was significantly higher than and comparable to that of the negative control (rhACE2). [The inventors gratefully acknowledge some of the technical data regarding enzyme activity in primary cell cultures kindly provided by the laboratory of Dr. Kevin Burns (OHRI, University of Ottawa, Ottawa, Canada)].

[0087] Example 6. Binding of polypeptide constructs to SARS-CoV-2 spike protein Dissociation rate of spike from RBD by SPR The binding kinetics of the polypeptides of the invention to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein was performed by surface plasmon resonance (SPR) experiments on a Biacore T200 instrument (GE Healthcare) in PBST pH 7.4 running buffer (PBS containing 0.05% Tween 20 and 3.4 mM EDTA) at 25 °C. In-house produced

[42] SARS-CoV-2 S-RBD Wuhan strain and B.1.315 variant as well as SARS-CoV-1 S-RBD were immobilized via amine coupling onto a CM-5 sensor chip at 200 RU. Single cycle kinetics were determined by injecting each polypeptide construct variant at 3-fold serial dilutions (maximum nominal concentrations of 30, 60 or 120 nM) and buffer blanks were simultaneously injected over the blank and S-RBD surfaces at 50 μL / min for 120 s with a dissociation phase of 900 s. Regeneration was performed for 30 seconds with 10 mM glycine pH 1.5 at a flow rate of 30 μL / min. Data analysis was performed using Biacore T200 evaluation software. Dual-referenced sensorgrams were baseline aligned and normalized to the end of the 60 nM injection for off-rate ranking. Off-rate k off (M -1 s -1 ) was determined from a 1:1 fit of the dual-reference data and is tabulated in Table 5. Avidity arises due to the bivalent nature of the flowed polypeptide construct, and the density of the immobilized S-RBD is proportional to the thermodynamic equilibrium constant K D This prevented the decision from being made.

[0088] The aligned normalized sensorgrams obtained for the tested polypeptide constructs of the present invention are shown in Figure 7A, which are useful in ranking the dissociation rates of these compounds from the SARS-CoV-2 RBD. The dissociation rate is typically considered to be the main criterion in selecting the best binders for further therapeutic development. It is immediately clear from Figure 7A that the ADAPT affinity changes at positions 27 and 330 in conjunction with the natural mutation at position 92 of the ACE2 catalytic domain (R1 region) result in a significantly reduced dissociation rate, thus reflecting an increased interaction with the SARS-CoV-2 domain, compared to variants with a non-optimized ACE2 catalytic domain: ACE2-SG4-Fc (SEQ ID NO: 15) and ACE2-SG4x3-Fc (SEQ ID NO: 17). The slowest dissociation rates were observed for the three ACE2m4 variants (SEQ ID NO: 21, 22 and 23), closely followed by the ACE2m1-SG4-Fc (SEQ ID NO: 18) and ACE2m3-SG4-Fc (SEQ ID NO: 20) variants, and then by ACE2m2-SG4-Fc (SEQ ID NO: 19), which showed a relatively fast k off and this k off is still much slower than for the unoptimized ACE2 variant. The structure-activity relationship of the dissociation rates in Table 5 points out a greater role of the N330Y substitution for improving the binding interaction to the SARS-CoV2 RBD than that of the T27Y substitution. The slowest dissociation rate obtained in this series of polypeptide constructs was 10 -6 s -1 This shows very tight binding.

[0089] Binding competition to spike RBD by snELISA A unique and clinically relevant surrogate neutralization (sn) ELISA-based assay

[43] was also used as an additional method for in vitro testing of the binding capacity of the polypeptide constructs of the invention against the SARS-CoV-2 spike protein. In this assay, spike RBD was immobilized on a multi-well plate and subsequently incubated with the subject samples to be tested for neutralization capacity. Biotinylated hACE2 was then added and detected by streptavidin-polyHRP. The ability of the sample to block the interaction of biotinylated hACE2 with the antigen can be measured by a dose-dependent decrease in signal. We used 100 ng of immobilized recombinant RBD on a 96-well Immulon HBX plate and incubated the plate overnight at 4 °C (2 μg / mL). All volumes added to the wells were 50 μL. Plates were washed three times with 200 μL PBS-T and blocked with 200 μL 3% BSA (BioShop Canada Inc., SKI400.1) for 1–1.5 h at room temperature. After 4 steps of washing as above, two-fold serial dilutions of patient serum or plasma (0.5–4 μL samples) were incubated for 1 h. Wells were washed as above and incubated with 50 ng biotinylated recombinant human ACE2 for 1 h. After washing as above, wells were incubated with 44 ng streptavidin-peroxidase polymer (MilliporeSigma, S2438). The resulting signal was developed with TMB and quantified. Due to interday variability in the signal, all ODs were 450 Values ​​were calculated from the OD of wells to which no sample was added for each sample. 450 All values ​​are expressed in this ratio space.

[0090] The IC90 values ​​obtained using the snELISA assay are listed in Table 5, and the dose-response curves are shown in Figure 7B. offThere is a quantitative agreement between the dissociation rates. The snELISA experiments thereby further confirm that the structural optimization performed in the ACE2 catalytic domain of the polypeptide constructs of the present invention significantly improves the binding properties to the SARS-CoV-2 spike RBD compared to the unoptimized variant. An important aspect that is evident from Figure 7B is that these compounds are able to reduce the binding to biotinylated ACE2, which is used as a competitor in the assay, to very low levels at relatively high doses. This strongly suggests that these compounds are able to completely neutralize the SARS-CoV-2 virus and prevent its binding to ACE2 on the host cell. This indicates that these compounds can be used as potent antiviral agents.

