EP4646222A1 - Protéines solubles de l'enzyme de conversion de l'angiotensine 2 (ace2) et protéines de fusion d'ace2 - Google Patents

Protéines solubles de l'enzyme de conversion de l'angiotensine 2 (ace2) et protéines de fusion d'ace2

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Publication number
EP4646222A1
EP4646222A1 EP23901407.9A EP23901407A EP4646222A1 EP 4646222 A1 EP4646222 A1 EP 4646222A1 EP 23901407 A EP23901407 A EP 23901407A EP 4646222 A1 EP4646222 A1 EP 4646222A1
Authority
EP
European Patent Office
Prior art keywords
substitution
protein
ace2
threonine
serine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23901407.9A
Other languages
German (de)
English (en)
Inventor
Savanna SKEETERS
Galina Stepanyuk
Kui K. CHAN
Benjamin DUTZAR
David THIEKER
Aaron AGUHOB
Yifan SONG
Eric TARCHA
Erik PROCKO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cyrus Biotechnology Inc
Original Assignee
Cyrus Biotechnology Inc
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Filing date
Publication date
Application filed by Cyrus Biotechnology Inc filed Critical Cyrus Biotechnology Inc
Publication of EP4646222A1 publication Critical patent/EP4646222A1/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • ACE2 modified angiotensin-converting enzyme 2
  • fusion proteins comprising the same having enhanced activity and pharmacokinetics as compared to wild-type ACE2.
  • SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
  • ACE2 angiotensin converting enzyme 2
  • ACE2 receptor angiotensin converting enzyme 2
  • ACE2 is a dimeric protease at the plasma membrane that catalyzes the turnover of vasoconstrictive and pro-inflammatory hormones.
  • Trimeric Spikes (S) on the viral surface bind with moderate affinity to ACE2 via a receptor-binding domain (RBD), triggering conformational changes in S that facilitate fusion of the viral envelope and host cell membrane, releasing the viral genome into the infected host cell.
  • RBD receptor-binding domain
  • Antibodies targeting the RBD may block ACE2 interactions or block the necessary conformational changes associated with membrane fusion, thereby neutralizing the SARS- CoV-2 virus.
  • Monoclonal anti-RBD antibodies have been developed as highly effective drugs and prophylactics for Coronavirus Disease 2019 (COVID-19), yet their efficacy against newly emerging SARS-CoV-2 variants of concern has waned. This was especially evident with sublineages of the omicron variant that were first reported in late 2021 in southern 1 IPTS/125367707.1 Attorney Docket No.: CYR-004WO Africa and within weeks had spread globally to become the dominant circulating variants, with many omicron variants now in co-circulation in what is colloquially called a “variant soup.” S proteins of omicron variants differ from original Wuhan isolates by approximately 30 mutations, with approximately 15 mutations in the RBD alone.
  • soluble ACE2 variant proteins and soluble ACE2 variant proteins fused to an immunoglobulin Fc polypeptide having one or more than one amino acid substitution that elevates the protein’s in vivo catalytic activity and improves its pharmacokinetic (PK) properties as compared to wild-type ACE2.
  • the present disclosure relates to human angiotensin-converting enzyme 2 (ACE2) polypeptides and ACE2 fusion proteins that exhibit enhanced activity and/or pharmacokinetics, that can be used as therapeutic agents for the prophylaxis (pre- or post- exposure prophylaxis), or treatment of COVID-19, or a disease caused by any coronavirus that utilizes ACE2 as a cellular receptor.
  • ACE2 angiotensin-converting enzyme 2
  • the present disclosure provides a protein including a modified ACE2 polypeptide incorporating at least one substitution relative to wild-type human ACE2 of SEQ ID NO:1.
  • the at least one substitution introduces at least one glycosylation site not present in wild-type human ACE2.
  • the present disclosure provides a protein including a modified ACE2 polypeptide incorporating a substitution or combination of substitutions relative to wild-type human ACE2 of SEQ ID NO:1 selected from: (a) a substitution of valine at position 491 to isoleucine; a substitution of methionine at position 662 to serine or threonine; and a substitution of asparagine at position 720 to serine or threonine; (b) a substitution of isoleucine at position 663 to tryptophan, and a substitution of alanine at position 673 to tyrosine; (c) a substitution of alanine at position 673 to tyrosine, and a substitution of isoleucine at position 694 to phenylalanine; (d) a substitution of valine at position 491 to isoleucine, and
  • the protein further incorporates a substitution of threonine at position 27 to tyrosine, a substitution of leucine at position 79 to threonine, and a substitution of asparagine at position 330 to tyrosine.
  • the modified ACE2 polypeptide incorporates a substitution of threonine at position 27 to tyrosine, a substitution of leucine at position 79 to threonine, a substitution of asparagine at position 330 to tyrosine, a substitution of valine at position 491 to isoleucine; a substitution of methionine at position 662 to threonine; and a substitution of asparagine at position 720 to serine.
  • the modified ACE2 polypeptide has a sequence having at least 85% 3 IPTS/125367707.1 Attorney Docket No.: CYR-004WO sequence identity to SEQ ID NO:12. In certain aspects, the modified ACE2 polypeptide has a sequence having 100% sequence identity to SEQ ID NO:12. [0012] In certain aspects, the modified ACE2 polypeptide comprises a signal peptide fused to the N-terminus of the modified ACE2 polypeptide. In certain aspects, the signal peptide has a sequence having at least 85% sequence identity to SEQ ID NO:11. [0013] In certain aspects, the modified ACE2 polypeptide is fused to an immunoglobulin Fc domain polypeptide, or functional fragment thereof.
  • the immunoglobulin Fc domain polypeptide is a human IgG1, IgG2, IgG3, or IgG4 Fc domain polypeptide, or functional fragment thereof.
  • the immunoglobulin Fc domain, or functional fragment thereof includes residues 221 to 447 of human IgG1 Fc, where positions are numbered according to EU numbering.
  • the human IgG1 Fc domain, or functional fragment thereof incorporates one or more than one substitution relative to wild- type human IgG1 Fc domain of SEQ ID NO:4 selected from: (a) a substitution of methionine at position 252 to tyrosine; (b) a substitution of serine at position 254 to threonine; and (c) a substitution of threonine at position 256 to glutamic acid, where positions are numbered according to the EU numbering.
  • the immunoglobulin Fc domain polypeptide, or functional fragment thereof is fused to the C-terminus of the modified ACE2 polypeptide.
  • the immunoglobulin Fc domain polypeptide, or functional fragment thereof is fused to the C-terminus of the modified ACE2 polypeptide via a linker.
  • the linker is a single serine residue.
  • the present disclosure provides a protein including: (a) a modified ACE2 polypeptide incorporating a substitution of threonine at position 27 to tyrosine, a substitution of leucine at position 79 to threonine, a substitution of asparagine at position 330 to tyrosine, a substitution of valine at position 491 to isoleucine, a substitution of methionine at position 662 to threonine, and a substitution of asparagine at position 720 to serine; and (b) an immunoglobulin Fc domain incorporating a substitution of methionine at position 252 to tyrosine, a substitution of serine at position 254 to threonine, and a substitution of
  • the protein has a sequence having at least 85% or 100% sequence identity to SEQ ID NO:10.
  • a protein of the present disclosure has increased levels of glycosylation as compared to wild-type ACE2.
  • a protein of the present disclosure has two to fifty-fold increased affinity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein 4 IPTS/125367707.1 Attorney Docket No.: CYR-004WO as compared to wild-type ACE2 as measured by biolayer interferometry (BLI).
  • a protein of the present disclosure has 1.2 to 2-fold increased catalytic activity as compared to wild-type ACE2 as measured in an in vitro fluorometric ACE2 activity assay. [0018] In certain aspects, at least 20% by moles of the glycosylations of a protein of the present disclosure are sialylated. [0019] In certain aspects, a protein of the present disclosure has a two to ten-fold decreased dissociation rate with FcRn at endosomal pH as compared to a protein comprising wild-type ACE2 fused to a wild-type human IgG1 Fc domain.
  • a protein of the present disclosure has an equilibrium dissociation constant for FcRn at pH 6.0 of 10 nM to 50 nM as measured by BLI. In certain aspects, a protein of the present disclosure has an off rate for FcRn at pH 6.0 of 5.0 x 10 -4 s -1 to 2.0 x 10 -3 s -1 as measured by BLI. In certain aspects, a protein of the present disclosure has a higher binding affinity to FcRn at pH 6.0 than at pH 7.0 as measured by BLI. [0020] In certain aspects, the plasma half-life of a protein of the present disclosure is 40 to 240 hours when administered to a subject.