[0091] [Table 5]

[0092] Binding to SARS-CoV variants In addition to evaluating the dissociation rate from the spike RBD of the original Wuhan strain SARS-CoV-2, a number of the polypeptides of the present invention were also evaluated for their ability to bind to the immobilized S-RBD of the interesting B.1.351 (beta) variant of SARS-CoV-2, as well as the S-RBD of the earlier SARS-CoV-1, which is phylogenetically more distant from the Wuhan strain SARS-CoV-2

[44] . As shown in the graph displayed in Figure 7C, all tested polypeptides of the present invention showed dissociation rates from the S-RBD of the B.1.351 (beta) variant, which were similar to those from the S-RBD of the original Wuhan strain virus variant. Notably, the polypeptide ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) of the present invention in this assay had an even slower dissociation rate from the S-RBD of the B.1.351 (beta) variant than from the S-RBD of the original Wuhan strain virus.

[0093] For the selected variants, the apparent K with rate constant and avidityD To derive a more accurate estimate of , replicate measurements were performed by immobilizing purified spiked RBD at approximately 500 RU and injecting 3, 30 and 300 nM of each polypeptide construct over the blank and S-RBD surfaces at 50 μL / min for 180 seconds with a relatively long dissociation phase of 3600 seconds. Figure 7D shows an SPR sensorgram for the inventive polypeptide ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) against the S-RBS of the Wuhan strain, B.1.351 (beta) and B.1.1.529 (Omicron) variants of SARS-CoV-2, and Figure 7E shows an SPR sensorgram for the inventive polypeptide ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) against the S-RBS of the Wuhan strain, B.1.351 (beta) and B.1.1.529 (Omicron) variants of SARS-CoV-2. The polypeptide ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) exhibits very slow kinetic dissociation rates (10 ) for the S-RBD of either of these two important SARS-CoV-2 variants, with an apparent 10-fold stronger binding to the B.1.135 variant. -6 M -1 s -1 Nearby k off ) and picomolar dissociation equilibrium constant (K D ) to the S-RBD of either of these important SARS-CoV-2 variants. However, SPR experiments reach the limit of detection (LOD) for these protein complexes due to very low dissociation rates. We also performed SPR binding experiments of the polypeptide ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) to the B.1.1.529 (Omicron) spike protein RBD variants and observed the same behavior, i.e., extremely strong binding that reached the LOD of the SPR method. The polypeptide ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) exhibits slow kinetic dissociation rates (10 -4 M -1 s -1 Nearby k off ) and nanomolar dissociation equilibrium constant (K D) binds to the B.1.135 virus variant. Binding was stronger (4-fold) to the B.1.1.529 (Omicron)S RBD variant. We also performed SPR binding experiments of the polypeptide ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) to the B.1.1.529 (Omicron)S RBD variant and observed the same behavior, i.e., approximately 4-fold improved binding affinity to the original Wuhan strain virus spike protein RBD. Taken together, these data clearly demonstrate the broad panspecificity of the polypeptide of the present invention, which efficiently binds to all currently circulating SARS-CoV-2 variants and, with a very high probability, to other mutated SARS-CoV-2 variants that may emerge in the future.

[0094] Example 7. SARS-CoV-2 pseudovirus neutralization by polypeptide constructs Pseudotyped VLP assay A virus-like particle (VLP)-based spike pseudotyped virus surrogate assay was used to evaluate the polypeptide constructs of the invention for inhibition of SARS-CoV-2 virus entry into host cells mediated by binding of the viral spike protein to the human ACE2 receptor present on the host cell surface. The assay uses pseudotyped lentiviral particles as a surrogate for live SARS-CoV-2 virus and measures the ability of compounds of interest to block VLP entry into host cell lines expressing ACE2

[45] . Pseudotyped VLPs contain the SARS-CoV-2 spike protein, a luciferase reporter, and a minimal set of lentiviral proteins required to assemble the VLP. Blockade of virus entry is detected by loss of luciferase reporter signal. Two slightly different pseudotyped VLP assay implementations, designated Method 1 and Method 2, were used to evaluate the polypeptides of the invention.

[0095] In method 1, the assay originally developed by the Bloom laboratory

[45] was optimized to increase robustness and reproducibility

[43] . The main modifications were: 1) co-expressing ACE2 and TMPRSS2 in HEK293T cells, which improved the efficiency of infection; 2) using a second generation lentiviral packaging system (which yielded higher VLP levels); and 3) adapting VLP production conditions, including lowering the temperature to 33°C for VLP production, which improved consistency in the quality of the VLPs produced. Entry vectors for the ACE2 and TMPRSS2 coding sequences were cloned into pLenti CMV Puro DEST (Addgene, 17452) and pLenti CMV Hygro DEST (Addgene, 17454), respectively. The resulting transfer vectors were used to generate lentiviruses via second generation psPAX2 and VSV-G (Addgene, 8454). HEK293T cells were transduced with ACE2 lentivirus at an MOI < 1 and selected with puromycin (1 μg / mL) to generate stable populations. These cells were then transduced with TMPRSS2 lentivirus and similarly selected with hygromycin (200 μg / mL). For VLP generation, HEK293T cells were transiently co-transfected with 8 μL JetPrime (Polyplus-transfection SA, 114-01) in 500 μL JetPrime buffer in 2 mL growth medium (10% FBS, 1% penicillin / streptomycin [Pen / Strep] in DMEM) with 1.3 μg psPAX2, 1.3 μg pHAGE-CMV-Luc2-IRES-ZsGreen-W (BEI, NR-52516; lentiviral backbone plasmid using the CMV promoter to express luciferase followed by IRES and ZsGreen), and 0.4 μg HDM-IDTSpike-fixK (BEI, NR-52514; codon-optimized Wuhan-Hu-1 spike expressed under the CMV promoter; GenBank, NC_045512).Eight hours after transfection, the medium was replaced with 3 mL of DMEM containing 5% heat-inactivated FBS and 1% Pen / Strep, and the cells were incubated at 37°C and 5% CO2 for 16 hours; subsequently, transferred to 33°C and 5% CO2 for an additional 24 hours. 48 hours after transfection, the supernatant was collected, spun at 500 x g for 5 minutes at room temperature, filtered through a 0.45 μm filter, and frozen at -80°C. Viral titers were assessed using HEK293T-ACE2 / TMPRSS2 cells at 10,000 cells per well on poly-L-lysine coated (5-10 μg / mL) 96-well plates using HI10 medium (10% heat-inactivated FBS, 1% Pen / Strep) with a virus dilution that yielded a relative luciferase unit (RLU) of >1000 relative to the control (approximately 1:100 virus stock dilution). For neutralization assays, 2.5-fold serial dilutions of serum samples were incubated with diluted virus at 1:1 ratio for 1 h at 37°C, then transferred to plated HEK293-ACE2 / TMPRSS2 cells and incubated for another 48 h at 37°C and 5% CO2. After 48 h, cells were lysed and Bright-Glo luciferase reagent (Promega, E2620) was added for 2 min, then read using a PerkinElmer Envision instrument. The 50% inhibitory concentration or dilution (IC50 or ID50) was calculated by nonlinear regression (log[inhibitor] vs. normalized response-variable slope) using GraphPad Prism 8 (GraphPad Software Inc.).

[0096] Method 2 is also based on the same published protocol

[45] and is therefore conceptually similar to method 1 with a few notable differences, notably (i) no co-expression of TMPRSS2 on the HEK293T cell line expressing human ACE2 and (ii) lentiviral VLPs generated at 37 °C by transient transfection of HEK293SF cells.

[0097] The IC50 data obtained by both methods for the polypeptide constructs of the invention are listed in Table 6, while the dose-response curves are shown in Figure 8. First, we found a linear correlation R of 1.0 between IC50 values. 2 and log 10 R between (IC50) values ​​of 0.99 2 We note good correlation between IC50 values ​​obtained using the two implementations, with k 0 = 0.001 and k 0 = 0.001. Furthermore, these pseudotype virus neutralization data correlate almost perfectly with the spike dissociation rates from the spike RBD obtained by SPR binding experiments, as previously described and listed in Table 5. off and IC50 between R 2 = 0.99 (neutralization of either pseudotype virus), and log 10 k off and log 10 R between IC50s is 0.96-0.99 2 values ​​(either pseudotype virus neutralization method). These extremely high correlations indicate the robustness of the data and strongly confirm the mechanism of action of the polypeptide constructs of the present invention, i.e., binding to the viral spike RBD prevents viral entry into host cells. We also note that although there is a good correlation between the IC50 data obtained using the two methods, the absolute IC50 translates to higher values ​​using method 1 than method 2. This effect may be due to the different implementations, and in particular the inclusion of TMPRSS2 co-expression on ACE2-expressing cells in method 1, but not in method 2. Co-expression of TMPRSS2 promotes viral entry and infection, and therefore may explain why neutralization occurs at relatively high doses of compound using method 1 compared to method 2, which does not include TMPRSS2.

[0098] The best pseudotype virus neutralization levels achieved are excellent using both methods, with some of the ACE2m4 variants (SEQ ID NO: 21, 23) reaching IC50 values ​​of approximately 25 ng / mL using method 1 and 3 ng / mL using method 2 (Table 6). It is immediately clear that the sequence and structure optimization performed during the polypeptide molecular engineering phase (see Example 1) results in significant improvements in pseudotype virus neutralization potency. These improvements in IC50 range from 8-100 fold using method 1 and at least 8 to at least 300 fold using method 2. A more detailed structure-activity relationship analysis of the data in Table 6 also reveals interesting patterns that are in full agreement with the analysis of the binding data as described in the preceding section (Table 5). Among these patterns, it is important to highlight the surprising and unexpected finding that the natural human variant Ile92 of the ACE2 catalytic domain (R1 region) confers a significant improvement in neutralization potency compared to the more common human variant with a Thr residue at position 92 and a carbohydrate structure linked to Asn90 as a result. This becomes readily apparent from the data in Table 6 and Figure 8 when comparing the variant ACE2m1-SG4-Fc (SEQ ID NO: 18) containing Thr92 with the variants ACE2m4-SG4-Fc (SEQ ID NO: 21) and ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) containing Ile92, but with the remainder of the ACE2 catalytic domain (R1 region) being identical between these variants. The surprising 4-5 fold (depending on the method used) decrease in IC50 is therefore directly attributable to the spontaneous mutation T92I in the human ACE2 catalytic domain.