  • a protein of the present disclosure has a plasma activity area under the curve over 48 hours (AUC 0-48h ) of 50 to 150 ( ⁇ M product/minute) x h when intravenously administered to a subject at a dose of 2 mg/kg. In certain aspects, a protein of the present disclosure has an area under the curve over 120 hours (AUC 0-120h ) of 100 to 9000 ⁇ g/ml x h when intravenously administered to a subject at a dose of 10 mg/kg. [0022] In certain aspects, a protein of the present disclosure maintains a plasma concentration above 18 pM at 100 to 150 hours post administration to a subject.
  • a protein of the present disclosure is nonimmunogenic when administered to a subject.
  • a protein of the present disclosure forms a stable homodimer.
  • the present disclosure also provides a pharmaceutical formulation including a protein as described herein and a pharmaceutically acceptable carrier.
  • the present disclosure additionally provides a nucleic acid vector encoding a protein as described herein.
  • the present disclosure provides a method of producing a protein as described herein where a vector encoding the protein is expressed in a cell line and at least 10% by moles of the expressed protein is glycosylated at each N-glycosylation motif.
  • glycosylation is measured by mass spectrometry.
  • the expressed protein is glycosylated with N-acetylhexosamine.
  • at least 20% of the glycosylations by moles are sialylated.
  • the cell line for expressing a protein of the present disclosure is Expi293F cells.
  • the cell line for expressing a protein of the present disclosure is a stable CHO line.
  • the percentage is a mole percentage.
  • the present disclosure provides a method of inhibiting SARS-CoV- 2 replication including administering to a subject a therapeutically or prophylactically effective amount of a protein or pharmaceutical formulation as disclosed herein.
  • the protein or pharmaceutical formulation is administered to the subject intravenously, subcutaneously, intratracheally, or by inhalation.
  • the SARS-CoV-2 variant is Wuhan, alpha, beta, gamma, delta, or omicron.
  • FIG.1A is a line graph showing catalytic activity in serum of soluble angiotensin- converting enzyme 2 v2.4-IgG1 fusion protein (sACE2 2 .v2.4-IgG1) expressed and purified from human Expi293F cell culture (broken line) or non-human ExpiCHO-S (solid line) cell culture and injected via tail vein at 10 mg/kg into human FcRn transgenic mice.
  • sACE2 2 .v2.4-IgG1 fusion protein sACE2 2 .v2.4-IgG1 fusion protein
  • FIG.2A and FIG.2B are annotated MALDI-TOF-MS spectra (950-2500 m/z and 2500-5000 m/z, respectively) of N-glycans released from sACE2 2 .v2.4-IgG1 expressed from ExpiCHO-S cells following 20-hour incubation with PNGase F at 37 °C, and permethylation.
  • FIG.3A and FIG.3B are annotated MALDI-TOF-MS spectra (950-2500 m/z and 2500-5000 m/z, respectively) of N-glycans released from sACE2 2 .v2.4-IgG1 expressed from Expi293F cells following 20-hour incubation with PNGase F at 37 °C, and permethylation.
  • N-glycan structures were assigned based on precursor masses and the common mammalian biosynthetic pathway. GlcNAc, squares; Man, open circles; Gal, filled circles; Fuc, triangles; Neu5Ac (sialic acid), diamonds.
  • FIG.4A and FIG.4B are pie graphs showing the relative abundance of N-glycan types released from sACE2 2 .v2.4-IgG1 expressed from ExpiCHO-S cells and Expi293F cells, respectively, following 48-hour incubation with PNGase F at 37 °C, permethylation, and analysis by MALDI-TOF-MS.
  • FIG.4C is a bar graph showing the relative abundance of sialylated (Neu5Ac) and fucosylated N-glycan structures released from sACE2 2 .v2.4-IgG1 expressed from ExpiCHO- S cells and Expi293F cells, respectively, following 48-hour incubation with PNGase F at 37°C, permethylation, and analysis by MALDI-TOF-MS.
  • FIG.4D and FIG.4E are bar graphs showing the relative abundance of O-glycans released from sACE2 2 .v2.4-IgG1 expressed from ExpiCHO-S cells and Expi293F cells, respectively, following 18-hour incubation with sodium hydroxide and sodium borohydride at 45 °C, permethylation, and analysis by MALDI-TOF-MS.
  • O-glycan structures were assigned based on precursor masses and the common mammalian biosynthetic pathway. Glycan structures are indicated on the x-axis by their m/z ratio. GlcNAc, filled squares; GalNAc, open squares; Gal, circles; Neu5Ac, diamonds.
  • FIG.5A is a ribbon diagram of the structure of dimeric ACE2 (PDB 6M17) bound to the receptor-binding domain (RBD) of spike protein (S) of SARS-CoV-2 virus.
  • ACE2 Chain ‘A’ dark green
  • ACE2 Chain ‘B’ light green
  • RBD gray
  • glycans orange
  • PD protease domain
  • CLD collectrin-like dimerization domain.
  • FIG.5B, FIG.5C, and FIG.5D are ribbon diagrams of the structure of a single ACE2 subunit having residues substituted to fill cavities (blue spheres), introduce disulfide bonds (yellow spheres), or add N-glycosylation motifs (purple spheres), respectively.
  • FIG.6A is a line graph showing the catalytic activity of purified sACE2 2 .v2.4-IgG1 and derivatives (0.5 nM) containing cavity filling mutations or mutations introducing disulfide bonds as measured by an in vitro fluorometric assay.
  • FIG.6B is a line graph showing the catalytic activity of purified sACE2 2 .v2.4-IgG1 derivatives (0.5 nM) containing mutations introducing consensus glycosylation motifs as measured by an in vitro fluorometric assay.
  • FIG.6C is a line graph showing the catalytic activity of purified sACE2 2 .v2.4-IgG1 derivatives (0.5 nM) having M662T and N720S mutations as measured by an in vitro 7 IPTS/125367707.1 Attorney Docket No.: CYR-004WO fluorometric assay.
  • FIG.7A is a line graph showing the catalytic activity of sACE2 2 .v2.4-IgG1 and derivatives having cavity-filling mutations or disulfides in retroorbital blood draw samples from human FcRn mice injected with 2 mg/kg of protein via tail vein injection.
  • FIG.7B is a line graph showing the catalytic activity of sACE2 2 .v2.4-IgG1 and derivatives having introduced consensus glycosylation motifs in retroorbital blood draw samples from human FcRn mice injected with 2 mg/kg of protein via tail vein injection.
  • FIG.7C is a line graph showing the catalytic activity of sACE2 2 .v2.4-IgG1 and derivatives having M662T and N720S mutations in retroorbital blood draw samples from human FcRn mice injected with 2 mg/kg of protein via tail vein injection.
  • FIG.7D is an image of a non-reducing polyacrylamide gel electrophoresis and anti- human IgG1 immunoblot of serum (1 ⁇ l each) sampled from a human FcRn mouse administered via tail vein injection with 2 mg/kg sACE2 2 .S14-IgG1.
  • FIG.8A and FIG.8B are annotated MALDI-TOF-MS spectra (950-2500 m/z and 2500-5000 m/z, respectively) of N-glycans released from sACE2 2 .S19-IgG1 expressed from Expi293F cells following 20-hour incubation with PNGase F at 37°C, and permethylation. Peak intensities are representative of relative N-glycan abundance.
  • N-glycan structures were assigned based on precursor masses and the common mammalian biosynthetic pathway. GlcNAc, squares; Man, open circles; Gal, filled circles; Fuc, triangles; Neu5Ac (sialic acid), diamonds.
  • FIG.9A is a pie graph showing the relative abundance of N-glycan types released from sACE2 2 .S19-IgG1 expressed from Expi293F cells, following 48-hour incubation with PNGase F at 37°C, permethylation, and analysis by MALDI-TOF-MS.
  • FIG.9B is a bar graph showing the relative abundance of sialylated (Neu5Ac) and fucosylated N-glycan structures released from sACE2 2 .S19-IgG1 expressed from Expi293F cells following 48-hour incubation with PNGase F at 37°C, permethylation, and analysis by MALDI-TOF-MS.
  • FIG.9C is a bar graph showing the relative abundance of O-glycans released from sACE2 2 .S19-IgG1 expressed from Expi293F cells following 18-hour incubation with sodium hydroxide and sodium borohydride at 45 °C, permethylation, and analysis by MALDI-TOF- MS.
  • O-glycan structures were assigned based on precursor masses and the common mammalian biosynthetic pathway. Glycan structures are indicated on the x-axis by their m/z ratio. GlcNAc, filled squares; GalNAc, open squares; Gal, circles; Neu5Ac, diamonds.
  • FIG.10A is a bar graph showing the percent occupancy of N-glycosylation sites in sACE2 2 .S19-IgG1 expressed in Expi293F (white) cells, sACE2 2 .v2.4-IgG1 expressed in Expi293F (dense diagonal hatching) cells, and sACE2 2 .v2.4-IgG1 expressed in ExpiCHO-S (sparse diagonal hatching) cells.