[0099] [Table 6]

[0100] Neutralization of VLPs pseudotyped with different SARS-CoV-2 variants The ability of neutralizing VLPs pseudotyped with the S protein from the original Wuhan strain SARS-CoV-2 prompted us to test select polypeptides of the invention for their ability to block cell entry of VLPs pseudotyped with S proteins from other SARS-CoV-2 variants. Method 1, co-expressing human ACE2 and TMPRSS2 on HEK293T cells, was utilized for this assay. Pseudotyped SARS-CoV-2 spike lentiviral particles were produced using plasmids expressing different variants of the SARS-CoV-2 spike protein according to the protocol and reagents described by Bloom's laboratory

[45] with the following modifications: (1) HEK293SF-3F6 cells

[46] were used for large-scale production of lentiviral particles at 300 mL; (2) after transfection, HEK293SF-3F6 cells were incubated at 33 °C for improved yield; (3) 72 h after infection, lentiviral particles were harvested and subjected to concentration by sucrose cushion centrifugation. Briefly, the supernatant was placed on a 20% sucrose cushion and spun at 37,000 × g for 3 h at 4 °C. The pellet containing the concentrated pseudotyped VLPs was then resuspended in DMEM with 10% FBS and divided into aliquots. Titer determinations were performed using HEK293T cells overexpressing human ACE2 and TMPRSS2 obtained from the ATCC and NIH BEI Resources repositories (NR-55293). The following plasmids were used: Wuhan strain: plasmid name="pHDM SARS-Cov-2" (BEI Resources, NIAID, NIH: SARS-Associated Coronavirus 2, Wuhan-Hu-1 Spike-Pseudotyped Lentivirus Kit, NR-52948). D614G: plasmid name="pHDM SARS2-Spike-Del21-D614G", HDM_SARS2_Spike_del21_D614G was a gift from Jesse Bloom (Addgene plasmid #: 158762; http: / / n2t.net / addgene:158762; RRID:Addgene_158762).B.1.1.7 (alpha): plasmid name = "pPACK-SPIKE N501Y", SARS-CoV-2 "S" pseudotype-N501Y mutant-lentivector packaging mix (SBI System Biosciences SBI, Cat#: CVD19-560A-1; mutations: N501Y). B.1.351 (beta): plasmid name = "pPACK-SPIKE B.1.351", RBD mutant lentivector packaging mix (SBI System Biosciences, Cat#: CVD19-580A-1; mutations: K417N, E484K and N501Y). B.1.617.2 (delta): plasmid name="pcDNA3.3-SARS2-B.1.617.2) pcDNA3.3-SARS2-B.1.617.2" was a gift from David Nemazee (Addgene plasmid #: 172320; http: / / n2t.net / addgene:172320; RRID:Addgene_172320).

[0101] Pseudovirus neutralization assays were performed according to a previously described protocol

[45] and adapted to 384-well plates. Briefly, 3-fold serial dilutions of samples containing selected polypeptides of the invention were incubated with the diluted virus in a 1:1 ratio for 1 h at 37°C and then added to HEK293-ACE2 / TMPRSS2 cells. Infectivity was then measured by luminescence readout per well. Bright-Glo luciferase reagent (Promega, E2620) was added to the wells for 2 min and read using a PerkinElmer Envision instrument. The 50% inhibitory concentration (IC50) was calculated by nonlinear regression (log[inhibitor] vs. normalized response - variable slope) using GraphPad Prism 8 (GraphPad Software Inc.).

[0102] As shown in Figure 8C, the tested polypeptides provided in the present invention provide good blocking of cell infection for VLPs pseudotyped with all investigated viral S protein variants. The relevant IC50 values ​​for various viral variants are listed in Table 6a (nM and ng / mL). It is directly evident that for each of the tested polypeptides of the present invention, there is a small variation in IC50 values ​​across viral variants. Notably, both tested polypeptides of the present invention are found to neutralize the main variant of interest, B.1.617.2 (delta), more potently (up to 2-fold) than the original Wuhan strain virus. Taken together, this pseudotype virus neutralization data further supports the pan-specificity of the class of polypeptides of the present invention, which may provide a robust approach to mitigate COVID-19 infection caused by current variants of SARS-CoV-2 as well as future emerging variants.

[0103] [Table 7]

[0104] Example 8. SARS-CoV-2 Genuine Virus Neutralization by Polypeptide Constructs The ability of the polypeptides of the invention to neutralize infection of human VERO-E6 cells with live replicating authentic viruses was also evaluated. This was done using a microneutralization assay. SARS-CoV-2 isolate Canada / ON / VIDO-01 / 2020 was obtained from the National Microbiology Laboratory (Winnipeg, MB, Canada), grown on Vero E6 cells, and quantified on Vero cells. Whole virus genome sequencing was performed to confirm the exact genetic identity to the original isolate. Third passage virus stocks were used. Neutralization activity was determined using a microneutralization assay. Briefly, 1:5 serial dilutions of 15 μg of each polypeptide were performed in DMEM, a high glucose medium supplemented with 1 mM sodium pyruvate, 1 mM non-essential amino acids, 100 U / mL penicillin-streptomycin, and 0.1% bovine serum albumin (BSA). SARS-CoV-2 was added at 125 plaque-forming units (PFU) in a 1:1 ratio to each antibody dilution and incubated for 1 h at 37 °C. After incubation, Vero E6 cells seeded in 96-well plates were infected with the virus / polypeptide mix and incubated at 37 °C in a humidified / 5% CO2 incubator for 72 hours post-infection (hpi). Cells were subsequently fixed overnight in 10% formaldehyde and viral infection was detected using a mouse anti-SARS-CoV-2 nucleocapsid antibody (R&D Systems, clone #1035111) and counterstained with rabbit anti-mouse IgG-HRP (Rockland Inc.). Colorimetric color development was acquired using o-phenylenediamine dihydrochloride peroxidate substrate (Sigma-Aldrich) and detected at 490 nm on a Biotek Synergy H1 plate reader. IC50s were determined from nonlinear regression using GraphPad Prism 9 software.

[0105] As shown in Figure 9 and listed in Table 7, the selected polypeptides tested inhibited the replication of live virus with IC50 values ​​in the range of 1-4 ng / mL, giving more than 10-fold improved efficacy compared to the positive control anti-SARS-CoV-2 monoclonal antibody REGN10933

[47] . For example, the polypeptide ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) inhibited the infection of human VERO-E6 cells with authentic SARS-CoV-2 virus in in vitro cell culture with an excellent IC50 of around 1 ng / mL or 6 pM. Also, the polypeptide ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) inhibited the infection of human VERO-E6 cells with authentic SARS-CoV-2 virus in in vitro cell culture with a good IC50 of around 4 ng / mL or 22 pM. In comparison, the control antibody REGN10933 exhibited an IC50 of 43 ng / mL or 287 pM in this assay. Taken together, these data demonstrate that ADAPT-guided optimization of the ACE2 interface with the SARS-CoV-2 spike RBD to improve binding affinity can translate into significant functional improvements at the cellular level.

[0106] [Table 8]

[0107] Example 9. In vivo evaluation in hypertensive mice [The inventors gratefully acknowledge that some of the technical data relating to the in vivo evaluation in hypertensive mice was kindly provided by the laboratory of Dr. Kevin Burns (OHRI, University of Ottawa, Ottawa, Canada)].

[0108] ethical statement All experiments performed on animals were in accordance with Canadian Council on Animal Care (CCAC) regulations and approved by the University of Ottawa Animal Care Committee (Protocol 3514).

[0109] Intervention allocation Mice were housed in the University of Ottawa Animal Care Facility with constant monitoring of humidity and temperature and free access to water and food. Cage allocation was randomized within shelves. Allocation to experimental groups was performed using an online randomization tool (randomizer.org).

[0110] Angiotensin II infusion 12-14 week old C57BL / 6 male and female mice were subjected to subcutaneous implantation of osmotic mini-pumps (Alzet®, model 1004). Mice were injected with 0.01 mg / kg buprenorphine 1 hour prior to mini-pump implantation. Mice were initially anesthetized with 5% isoflurane and maintained with 2.5% isoflurane throughout the procedure. Animals were shaved and the skin was sterilized with a solution containing chlorhexidine gluconate 4%. A 0.5 cm wide incision was made perpendicular to the spine in the subcapsular region and a subcutaneous pocket was created with a hemostat. The mini-pump was inserted into the pocket and the incision was closed with two wound clips. Topical bupivacaine was applied to the incision and the animals were transferred to a heated recovery area until they recovered from anesthesia. Mice were given an additional buprenorphine infusion 4 hours after surgery and monitored twice daily for 72 hours. Angiotensin II was infused at 1000 ng / kg / min for 3 weeks, and control mice were infused with saline solution via a minipump.

[0111] Intravenous Administration of Polypeptides of the Invention Two polypeptides of the invention were administered intravenously to mice: ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) and ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28). Two classes of mice were studied: normotensive and hypertensive. Mice were assigned to the following groups: i) saline, ii) Ang II, iii) Ang II+rhACE2, iv) Ang II+ACE2m4-hinge2CS-SG4-Fc, v) Ang II+ACE2I92-hinge2CS-SG4-Fc. 2.5 weeks after osmotic minipump insertion, mice were administered a single intravenous injection (via tail vein) of variants ACE2m4-hinge2CS-SG4-Fc (10 mg / kg), ACE2I92-hinge2CS-SG4-Fc (10 mg / kg), or rhACE2 (2.5 mg / kg-BioLegend, Cat#:792008). Control mice were administered PBS. 72 hours after injection of the polypeptide of the invention or rhACE2, mice were euthanized and plasma, kidney, heart, lung, liver, and spleen were collected for analysis.

[0112] Blood pressure measurement Systolic blood pressure (SBP) was measured by tail cuff plethysmography (BP-2000; Visitech Systems, Apex, NC) as previously described

[48] . Mice were trained and mock measurements were taken over 5 consecutive days. SBP was assessed at baseline (3 measurements), 2.5 weeks after osmotic minipump implantation (2-3 measurements), and 3, 6, 24, 48, and 72 hours after injection of the polypeptide of the invention. Each mouse was subjected to 10 SBP readings per time point, and the mean SBP was calculated from these readings.

[0113] Hypertension induced by angiotensin II infusion was confirmed by a significant increase in SBP 2.5 weeks after osmotic minipump implantation. rhACE2 transiently reduced SBP up to 6 hours after infusion, while ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) had no significant effect on SBP (Figure 10A). Notably, ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) significantly reduced SBP for up to 24 hours, and this reducing effect was partially sustained for 48 hours (Figure 10A).