  • FIG.10B is a bar graph showing the percentage of glycoforms at each N- glycosylation site having at least one sialic acid in sACE2 2 .S19-IgG1 expressed in Expi293F (white) cells, sACE2 2 .v2.4-IgG1 expressed in Expi293F (dense diagonal hatching) cells, and sACE2 2 .v2.4-IgG1 expressed in ExpiCHO-S (sparse diagonal hatching) cells.
  • FIGs.11A-11D are sensogram plots of sACE2 2 .v2.4-IgG1 (FIG.11A), sACE2 2 .v2.4- IgG1 (YTE) (FIG.11B), sACE2 2 .S19-IgG1 (FIG.11C), and sACE2 2 .S19-IgG1 (YTE) (FIG.11D) binding to FcRn immobilized on biolayer interferometry (BLI) biosensors equilibrated at pH 6.0.
  • BKI biolayer interferometry
  • FIGs.11E-11H are sensogram plots of sACE2 2 .v2.4-IgG1 (FIG.11E), sACE2 2 .v2.4- IgG1 (YTE) (FIG.11F), sACE2 2 .S19-IgG1 (FIG.11G), and sACE2 2 .S19-IgG1 (YTE) (FIG.11H) binding to FcRn immobilized on biolayer interferometry (BLI) biosensors equilibrated at pH 7.4.
  • BKI biolayer interferometry
  • FIG.12A is a line graph showing the serum concentrations of sACE2 2 .v2.4-IgG1, sACE2 2 .S19-IgG1, and sACE2 2 .S19-IgG1 (YTE) over 48 hours in retroorbital blood samples collected from human FcRn mice following 10 mg/kg administration into the tail vein.
  • FIG.12B is a line graph showing the catalytic activity of sACE2 2 .v2.4-IgG1 (10 mg/kg), sACE2 2 .S19-IgG1 (10 mg/kg), sACE2 2 .S19-IgG1 (100 mg/kg), and sACE2 2 .S19- IgG1 (YTE) (10 mg/kg) over 120 hours in retroorbital blood samples collected from human FcRn mice following subcutaneous administration into the flank.
  • YTE sACE2 2 .S19- IgG1
  • FIG.12C is a line graph showing the serum concentrations of sACE2 2 .v2.4-IgG1 (10 mg/kg), sACE2 2 .S19-IgG1 (10 mg/kg), sACE2 2 .S19-IgG1 (100 mg/kg), and sACE2 2 .S19- IgG1 (YTE) (10 mg/kg) over 120 hours in retroorbital blood samples collected from human FcRn mice following subcutaneous administration into the flank.
  • YTE sACE2 2 .S19- IgG1
  • FIG.13A is an image of a Coomassie-stained, non-reducing, SDS-polyacrylamide gel electrophoresis of 20 ⁇ g of sACE2 2 .S19-IgG1 (YTE) treated with PNGase F or neuraminidase.
  • FIG.13B is an image of a Coomassie-stained, isoelectric focusing gel of 10 ⁇ g of sACE2 2 .S19-IgG1 (YTE) treated with PNGase F or neuraminidase (NA) in the presence (+) or absence (-) of a gel filtration purification step.
  • FIG.13C is a line graph showing the serum concentrations of untreated sACE2 2 .v2.4- 9 IPTS/125367707.1 Attorney Docket No.: CYR-004WO IgG1 (YTE), untreated sACE2 2 .S19-IgG1 (YTE), PNGase F treated sACE2 2 .S19-IgG1 (YTE), and NA treated sACE2 2 .S19-IgG1 (YTE) across 120 hours in retroorbital blood samples collected from human FcRn mice following 10 mg/kg administration into the tail vein.
  • FIG.14A is a gel image of Capillary Electrophoresis-SDS analysis of sACE2 2 .v2.4- IgG1 (YTE) and sACE2 2 .S19-IgG1 (YTE) purified from CHOK1SV GS-KO stable pools (left lanes) versus transiently transfected Expi293F (center-right lanes). Samples were analyzed under reducing (R) and non-reducing (NR) conditions, with accurate separation between the markers for 4.5 kD and 240 kD. IgG1 control is shown in the far right lanes.
  • FIGs.14B-14E show traces from capillary Isoelectric Focusing analysis of sACE2 2 .v2.4-IgG1(YTE) purified from Expi293F (FIG.14B), sACE2 2 .S19-IgG1(YTE) purified from Expi293F (FIG.14C), sACE2 2 .v2.4-IgG1(YTE) purified from CHOK1SV GS- KO (FIG.14D), and sACE2 2 .S19-IgG1(YTE) purified from CHOK1SV GS-KO (FIG.14E).
  • FIG.16A is a line graph showing plasma concentrations of sACE2 2 .v2.4-IgG1(YTE) (solid line) and sACE2 2 .S19-IgG1(YTE) (broken line) purified from stable CHOK1SV GS- KO pools after IV administration to human FcRn mice at a single dose of 10 mg/kg.
  • FIG.16B is a line graph showing plasma concentrations of sACE2 2 .S19-IgG1(YTE) purified from stable CHOK1SV GS-KO pools after IV (solid line) or SC (broken line) administration to human FcRn mice at a single dose of 10 mg/kg. Concentrations in plasma were measured by ELISA for 14 days.
  • FIG.17 is an immunogenicity heat map of sACE2 2 -IgG1 (WT), sACE2 2 .v2.4-IgG1, sACE2 2 .S19-IgG1 (YTE), 3N39v4 decoy, and 3J320v3 decoy for peptides predicted to have affinity for at least four HLA-II allotypes (Nhits ⁇ 4).
  • sACE2 2 -IgG1 derivatives are shaded 10 IPTS/125367707.1 Attorney Docket No.: CYR-004WO gray except for regions where mutations are introduced.
  • FIG.18 is a line graph showing the binding of wild type sACE2 2 -IgG1 (circles), sACE2 2 .v2.4-IgG1(YTE) (squares), or sACE2 2 .S19-IgG1(YTE) (triangles) to cells expressing the S proteins of omicron sublineages (clockwise from top-left) BF.7, XBB, BA.2.75.2, and BQ.1.1. Bound proteins were detected by flow cytometry.
  • the present application provides modified human angiotensin-converting enzyme 2 (ACE2) proteins and fusion proteins comprising modified human ACE2 proteins having enhanced activity and pharmacokinetics as compared to wild-type ACE2. Also provided are methods of producing modified human ACE2 proteins and fusion proteins, and methods of treating SARS-CoV-2 infection in a subject using the same. [0068] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below. [0069] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate. [0070] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein.
  • Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans.
  • the term “effective amount” refers to the amount of a protein or fusion protein sufficient to effect beneficial or desired results (e.g., a desired prophylactic or therapeutic effect).
  • An effective amount can be administered in one or more administration(s), application(s) or dosage(s) and is not intended to be limited to a particular formulation or administration route.
  • the term “treating” includes any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.
  • the term “therapeutically effective amount” refers to a quantity of a specific substance (such as a modified human ACE2 polypeptide) sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit CoV replication or reduce CoV titer in a subject.
  • a therapeutically effective amount is the amount necessary to inhibit CoV replication by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% (as compared to the absence of treatment).
  • a 11 IPTS/125367707.1 Attorney Docket No.: CYR-004WO therapeutically effective amount is the amount necessary to reduce CoV titer in a subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% (as compared to the absence of treatment).
  • the therapeutically effective amount can also be the amount necessary to reduce or eliminate one of more symptoms of CoV infection, such as the amount necessary to reduce or eliminate fever, cough or shortness of breath.
  • a prophylactically effect amount is the amount necessary to reduce the risk of becoming infected with a CoV or developing disease, such as COVID-19, by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% (as compared to the absence of treatment).
  • the term “pharmaceutical formulation” refers to the combination of an active agent (e.g., protein or fusion protein) with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
  • an active agent e.g., protein or fusion protein
  • a carrier inert or active
  • pharmaceutically acceptable carrier refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • proteins or polypeptide are used interchangeably herein and refer to a polymer of repeating structural units connected by a peptide bond.
  • the repeating structural units of the peptide are amino acids including naturally occurring amino acids, non- naturally occurring amino acids, analogues of amino acids or any combination of these.
  • proteins or polypeptides may be post-translationally modified (e.g., glycosylated, phosphorylated, lapidated, acetylated, or conjugation with a labeling component).
  • sequence identity means the proportion of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 85%, the percentage denotes the fraction of matches over the length of sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; for example, wherein gap lengths of 5 amino acids or less, optionally 3 amino acids or less, are usually used. [0077] Percent sequence identity can be any integer from 60% to 100%.