[0114] Plasma and tissue ACE2 activity ACE2 enzyme activity was assessed in plasma and tissues (kidney, heart, lung, liver, and spleen) collected at the end point (72 hours). Activity was determined using the fluorogenic substrate Mca-APK (Dnp) from Anaspec with or without the ACE2-specific inhibitor MLN-4760 as described

[40] . Plasma ACE2 activity was determined in 5 μL of plasma. For tissue ACE2 activity, samples were homogenized in 500 μL of lysis buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% TritonX-100, 0.025 mM ZnCl2, and a protease inhibitor cocktail (Sigma-Aldrich, Cat#: P8340). Lysates were centrifuged at 12,000×g for 10 min at 4° C. to remove debris. Supernatants were stored at −80° C. Protein concentrations were determined using a DC protein assay kit (Bio-Rad Laboratories, Cat#:5000112). Due to differences in ACE2 abundance, kidney tissue was assayed at 1 μg total protein and incubated with fluorogenic substrate for 1 h. Heart, lung, liver, and spleen ACE2 activity was assayed at 10 μg and incubated with fluorogenic substrate (11.25 μM) for 16 h. Data are presented as mean ± SEM. Statistical analysis was performed using Prism 9.3.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com. Comparisons between groups were performed by one-way ANOVA followed by either Tukey or Dunnett's post-hoc test. P<0.05 was considered statistically significant.

[0115] ACE2 activity was evaluated in plasma collected from mice at the end point (72 hours after administration of the polypeptide of the present invention). In this assay, ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) was almost inactive in plasma at the end point, while the activity of ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) was significantly increased compared to all groups (Figure 10B).

[0116] Tissue ACE2 activity was analyzed in lysates of kidney, heart, lung, liver, and spleen (Figure 10B). There was no significant difference in activity in kidney lysates among all samples. ACE2 activity was significantly increased in heart lysates of the Ang II+ACE2m4-hinge2CS-SG4-Fc group. ACE2 activity was significantly higher in liver lysates of group Ang II+ACE2m4-hinge2CS-SG4-Fc. Finally, there was no difference in ACE2 activity between groups when samples from the spleen were analyzed (Figure 8A).

[0117] Immunoblotting Tissue lysates were obtained as described above. The amount of lysate varied according to tissue / sample type as follows: 1 μL plasma, 20 μg kidney, 30 μg heart and lung, 40 μg liver and spleen. Lysates were added to 4× Laemmli sample buffer, loaded onto gradient SDS-PAGE gels (5-15%) and subjected to electrophoresis. Proteins were transferred from the gels to nitrocellulose membranes (Bio-Rad Laboratories, Cat#: 1620112) and blocked for 1 h at room temperature in Tris-buffered saline solution (pH 7.6) (TBS-T) containing 0.1% Tween 20 (TBS-T) and 3% bovine serum albumin (BSA). The membrane was probed with goat anti-ACE2 (1:1000 dilution, R&D systems, Cat#:AF933) overnight at 4°C, followed by incubation with 1:5,000 HRP-donkey anti-goat IgG (Jackson ImmunoResearch, Cat#:705-035-147). Probing for IgG Fc was performed with HRP-donkey anti-human IgG Fcγ 1:10,000 (Jackson ImmunoResearch, Cat#:709-035-098) overnight at 4°C. Chemiluminescence was induced by addition of Amersham ECL Western blotting detection reagent to the membrane (GE Healthcare, Cat#:CA95038-564L) and detected on an Alpha Innotech FluorChem Q quantitative Western blot imaging system.

[0118] ACE2 was detected by immunoblotting in plasma of rhACE2 and Ang II+ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) groups, and at very low levels in plasma of Ang II+ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) group (Figure 10C). In addition, ACE2 was detected in lysates from multiple organ tissues from several groups (Figure 10C). In the majority of samples, immunoblotting detection (Figure 10C) correlated with the measured ACE2 activity (Figure 10B). Of note, immunoblotting for ACE2 in kidney showed substantial levels of endogenous ACE2 (approximately 100 kDa), with a less prominent high molecular weight band (just below 250 kDa; likely corresponding to the ACE2-Fc homodimer of the present invention) only detected in lysates from the Ang II+ACE2I92-hinge2CS-SG4-Fc group. The same band was detected when probed with anti-human IgG Fcγ, indicating the presence of ACE2I92-hinge2CS-SG4-Fc in kidney tissue lysates. Immunoblots for ACE2 and IgG Fcγ were consistent with the ACE2 activity data in heart lysates, indicating significant retention of ACE2I92-hinge2CS-SG4-Fc in heart tissue. Lung lysates also demonstrated persistent presence of ACE2I92-hinge2CS-SG4-Fc. Furthermore, the ratio of ACE2I92-hinge2CS-SG4-Fc to endogenous ACE2 was elevated. In liver lysates, a high MW band for ACE2I92-hinge2CS-SG4-Fc was again detected on immunoblots. In immunoblots from spleen, a strong signal for the high MW band corresponding to ACE2I92-hinge2CS-SG4-Fc was seen.

[0119] Albumin-creatinine ratio Spot urine was collected at the endpoint (72 hours). Urine samples were diluted 1:200-1:500 according to the manufacturer's instructions. Albumin was measured by ELISA (Bethyl Laboratories Inc., Cat#: E99-134). Creatinine was quantified in urine using a colorimetric-based kit from Ethos Bioscience (Cat#: 1012). Albumin values ​​were converted to μg / dL, and albumin to creatinine ratios were calculated by dividing albumin levels by creatinine levels (mg / dL).