  • Exemplary 12 IPTS/125367707.1 Attorney Docket No.: CYR-004WO embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below.
  • a reference sequence preferably BLAST using standard parameters, as described below.
  • One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.
  • For sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • sequence comparison algorithm When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. [0079] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1977) Nucleic Acids Res.25: 3389- 3402, respectively.
  • HSPs high scoring sequence pairs
  • T some positive-valued threshold score
  • These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc.
  • BLAST algorithm One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10 -5 , and most preferably less than about 10 -20 .
  • treating refers to any indicia of success in the treatment or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to a subject, such as a patient in need of treatment; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a subject's physical or mental well-being.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation.
  • the terms “treating” or “treatment” include management, slowing the progression, and abating the symptoms of a disease, pathology or condition.
  • the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value.
  • “about” means within a standard deviation using measurements generally acceptable in the art.
  • the term “about,” as used herein when referring to a measurable value such as an amount of mass, weight, time, volume, concentration, or percentage, is meant to encompass variations of in certain aspects ⁇ 20%, in certain aspects ⁇ 10%, in certain aspects ⁇ 9%, in certain aspects ⁇ 8%, in certain aspects ⁇ 7%, in certain aspects ⁇ 6%, in certain aspects ⁇ 5%, in certain aspects ⁇ 4%, in certain aspects ⁇ 3%, in certain aspects ⁇ 2%, in certain aspects ⁇ 1%, in certain aspects ⁇ 0.5%, and in certain 14 IPTS/125367707.1 Attorney Docket No.: CYR-004WO aspects ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods and/or employ the disclosed compositions.
  • the term “functional fragment thereof” refers to a portion of a protein or polypeptide that maintains the ability to perform a biological function of the whole protein or polypeptide.
  • a functional fragment of a polypeptide or protein of the present application maintains its ability to bind its cognate binding partner or ligand.
  • the terms “mutation” and “substitution” are used interchangeably and refer to the alteration of an amino acid, in the context of a reference amino acid sequence, to another amino acid.
  • an alteration of an amino acid in a reference amino acid sequence can occur at the N-terminal or C-terminal position or anywhere between those terminal positions. Substitutions may be interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. [0086] A substitution can be, but need not necessarily be, a conservative substitution.
  • substitutions typically include substitutions within the following groups: (i) alanine and glycine; (ii) isoleucine, leucine, and valine; (iii) aspartic acid and glutamic acid; (iv) asparagine, glutamine, serine, and threonine; (v) arginine and lysine; and (vi) phenylalanine and tyrosine.
  • the term “vector” refers to a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • the vector is a virus vector, such as a lentivirus vector.
  • the spike (S) glycoprotein of SARS-CoV-2 binds angiotensin-converting enzyme 2 (ACE2) on host cells.
  • S is a trimeric class I viral fusion protein that is proteolytically processed into S1 and S2 subunits that remain noncovalently associated in a prefusion state.
  • the virus has limited potential to escape sACE2-mediated neutralization without simultaneously decreasing affinity for native ACE2 receptors, thereby attenuating virulence.
  • fusion of sACE2 to the Fc region of human immunoglobulin can provide an avidity boost while recruiting immune effector functions and increasing serum stability.
  • Recombinant sACE2 has proven safe in healthy human subjects and patients with lung disease.
  • Modified ACE2 polypeptides of the present disclosure function to bind S and neutralize SARS-CoV2 infection.
  • modified ACE2 polypeptides of the present disclosure bind to a protein having a sequence at least 85% identical (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical) to SEQ ID NO:32.
  • SARS-CoV2 S Spike SARS-CoV2 S Spike (S) Glycoprotein MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQD LFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFG TTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRV YSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINL VRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYY VGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFR VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSA SFSTFKCYGVSPTKLND
  • modified ACE2 polypeptides of the present disclosure have a sequence at least 85% identical (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical) to SEQ ID NO:1.
  • ACE2 Human Angiotensin-Converting Enzyme 2 (ACE2) (without signal peptide) QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDK WSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTIL NTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEV GKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQ LIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRF WTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQ GFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHE MGHIQYDMAYAAQPFLLRNGANEGFHEAV
  • modified ACE2 polypeptides of the present disclosure include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty substitution(s) introducing one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty glycosylation site(s) not present in SEQ ID NO:1.
  • all position numbering with reference to modified ACE2 polypeptides is relative to SEQ ID NO:2, unless otherwise stated.
  • modified ACE2 polypeptides of the present disclosure include 18 IPTS/125367707.1 Attorney Docket No.: CYR-004WO T27Y, L79T, and N330Y substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, and N330Y substitutions and one or more substitutions selected from E166C, S167I, A246L, K288C, E433C, V491I, H535F, E536N, P538S/T, N580S/T, D615N, L624C, K631N, A632C, Y633S/T, M662S/T, I663W, A673V, A673Y, I679Y, N682W, A687F, V691C, I694F, T698E, N720S/T, and Q728N.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, and N330Y substitutions and one or more substitutions selected from S167I, A246L, V491I, H535F, I663W, A673V, A673Y, I679Y, N682W, A687F, I694F, and T698E.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, I663W, and A673Y substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, A673Y, and I694F substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, V491I, and I679Y substitutions. In other aspects, modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, N682W, and A687F substitutions. In other aspects, modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, S167I, A246L, and I694F substitutions. In other aspects, modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, V491I, A673Y, A687F, and T698E substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, S167I, V491I, H535F, and A673V substitutions.
  • modified ACE2 polypeptides of the present disclosure have T27Y, L79T, and N330Y substitutions and one or more substitutions that introduce a disulfide bond not present in wild-type human ACE2.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, and N330Y substitutions and one or more substitutions selected from K288C, E433C, L624C, A632C, E166C, and V691C.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, K288C and E433C substitutions. In other aspects, modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, L624C and A632C substitutions. In other aspects, modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, E166C and V691C substitutions. [0099] In certain aspects, modified ACE2 polypeptides of the present disclosure have T27Y, L79T, and N330Y substitutions and one or more substitutions that introduce a glycosylation site not present in wild-type human ACE2.
  • modified ACE2 19 IPTS/125367707.1 Attorney Docket No.: CYR-004WO polypeptides of the present disclosure include T27Y, L79T, and N330Y substitutions and one or more substitutions selected from E536N, P538S/T, N580S/T, D615N, K631N, Y633S/T, M662S/T, N720S/T, and Q728N.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, and M662S/T substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, and M662T substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, E536N, P538S/T, and M662S/T substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, E536N, P538S, and M662T substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, E536N, P538S/T, N580S/T and M662S/T substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, E536N, P538S, N580T, and M662T substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, M662S/T, and N720S/T substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, M662T, and N720S substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, D615N, M662S/T, and N720S/T substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, D615N, M662T, and N720S substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, E536N, P538S/T, M662S/T, and Q728N substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, E536N, P538S, M662T, and Q728N substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, K631N, Y633S/T, M662S/T, and Q728N substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, K631N, Y633T, M662T, and Q728N substitutions.
  • modified ACE2 polypeptides of the present disclosure have T27Y, L79T, N3307, M662S/T, and I694F substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, K631N, M662T, and I694F substitutions.
  • modified ACE2 polypeptides of the present disclosure have T27Y, L79T, N330Y, V491I, M662S/T, and N720S/T substitutions.
  • modified ACE2 polypeptides of the present disclosure include T27Y, L79T, N330Y, 20 IPTS/125367707.1 Attorney Docket No.: CYR-004WO V491I, M662T, and N720S substitutions.
  • modified ACE2 polypeptides of the present disclosure includes a sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to SEQ ID NO:12.
  • modified ACE2 polypeptides of the present disclosure include a sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to any one of the sequences listed in TABLE 1.
  • modified ACE2 polypeptides of the present disclosure include a signal peptide fused to the N-terminus of the modified ACE2 polypeptide.
  • the signal peptide includes a sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to SEQ ID NO:11.
  • Human ACE2 Signal Peptide MSSSSWLLLSLVAVTAA SEQ ID NO:11
  • Modified ACE2 Fusion Polypeptides [00104] The present disclosure also provides modified ACE2 fusion proteins including a modified ACE2 polypeptide as previously described and a heterologous polypeptide.
  • the heterologous polypeptide is an Fc domain polypeptide, for example, a human Fc domain polypeptide.
  • the heterologous polypeptide is a human IgG1 Fc domain polypeptide, a human IgG2 Fc domain polypeptide, a human IgG3 Fc domain polypeptide, or a human IgG4 Fc domain polypeptide.
  • the heterologous polypeptide includes residues 221 to 447 of a human IgG1 Fc domain, wherein residue positions are numbered according to EU numbering.
  • the modified ACE2 fusion protein incorporates an Fc domain polypeptide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to SEQ ID NO:4.
  • modified ACE2 fusion proteins of the present disclosure include a human IgG1 Fc domain incorporating one or more substitution selected from: a substitution of methionine at position 252 to tyrosine; a substitution of serine at position 254 to threonine; and a substitution of threonine at position 256 to glutamic acid, wherein residue positions are numbered according to EU numbering.
  • modified ACE2 fusion proteins of the present disclosure include a human IgG1 Fc domain incorporating a substitution of methionine at position 252 to tyrosine; a substitution of serine at position 254 to threonine; and a substitution of threonine at position 256 to glutamic acid, wherein residue positions are numbered according to EU numbering.
  • the modified ACE2 fusion protein incorporates an Fc domain polypeptide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to SEQ ID NO:5.
  • modified ACE2 fusion proteins of the present disclosure include a 29 IPTS/125367707.1 Attorney Docket No.: CYR-004WO human IgG1 Fc domain having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to any one of the sequences listed in TABLE 2.
  • sequence identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%
  • a modified ACE2 fusion protein of the present disclosure includes a human IgG1 Fc domain polypeptide fused to the C-terminus of the modified ACE2 polypeptide.
  • a modified ACE2 fusion protein of the present disclosure includes a human IgG1 Fc domain polypeptide fused to the N-terminus of the modified ACE2 polypeptide.
  • a modified ACE2 fusion protein of the present disclosure includes 1 All references to accession numbers (i.e., GenBank numbers) throughout the application refer to the versions that were current in the database as of the filing date of the application.
  • the linker is one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, or twenty-five amino acid(s) in length.
  • the linker is a single amino acid in length.
  • the linker is a single serine residue.
  • a modified ACE2 fusion protein of the present disclosure includes a human IgG1 Fc domain polypeptide fused to the modified ACE2 polypeptide via a linker having a sequence selected from any one of the sequences listed in TABLE 3.
  • a modified ACE2 fusion protein of the present disclosure includes, from N-terminus to C-terminus, a modified ACE2 polypeptide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 31 IPTS/125367707.1 Attorney Docket No.: CYR-004WO 98%, 99%, 99.5%, or 100%) to any one of the sequences listed in TABLE 1, a linker sequence according to any one of the sequences listed in TABLE 3, and a human IgG1 Fc domain polypeptide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to any one of the sequences listed in
  • a modified ACE2 fusion protein of the present disclosure includes a sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to SEQ ID NO:6.
  • a modified ACE2 fusion protein of the present disclosure has a sequence 100% identical to SEQ ID NO:6.
  • a modified ACE2 fusion protein of the present disclosure includes a sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to SEQ ID NO:7.
  • a modified ACE2 fusion protein of the present disclosure has a sequence 100% identical to SEQ ID NO:7.
  • a modified ACE2 fusion protein of the present disclosure includes a sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to SEQ ID NO:8.
  • a modified ACE2 fusion protein of the present disclosure has a sequence 100% identical to SEQ ID NO:8.
  • a modified ACE2 fusion protein of the present disclosure includes a sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to SEQ ID NO:9.
  • a modified ACE2 fusion protein of the present disclosure has a sequence 100% identical to SEQ ID NO:9.
  • a modified ACE2 fusion protein of the present disclosure includes a sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to SEQ ID NO:10.
  • a modified ACE2 fusion protein of the present disclosure has a sequence 100% identical to SEQ ID NO:10.
  • a modified ACE2 fusion protein of the present disclosure includes a sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to any one of the sequences listed in TABLE 4.
  • IPTS/125367707.1 Attorney Docket No.: CYR-004WO TABLE 4: Exemplary Modified ACE2 Fusion Protein Sequences
  • IPTS/125367707.1 Attorney Docket No.: CYR-004WO
  • IPTS/125367707.1 Attorney Docket No.: CYR-004WO
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure have increased levels of glycosylation as compared to wild-type ACE2.
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides have 1 to 50% increased levels of glycosylation as compared to wild-type ACE2 (e.g., 1 to 50%, 5 to 50%, 10 to 50%, 15 to 50%, 20 to 50%, 25 to 50%, 30 to 50%, 35 to 50%, 40 to 50%, 45 to 50%, 1 to 45%, 5 to 45%, 10 to 45%, 15 to 45%, 20 to 45%, 25 to 45%, 30 to 45%, 35 to 45%, 40 to 45%, 1 to 40%, 5 to 40%, 10 to 40%, 15 to 40%, 20 to 40%, 25 to 40%, 30 to 40%, 35 to 40%, 1 to 35%, 5 to 35%, 10 to 35%, 15 to 35%, 20 to 35%, 25 to 35%, 30 to 35%, 1 to 30%, 5 to 30%, 10 to 30%, 15 to 30%, 20 to 30%, 25 to 30%, 1 to 25%, 5 to 25%, 10 to 25%, 15 to 25%, 20 to 25%, 1 to 20%, 5 to 20%, 5 to 20%
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure have 2 to 50-fold increased affinity (e.g., 2 to 50-fold, 4 to 50-fold, 6 to 50-fold, 8 to 50-fold, 10 to 50-fold, 15 to 50-fold, 20 to 50-fold, 25 to 50- fold, 30 to 50-fold, 35 to 50-fold, 40 to 50-fold, 45 to 50-fold, 2 to 40-fold, 4 to 40-fold, 6 to 40-fold, 8 to 40-fold, 10 to 40-fold, 15 to 40-fold, 20 to 40-fold, 25 to 40-fold, 30 to 40-fold, 35 to 40-fold, 2 to 35-fold, 4 to 35-fold, 6 to 35-fold, 8 to 35-fold, 10 to 35-fold, 15 to 35- fold, 20 to 35-fold, 25 to 35-fold, 30 to 35-fold, 2 to 30-fold, 4 to 30-fold, 6 to 30-fold, 8 to 30-fold, 10 to 30-fold,
  • the S protein is of a strain selected from Wuhan, alpha, beta, gamma, delta, and omicron. In some embodiments, the S protein is of BF.7, XBB, BA.2.75.2, or BQ.1.1 omicron sublineages.
  • affinity can be measured by any method known by persons of skill in the art. For example, in certain aspects, affinity is measured by biolayer interferometry (BLI).
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure have 2 to 50-fold increased avidity (e.g., 2 to 50-fold, 4 to 50-fold, 6 to 50-fold, 8 to 50-fold, 10 to 50-fold, 15 to 50-fold, 20 to 50-fold, 25 to 50-fold, 30 to 50-fold, 35 to 50-fold, 40 to 50-fold, 45 to 50-fold, 2 to 40-fold, 4 to 40-fold, 6 to 40- fold, 8 to 40-fold, 10 to 40-fold, 15 to 40-fold, 20 to 40-fold, 25 to 40-fold, 30 to 40-fold, 35 to 40-fold, 2 to 35-fold, 4 to 35-fold, 6 to 35-fold, 8 to 35-fold, 10 to 35-fold, 15 to 35-fold, 20 to 35-fold, 25 to 35-fold, 30 to 35-fold, 2 to 30-fold, 4 to 30-fold, 6 to 30-fold, 8 to 30- fold, 10 to 30-fold, 10 to 30-fold,
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding affinity to SARS- CoV-2 of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 11 36 IPTS/125367707.1 Attorney Docket No.: CYR-004WO nm, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, less than 30 nM, less than 35 nM, less than 40 nM, less than 45 nM, or less than 50 nM.
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding affinity of less than 1 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding affinity of less than 1.2 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding affinity of less than 2 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding affinity of less than 5 nM.
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding affinity of less than 10 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding affinity of less than 15 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding affinity of less than 20 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding affinity of less than 25 nM.
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding affinity of less than 30 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding affinity of less than 50 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding affinity of less than 75 nM.
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding avidity to SARS- CoV-2 of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 11 nm, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, less than 30 nM, less than 35 nM, less than 40 nM, less than 45 nM, or less than 50 nM.
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding avidity of less than 1 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding avidity of less than 1.2 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding avidity of less than 2 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding avidity of less than 5 nM.
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding avidity of less than 10 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding avidity of less than 15 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding avidity of less than 20 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding avidity of less than 25 nM.
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding avidity of less than 30 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding avidity of less than 50 nM. In some instances, modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure comprise a binding avidity of less than 75 nM.