[0120] Increased urinary excretion of albumin can indicate kidney damage. Analysis of urine collected at the end point (72 hours) showed no significant change in albumin-creatinine ratio (ACR). However, there was a clear trend of increase in the hypertensive group compared to the saline group. Hypertensive mice treated with ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) had the lowest values ​​for albumin excretion, comparable to the saline group (Figure 10D).

[0121] In conclusion, many of the novel polypeptides of the present invention showed sustained in vitro ACE2 activity with comparable performance to rhACE2, while the polypeptide ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) lost activity rapidly in vivo. The candidate polypeptide ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) had enhanced performance in vivo in this model and transiently reduced SBP. The sustained detection of ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) in various tissue lysates 72 hours after iv injection, together with the increased ACE2 activity and long-term blood pressure lowering effect, suggests that ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) may have significant therapeutic potential. Taken together, ACE2 enzymatic activity and immunoblotting in plasma and various tissues are consistent and support the SBP lowering effect as well as reduced albuminuria observed with ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28), strongly suggesting the therapeutic effect of the selected polypeptides of the present invention as protective agents against major organ injury caused by COVID-19.

[0122] Example 10. In vivo evaluation in SARS-CoV-2 infected hamsters Intranasal Considering the instability of some of the polypeptides of the present invention in plasma observed in an in vivo model of hypertensive mice, the intranasal administration route was first adopted to test in vivo recovery from COVID-19 using a hamster model

[49] . Male Syrian golden hamsters (81–90 g) were obtained from Charles River Laboratories (Saint-Constant, QC, Canada). Animals were maintained in the animal facility of the National Research Council Canada (NRC) in accordance with the guidelines of the Canadian Council on Animal Care. All procedures performed on animals in this study were in accordance with the regulations and guidelines reviewed and approved by the NRC Human Health Therapeutics Animal Care Committee in the Animal Use Protocol 2020.06. Male hamsters were inoculated with 8 × 10 3 Animals were challenged intranasally with PFU of SARS-CoV-2 isolate Canada / ON / VIDO-01 / 2020. Two dosing regimens were tested. In combined prophylactic and therapeutic dosing, animals were administered intranasally with the polypeptide of the invention 4 hours prior to viral challenge and also with a repeat dose 24 hours after viral challenge. In therapeutic dosing alone, animals were administered intranasally with the polypeptide of the invention 6 hours after viral challenge and with repeat doses administered at 24-hour intervals. All intranasal dosing was performed with a fixed total volume of 150 μL, alternating between the left and right nostrils until a total of 75 μL of test article solution had been administered to each nostril. Control animals received PBS vehicle only. Daily body weights were measured. Data presented as mean ± SEM. n=6-7 animals / group.

[0123] The weight changes for the various groups after combined prophylactic and therapeutic administration are shown in FIG. 11A. Both tested polypeptides of the invention, ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) and ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28), had a significant effect on the treatment group compared to the control group (vehicle only), as shown by the reduced weight loss. For both tested articles, recovery actually resulted in weight gain 3 days after challenge. Furthermore, a similar recovery with the ACE2m4-hinge2CS-SG4-Fc polypeptide construct was achieved at the two tested doses of 4 mg / kg high dose (administered twice) as well as 1 mg / kg low dose (administered twice). These results demonstrate the feasibility of treating COVID-19 with polypeptides of the invention, including but not limited to ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23), via non-systemic administration routes.

[0124] Body weight changes for the various groups after therapeutic administration alone are shown in Figure 11B. Both polypeptides of the present invention tested, ACE2m4-hinge2CS-SG4-Fc (SEQ ID NO: 23) and ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28), had a significant effect on the treatment group compared to the control group (vehicle only), as shown by reduced body weight loss. The ACE2I92-hinge2CS-SG4-Fc polypeptide construct resulted in a more significant recovery even with fewer doses than the ACE2m4-hinge2CS-SG4-Fc polypeptide construct (3 vs. 5 therapeutic doses of 1 mg / kg, respectively). Furthermore, recovery actually resulted in weight gain after 2 days post-challenge with ACE2I92-hinge2CS-SG4-Fc treatment and after 4 days post-challenge with ACE2m4-hinge2CS-SG4 treatment. These results demonstrate further support for the intended use of these inventive polypeptides as therapeutic agents for SARS-CoV-2 viral infection and recovery from COVID-19.

[0125] Intravenous Since ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) showed excellent stability in plasma and organ tissues, including lungs and other organs (Figure 10), it was also evaluated using a systemic route of administration for in vivo in the COVID-19 hamster model

[49] . Female Syrian golden hamsters weighing 81–90 g (Charles River Labs) were administered 10 4 The animals were intranasally infected with PFU of live SARS-CoV-2 virus. The selected polypeptide of the invention in PBS solution was administered in the retro-orbital vein at a dose of 10 mg / Kg at the time of viral challenge. Repeated administration with the same dose was performed 24 hours after viral challenge. A control group of animals received PBS only. Each group consisted of 7 animals. Three days after challenge, the animals were euthanized and the viral load was determined in lung tissue by plaque assay. Three days after challenge, the animals were euthanized and the viral load in lung tissue was determined using a plaque assay by infection of cultured VERO-E6 cells as previously described

[50] .