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure have 1.2 to 2-fold increased catalytic activity as compared to wild-type ACE2 (e.g., 1.2 to 2-fold, 1.3 to 2-fold, 1.4 to 2-fold, 1.5 to 2-fold, 1.6 to 2-fold, 1.7 to 2-fold, 1.8 to 2-fold, 1.9 to 2-fold, 1.2 to 1.8-fold, 1.3 to 1.8-fold, 1.4 to 1.8- fold, 1.5 to 1.8-fold, 1.6 to 1.8-fold, 1.7 to 1.8-fold, 1.2 to 1.6-fold, 1.3 to 1.6-fold, 1.4 to 1.6- fold, 1.5 to 1.6-fold, 1.2 to 1.4-fold, or 1.3 to 1.4-fold).
  • wild-type ACE2 e.g., 1.2 to 2-fold, 1.3 to 2-fold, 1.4 to 2-fold, 1.5 to 2-fold, 1.6 to 2-fold, 1.7 to 2-fold, 1.8 to 2-fold, 1.9 to 2-fold,
  • catalytic activity can be measured by any method known by persons of skill in the art.
  • catalytic activity is measured by an in vitro fluorometric ACE2 activity assay.
  • at least 10% e.g. at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%
  • the glycosylation sites of a modified ACE2 polypeptide or modified ACE2 fusion polypeptide of the present disclosure are sialylated.
  • At least 20% of the glycosylation sites of a modified ACE2 polypeptide or modified ACE2 fusion polypeptide of the present disclosure are sialylated.
  • the percentage is a mole percentage.
  • sialylation can be measured by any method known by persons of skill in the art.
  • sialylation is measured by mass spectroscopy.
  • modified ACE2 fusion polypeptides of the present disclosure have a 2 to 10-fold (e.g., 2 to 10-fold, 3 to 10-fold, 4 to 10-fold, 5 to 10-fold, 6 to 10-fold, 7 to 10- fold, 8 to 10-fold, 9 to 10-fold, 2 to 9-fold, 3 to 9-fold, 4 to 9-fold, 5 to 9-fold, 6 to 9-fold, 7 38 IPTS/125367707.1
  • dissociation rates can be measured by any method known by persons of skill in the art.
  • dissociation rates are measured by BLI.
  • modified ACE2 fusion polypeptides of the present disclosure have an equilibrium dissociation constant for FcRn at pH 6.0 of 10 nM to 50 nM (e.g., 10 nM to 50 nM, 15 nM to 50 nM, 20 nM to 50 nM, 25 nM to 50 nM, 30 nM to 50 nM, 35 nM to 50 nM, 40 nM to 50 nM, 45 nM to 50 nM, 10 nM to 45 nM, 15 nM to 45 nM, 20 nM to 45 nM, 25 nM to 45 nM, 30 nM to 45 nM, 35 nM to 45 nM, 40 nM to 45 nM, 10 nM to 40 nM, 15
  • modified ACE2 fusion polypeptides of the present disclosure have an off-rate for FcRn at pH 6.0 of 5.0 x 10 -4 s -1 to 2.0 x 10 -3 s -1 (e.g., 5.0 x 10 -4 s -1 to 2.0 x 10 -3 s- 1 , 6.0 x 10 -4 s -1 to 2.0 x 10 -3 s -1 , 7.0 x 10 -4 s -1 to 2.0 x 10 -3 s -1 , 8.0 x 10 -4 s -1 to 2.0 x 10 -3 s -1 , 9.0 x 10 -4 s -1 to 2.0 x 10 -3 s -1 , 1.0 x 10 -3 s -1 to 2.0 x 10 -3 s -1 , 5.0
  • off-rate for FcRn can be measured by any method known by persons of skill in the art.
  • off-rate for FcRn is measured by BLI.
  • modified ACE2 fusion polypeptides of the present disclosure have a higher binding affinity to FcRn at pH 6.0 than at pH 7.0.
  • binding affinity can be measured by any method known by persons of skill in the art. For example, in certain 39 IPTS/125367707.1 Attorney Docket No.: CYR-004WO aspects, binding affinity is measured by BLI.
  • modified ACE2 fusion polypeptides of the present disclosure have a plasma half-life of 40 to 80 hours (e.g., 40 to 80 hours, 42 to 80 hours, 44 to 80 hours, 46 to 80 hours, 48 to 80 hours, 50 to 80 hours, 52 to 80 hours, 54 to 80 hours, 54 to 80 hours, 56 to 80 hours, 58 to 80 hours, 60 to 80 hours, 62 to 80 hours, 64 to 80 hours, 66 to 80 hours, 68 to 80 hours, 70 to 80 hours, 72 to 80 hours, 74 to 80 hours, 76 to 80 hours, 78 to 80 hours, 40 to 70 hours, 42 to 70 hours, 44 to 70 hours, 46 to 70 hours, 48 to 70 hours, 50 to 70 hours, 52 to 70 hours, 54 to 70 hours, 54 to 70 hours, 56 to 70 hours, 58 to 70 hours, 60 to 70 hours, 62 to 70 hours, 64 to 70 hours, 66 to 70 hours, 68 to 70 hours, 40 to 60 hours, 42 to 60 hours, 44 to 60 hours, 46 to 60 hours, 46 to 60 hours, 42
  • modified ACE2 fusion polypeptides of the present disclosure have a plasma half-life of about 75 hours. In certain aspects, modified ACE2 fusion polypeptides of the present disclosure have a plasma half-life in a range of about 70 to about 80 hours. In certain aspects, modified ACE2 fusion polypeptides of the present disclosure have a plasma half-life of about 125 hours. In certain aspects, modified ACE2 fusion polypeptides of the present disclosure have a plasma half-life in a range of about 110 to about 140 hours. In certain aspects, modified ACE2 fusion polypeptides of the present disclosure have a plasma half-life of about 225 hours.
  • modified ACE2 fusion polypeptides of the present disclosure have a plasma half-life in a range of about 210 to about 240 hours.
  • plasma half-life can be measured by any method known by persons of skill in the art.
  • plasma half-life is measured by ELISA or an in vitro fluorometric ACE2 activity assay.
  • modified ACE2 fusion polypeptides of the present disclosure have a plasma half-life of 40 to 240 hours (e.g., 40 to 240 hours, 50 to 240 hours, 60 to 240 hours, 70 to 240 hours, 80 to 240 hours, 90 to 240 hours, 100 to 240 hours, 110 to 240 hours, 120 to 40 IPTS/125367707.1 Attorney Docket No.: CYR-004WO 240 hours, 130 to 240 hours, 140 to 240 hours, 150 to 240 hours, 160 to 240 hours, 170 to 240 hours, 180 to 240 hours, 190 to 240 hours, 200 to 240 hours, 210 to 240 hours, 220 to 240 hours, 230 to 240 hours, 40 to 200 hours, 50 to 200 hours, 60 to 200 hours, 70 to 200 hours, 80 to 200 hours, 90 to 200 hours, 100 to 200 hours, 110 to 200 hours, 120 to 200 hours, 130 to 200 hours, 140 to 200 hours, 150 to 200 hours, 160 to 200 hours, 170
  • modified ACE2 fusion polypeptides of the present disclosure have a plasma activity area under the curve over 48 hours (AUC 0-48h ) of 50 to 150 ( ⁇ M product/minute) x h (e.g., 50 to 150 ( ⁇ M product/minute) x h, 60 to 150 ( ⁇ M product/minute) x h, 70 to 150 ( ⁇ M product/minute) x h, 80 to 150 ( ⁇ M product/minute) x h, 90 to 150 ( ⁇ M product/minute) x h, 100 to 150 ( ⁇ M product/minute) x h, 110 to 150 ( ⁇ M product/minute) x h, 120 to 150 ( ⁇ M product/minute) x h, 130 to 150 ( ⁇ M product/minute) x h, 140 to 150 ( ⁇ M product/minute) x h, 50 to 140 ( ⁇ M product/minute) x h, 60 to 140 ( ⁇ M product/minute) x h, 70 to 140 ( ⁇ M product/minute)
  • modified ACE2 fusion polypeptides of the present disclosure have an area under the curve (AUC 0-120h ) of 100 to 600 ⁇ g/ml x h (e.g., 100 to 600 ⁇ g/ml x h, 125 to 600 ⁇ g/ml x h, 150 to 600 ⁇ g/ml x h, 175 to 600 ⁇ g/ml x h, 200 to 600 ⁇ g/ml x h, 225 to 600 ⁇ g/ml x h, 250 to 600 ⁇ g/ml x h, 275 to 600 ⁇ g/ml x h, 300 to 600 ⁇ g/ml x h, 325 to 600 ⁇ g/ml x h, 350 to 600 ⁇ g/ml x h, 375 to 600 ⁇ g/ml x h, 400 to 600 ⁇ g/ml x h, 425 to 600 ⁇ g/ml x h, 425
  • modified ACE2 fusion polypeptides of the present disclosure have an area under the curve (AUC 0-t , where t is 120 to 336 hours) of 100 to 9000 ⁇ g/ml x h (e.g., 100 to 9000 ⁇ g/ml x h, 500 to 9000 ⁇ g/ml x h, 1000 to 9000 ⁇ g/ml x h, 1500 to 9000 ⁇ g/ml x h, 2000 to 9000 ⁇ g/ml x h, 2500 to 9000 ⁇ g/ml x h, 3000 to 9000 ⁇ g/ml x h, 3500 to 9000 ⁇ g/ml x h, 4000 to 9000 ⁇ g/ml x h, 4500 to 9000 ⁇ g/ml x h, 5000 to 9000 ⁇ g/ml x h, 5500 to 9000 ⁇ g/ml x h,
  • modified ACE2 fusion polypeptides of the present disclosure maintain a plasma concentration above 18 pM (e.g., above 18 pM, above 20 pM, above 22 pM, above 24 pM, above 26 pM, above 28 pM, above 30 pM, above 32 pM, above 34 pM, above 36 pM, above 38 pM, above 40 pM, above 42 pM, above 44 pM, above 46 pM, above 48 pM, above 50 pM, above 75 pM, or above 100 pM) at 100 to 150 hours post- administration to a subject.