[0126] As shown in FIG. 11C, iv administration of ACE2I92-hinge2CS-SG4-Fc (SEQ ID NO: 28) reduced SARS-CoV-2 infection in female hamsters, as evidenced by a nearly order of magnitude reduction in the median level of viable virus as determined by plaque assay compared to the median level in the PBS control group. Importantly, it is evident from the data shown in FIG. 11C that the T92I mutation in the ACE2 ectodomain region of the polypeptide of the invention is important for achieving reduced viral titers. This is seen in comparison with the ACE2-hinge2CS-SG4-Fc (SEQ ID NO: 27) polypeptide incorporating the parent non-restricted mutated human ACE2 ectodomain, which did not result in a median reduction in viral titers compared to the median viral titers in the PBS control group, and in contrast appears to have the exact opposite effect. This data highlights the unique structure of the polypeptide fusion of the invention, based on the naturally occurring T92I mutation of human ACE2, as a key component with a surprising and unexpected efficacy profile against COVID-19 infection and associated major organ injury.

[0127] Sequence Listing

[0128] [Table 9] JPEG2024505203000011.jpg194149 JPEG2024505203000012.jpg159149 JPEG2024505203000013.jpg183149 JPEG2024505203000014.jpg187149 JPEG2024505203000015.jpg179149 JPEG2024505203000016.jpg179149 JPEG2024505203000017.jpg180149 JPEG2024505203000018.jpg183149 JPEG2024505203000019.jpg183149 JPEG2024505203000020.jpg209149 JPEG2024505203000021.jpg164149 JPEG2024505203000022.jpg205149 JPEG2024505203000023.jpg87149

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Claims

1. A polypeptide construct capable of neutralizing SARS-CoV-2 and converting Ang-II to Ang-(1-7), said polypeptide construct comprising four regions and having the general formula: R1 [hACE2I ９２ (18-614),X ２７ , X ２６１ , X ３３０ ]-R2-R3[Hinge S ２２０ , X ２２６ , X ２２９ ]-R4[C Ｈ 2G ２７０ -C Ｈ 3] having In the formula, hACE2I ９２ (18-614),X ２７ , X ２６１ , X ３３０ R1, represented as: ２７ , X ２６１ and X ３３０ a first N-terminal region comprising the naturally occurring variant Ile92 (I92) of the human angiotensin-converting enzyme 2 (hACE2) receptor catalytic domain residues 18-614, comprising: ２７ is the amino acid residue at position 27, which is either Thr or Tyr; ２６１ is the amino acid residue at position 261, which is either Cys or Ser, and X ３３０ is the amino acid residue at position 330, which is either Asn or Tyr; R2 is a second region comprising a flexible peptide spacer; R3 is a third region comprising the hinge region of a human IgG1 heavy chain antibody, said hinge region being comprised of residues S ２２０ , X ２２６ and X ２２９ the amino acid residue at position 220 of the human IgG1 hinge is Ser, and the amino acid residues at positions 226 and 229 of the human IgG1 hinge are either Cys or Ser; and R4 is a fourth region at the C-terminus of the polypeptide; R4 is Ｈ 2G ２７０ -C Ｈ 3 and the second constant domain of the human IgG1 antibody heavy chain (C Ｈ 2) and the third constant domain (C Ｈ 3) Human IgG1 C Ｈ The amino acid residue at position 270 in domain 2 is glycine. Polypeptide constructs.

2. The polypeptide construct of claim 1 , wherein the flexible peptide spacer comprises Gly and Ser residues.

3. 2. The polypeptide construct of claim 1, which neutralizes cell infection mediated by SARS-CoV-2 variants with an IC50 of less than 2500 ng / mL (12.5 nM).

4. The catalytic efficiency (k ｃａｔ / K Ｍ 4. The polypeptide construct according to claim 1, wherein the polypeptide construct retains at least 30% of the total protein content and at least 60% of the enzymatic activity.

5. R1 comprises a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and sequences at least 90% identical thereto; R2 comprises a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, and sequences at least 90% identical thereto; R3 comprises a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, and sequences at least 90% identical thereto; or 2. The polypeptide construct of claim 1, wherein R4 comprises a sequence having SEQ ID NO: 14, or a sequence at least 90% identical thereto.

6. 2. The polypeptide construct of claim 1, comprising a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:28, and sequences that are at least 90% identical thereto.

7. The polypeptide construct according to any one of claims 1 to 6, which is a dimeric polypeptide.

8. 8. The polypeptide construct of claim 7, wherein the dimeric polypeptides are linked or dimerized via their respective R3 hinge regions by disulfide bridges.

9. A nucleic acid molecule encoding a polypeptide construct according to any one of claims 1 to 8.

10. A vector comprising the nucleic acid molecule of claim 9.

11. A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 8 in a form which is secretable by a selected expression host.

12. A composition comprising a polypeptide construct according to any one of claims 1 to 8 and a pharma- ceutically acceptable carrier, diluent or excipient.

13. A transgenic cell host comprising the nucleic acid molecule of claim 9 or the vector of claim 10.

14. A method for producing a dimeric polypeptide comprising culturing the host described in claim 13 and recovering the dimeric polypeptide construct described in claim 7 from medium conditioned by growth of the host.

15. A composition for the treatment of coronavirus infection, acute respiratory distress syndrome (ARDS), or major organ failure associated with COVID-19, comprising a polypeptide construct described in any one of claims 1 to 8.

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