  • 18 pM e.g., above 18 pM, above 20 pM, above 22 pM, above 24 pM, above 26 pM, above 28 pM, above 30 pM, above 32 pM, above 34 pM, above 36 pM, above 38 pM, above 40 pM, above 42 pM, above 44 pM,
  • modified ACE2 fusion polypeptides of the present disclosure maintain a plasma concentration above 18 pM (e.g., above 18 pM, above 100 pM, above 300 pM, above 1 nM, above 3 nM, above 10 nM, above 30 nM, or above 100 nM) at 100 to 150 hours post-administration to a subject.
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure have reduced immunogenicity when administered to a subject as compared to a wild-type ACE2 protein administered to the same subject.
  • modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure are non-immunogenic when administered to a subject.
  • modified ACE2 fusion polypeptides of the present disclosure form stable homodimers at physiological pH.
  • dimerization of modified ACE2 fusion polypeptides is measured by gel filtration chromatography and/or size exclusion chromatography.
  • Modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure can be used in the manufacture of pharmaceutical formulations.
  • pharmaceutical formulations disclosed herein include modified ACE2 polypeptides and modified ACE2 fusion polypeptides of the present disclosure and a pharmaceutically 43 IPTS/125367707.1 Attorney Docket No.: CYR-004WO acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc.
  • pharmaceutically acceptable it is meant a material that is not toxic or otherwise undesirable, i.e. the material may be administered to a subject without causing any undesirable biological effects.
  • pharmaceutical formulations can comprise sterile aqueous and non-aqueous injection solutions, which are optionally isotonic with the blood of the subject to whom the pharmaceutical formulation is to be delivered.
  • Pharmaceutical formulations can contain anti-oxidants, buffers, bacteriostats and solutes, which render the composition isotonic with the blood of the intended subject to be administered.
  • Aqueous and non-aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • pharmaceutical formulations comprise pharmaceutically acceptable vehicles and can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • pharmaceutical formulations can be presented in unit/dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.
  • pharmaceutical formulations disclosed herein can be formulated for intravenous, intramuscular, subcutaneous, intraperitoneal, or inhalatory administration. V.
  • a nucleic acid molecule encoding a modified ACE2 polypeptide or modified ACE2 fusion polypeptide described herein can be incorporated into a vector and introduced into a cell.
  • a cell has one or more than one nucleic acid encoding a modified ACE2 polypeptide or modified ACE2 fusion polypeptide described herein.
  • Vectors can be introduced into a cell by a variety of methods, including transformation, transfection, direct uptake, projectile bombardment, and by encapsulation of 44 IPTS/125367707.1 Attorney Docket No.: CYR-004WO the vector in a liposome or nanoparticle.
  • suitable methods of transfecting or transforming cells are calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake.
  • Expression vectors for use in the compositions and methods described herein contain a polynucleotide sequence that encodes a modified ACE2 polypeptide or modified ACE2 fusion polypeptide as previously described as well as, e.g., additional sequence elements used for the expression of the polypeptide and/or the integration of the polynucleotide sequence into the genome of a mammalian cell.
  • Certain vectors that can be used include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.
  • Other useful vectors contain polynucleotide sequences that enhance the rate of translation or improve the stability or nuclear export of mRNA.
  • sequence elements include, e.g., 5’ and 3’ UTR regions, an internal ribosomal entry site (IRES), and polyA in order to direct efficient transcription of the gene carried on the expression vector.
  • the expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker are a gene that encodes green fluorescent protein or a gene that encodes resistance to an antibiotic. VI. Methods of Production [00144] Also provided in the present disclosure are methods of producing modified ACE2 polypeptides and modified ACE2 fusion polypeptides described herein.
  • a method for producing a modified ACE2 polypeptide or modified ACE2 fusion polypeptide of the present disclosure by expressing a nucleic acid vector in a cell line.
  • the expression cell line is ExpiCHO-S, Expi293F, or CHOK1SV GS-KO cells.
  • expression methods of the present disclosure provide modified ACE2 polypeptides or modified ACE2 fusion polypeptides in which at least 10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at 45 IPTS/125367707.1 Attorney Docket No.: CYR-004WO least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the polypeptides are glycosylated.
  • the percentage is a mole percentage.
  • glycosylation is measured by mass spectrometry.
  • expression methods of the present disclosure provide modified ACE2 polypeptides or modified ACE2 fusion polypeptides in which at least 10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the polypeptides are glycosylated with N-acetylhexosamine.
  • the percentage is a mole percentage.
  • glycosylation is measured by mass spectrometry.
  • expression methods of the present disclosure provide modified ACE2 polypeptides or modified ACE2 fusion polypeptides in which at least 20% (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the glycosylations of the polypeptides are sialylated.
  • the percentage is a mole percentage.
  • sialylation is measured by mass spectrometry. VII.
  • kits for treating SARS CoV-2 infection in a subject by administering a modified ACE2 polypeptide, a modified ACE2 fusion polypeptide, or a pharmaceutical formulation as described herein to the subject.
  • the present disclosure provides a method of inhibiting SARS CoV-2 replication in a subject by administering to the subject a therapeutically or prophylactically effective amount of a modified ACE2 polypeptide, a modified ACE2 fusion polypeptide, or a pharmaceutical formulation as described herein.
  • the modified ACE2 polypeptide, the modified ACE2 fusion polypeptide, or the pharmaceutical formulation as described herein is administered to the subject intravenously, subcutaneously, intramuscularly, intratracheally, or by inhalation.
  • the SARS CoV-2 strain is selected from Wuhan, alpha, beta, gamma, delta, or omicron variants.
  • EXAMPLE 1 Low sialylation of sACE22.v2.4-IgG1 is correlated with rapid clearance in vivo
  • This example describes the in vivo clearance of modified ACE2 fusion proteins of the present disclosure.
  • Modified ACE2 fusion protein (sACE2 2 .v2.4-IgG1) produced in either non-human ExpiCHO-S or human Expi293F cells, was intravenously (IV) administered to transgenic mice expressing human FcRn under the control of a human promoter (B6.Cg-Fcgrt tm1Dcr Tg(FCGRT)32Dcr/DcrJ mice).
  • sACE2 2 .v2.4-IgG1 produced in ExpiCHO-S cells was rapidly cleared.
  • plasma ACE2 catalytic activity of sACE2 2 .v2.4-IgG1 produced in ExpiCHO-S cells fell to levels close to background within 2 hours.
  • plasma ACE2 activity rapidly fell during the initial distribution phase for the first hour, but then remained elevated during a slower elimination phase (FIG 1A).
  • the plates were washed 4x with PBS containing 0.05% Tween 20 (PBS-T).
  • Purified protein standards were prepared in mouse plasma. Samples and standards were diluted in PBS and incubated in wells for 1 h at room temperature. Plates were washed 4x with PBS-T and wells were incubated 1 h at room temperature with 0.3-1.0 ⁇ g/ml polyclonal goat anti-human/mouse/rat/hamster ACE2 antibody in PBS. Plates were washed 4x with PBS-T and incubated 1 h at room temperature with 1:25,000 donkey anti-goat (H+L) antibody-HRP conjugate in PBS.
  • H+L donkey anti-goat
  • glycoproteins were diluted with 50 mM ammonium bicarbonate buffer, reduced with DTT, alkylated with iodoacetamide and desalted with 10 kDa molecular-weight cutoff centrifugal filtration devices.
  • N-Glycans were released with PNGaseF (37 °C for 48 h) and permethylated.
  • the O-glycans were removed by beta elimination and samples were treated with 50 mM sodium hydroxide and sodium borohydride for 18 h at 45 °C.
  • the released O-glycans were purified by Dowex H+ form ion exchange resin, lyophilized, borates were removed by a stream of nitrogen gas, and O-glycans were permethylated. Permethylated N- and O-glycans were analyzed by MALDI-TOF-MS. N- and O-glycan structures were assigned based on precursor masses (sodiated) and the common mammalian biosynthetic pathway. [00160] Glycopeptide analysis and site mapping were performed using LC-MS/MS. Glycoprotein samples were reduced with DTT, alkylated with iodoacetamide, and digested with 0.5 ⁇ g/ ⁇ l sequencing-grade trypsin at 37 °C for 16 h.
  • Nano-LC nanoscale liquid chromatography
  • a mass spectrometer connected to a mass spectrometer.
  • Nano-LC columns of 15 cm length with 75 ⁇ m internal diameter, filled with 3 ⁇ m C18 reverse phase material were used for chromatographic separation of the samples.
  • the separation conditions were low to high acetonitrile in a solution containing 0.1% formic acid, and the separation time was 1 h.
  • the precursor ion scan was acquired at 120,000 resolution in an ion trap mass analyzer and precursors at a time frame of 3 s were selected for subsequent fragmentation using HCD.
  • Charge state screening was enabled, and precursors with unknown charge state or a charge state of +1 were excluded. Dynamic exclusion was enabled (exclusion duration of 30 s).
  • the fragment ions were analyzed on an ion trap mass analyzer for HCD at 30,000 resolution.
  • the glycoproteomic data were processed with peptide and protein identification software and searched against the sequences and a catalogue of more than 180 N-glycans and 9 common O-glycans.
  • the precursor mass tolerance and fragment mass tolerances were set to 5 ppm and 10 ppm respectively. Additional modifications including deamidation of N and Q, carboxymethylation of C, and oxidation of precursor were also included in the search. Assignments were made using peptide and protein identification software (Delta Mod. Score ⁇ 10, Log Prob>3) and manual interpretation.
  • Raw files retrieved from nanoLC-MS were deconvoluted, and monoisotopic peak areas for each glycopeptide 48 IPTS/125367707.1 Attorney Docket No.: CYR-004WO found in the peptide and protein identification software were manually pooled.
  • the relative percentages of each glycoform were determined by deconvolution of the LC-MS data at the full MS level, then determining the area under the curve for each full MH+. Relative abundance was then calculated. Suggested glycan structures were predicted. Glycoforms are based on the assumed biosynthetic pathway.
  • O-glycans at Thr730 of ExpiCHO-S produced sACE2 2 .v2.4-IgG1 are almost exclusively monosialylated core 1 structures (FIG.4D), whereas O-glycans decorating sACE2 2 .v2.4-IgG1 produced in Expi293F cells are more complex and 80% disialylated (FIG. 4E). High sialylation is thus correlated with extended activity of sACE2 2 .v2.4-IgG1 in vivo.
  • EXAMPLE 2 Derivatives of sACE22.v2.4-IgG1 have higher catalytic activity in vitro and in vivo
  • This example describes the in vitro and in vivo catalytic activities of modified ACE2 fusion proteins derived from sACE2 2 .v2.4-IgG1.
  • Rosetta-Based Stability Design [00163] Stability was optimized using an in silico rational protein engineering approach based on the cryo-EM structure of human ACE2 (PDB 6M17). Stabilizing mutations in ACE2 were identified using the Rosetta software package (Leman et al. (2020) Nat Methods 17:665-680, incorporated by reference in its entirety).
  • truncated models of ACE2 monomers (amino acids [a.a.] 21-615 and 21-732) underwent backbone and sidechain optimization via Rosetta Relax with score function ref2015.
  • the protocol performed site saturation mutagenesis for both relaxed construct models, allowing neighboring amino acids to repack around a mutated residue (for example, as described in Frenz et al. (2020) Front Bioeng Biotechnol 8, incorporated by reference in its entirety).
  • the mutants were ranked according to the difference in score between the wild type amino acid and the substitutions.
  • Disulfide Design [00164] Disulfide bonds require specific geometric constraints for formation.
  • sACE2 2 .v2.4-IgG1 encompasses human ACE2 residues 18- 732 and is a stable dimer in both the ACE2 and IgG1 Fc moieties.
  • FIGs.5B, 5C, and 5D three additional design strategies were explored. First, human and mouse ACE2 sequences were compared to identify polymorphic residues.
  • Mouse ACE2 fused to murine IgG1 Fc has a long serum half-life (elimination phase t 1/2 ⁇ ) of 174 hours following intravenous (IV) administration (Liu et al. (2016) Kidney Int 94:114-125).
  • the polymorphic residues were selected for computational saturation mutagenesis and modelling using ROSETTA software (Leman et al. (2020) Nat Methods 17: 665-680). Multiple mutations were identified that decrease the computed ⁇ G for folding. Second, residue pairs were identified that when mutated to cysteine have a high probability (based on ROSETTA modeling) of oxidizing to form disulfides that constrain conformational dynamics.
  • sACE2 2 .v2.4-IgG1 Derivatives Designed with Additional Glycosylation Sites Construct Name Class Mutations sACE2 2 .S11-IgG1 Glycosylation M662T sACE2 2 .S12-IgG1 Glycosylation E536N, P538S, M662T sACE2 2 .S13-IgG1 Glycosylation E536N, P538S, N580T, M662T sACE2 2 .S14-IgG1 Glycosylation M662T, N720S sACE2 2 .S15-IgG1 Glycosylation D615N, M662T, N720S sACE2 2 .S16-IgG1 Glycosylation E536N, P538S, M662T, Q728N sACE2 2 .S17-IgG1 Glycosylation K631N, Y633T, M662
  • sACE2 2 .v2.4-IgG1 Derivatives Designed for Serum Stability and Additional Glycosylation Sites Construct Name Class Mutations sACE2 2 .S18-IgG1 Mixed M662T, I694F sACE2 2 .S19-IgG1 Mixed V491I, M662T, N720S Cell Lines and Transfection [00169] Expi293F cells were cultured at 37 °C, 125 r.p.m., 8% CO 2 , in Expi293 Expression Medium.
  • Expi293F were transfected at a density of 2 x 10 6 ml -1 with 0.5-1.0 ⁇ g plasmid 51 IPTS/125367707.1 Attorney Docket No.: CYR-004WO DNA per mL of culture.
  • Transfection enhancers 1 (5 ⁇ l per ml of culture) and 2 (50 ⁇ l per ml of culture) were added ⁇ 18 h post-transfection and medium was collected on day 5-7.
  • ExpiCHO-S cells were cultured at 37 °C, 125 r.p.m., 8% CO 2 , in ExpiCHO Expression Medium.
  • ExpiCHO-S were transfected at a density of 6 x 10 6 ml -1 with 1.0 ⁇ g plasmid DNA per ml of culture using Expifectamine CHO Reagent according to the manufacturer’s directions.
  • Expifectamine CHO Enhancer (6 ⁇ l per ml of culture) and ExpiCHO Feed (240 ⁇ l per ml of culture) were added ⁇ 20 h later and the temperature was lowered (33 °C).
  • ExpiCHO Feed (240 ⁇ l per ml of culture) was added again on day 5.
  • CO 2 was decreased step-wise on days 9-12 to 5% final.
  • the medium was harvested on days 12-14.
  • Plasmids [00170] The expression plasmid for sACE2 2 .v2.4-IgG1 is previously described (Chan et al. (2020) Science (1979)369: 1261-1265) and deposited. Briefly, the coding sequence is ligated into the NheI-XhoI sites of pcDNA3.1(+) and encompasses human ACE2 (GenBank NM_021804.1) amino acids (a.a.) M1-G732 fused via a 1x serine linker to human IgG1 Fc a.a. D221-K447 (nG1m1 isoallotype; GenBank KY432415.1). Targeted mutations were introduced by overlap extension PCR and confirmed by Sanger sequencing.
  • Expression medium was centrifuged (800 g, 4 °C, 10 min) to remove cells and the pH adjusted to 7.5 with 1M Tris base. Insoluble particulates were removed by centrifugation (15,000 g, 4 °C, 20 min). The supernatant was incubated with Protein A affinity chromatography resin (2 ml resin per 100 ml medium) for 1-2 h at 4 °C. Resin was collected by passing through a chromatography column and washed with 10 column volumes (CV) of Dulbecco’s phosphate-buffered saline (PBS).
  • CV column volumes
  • mice were administered protein solutions IV (tail vein) or SC (flank) diluted in PBS and sterile filtered for a final injection volume of 4 ml / kg.
  • blood was collected via retroorbital route into heparin tubes.
  • mice were euthanized via CO 2 asphyxiation and blood was collected via cardiocentesis into heparin.
  • Processed plasma samples were stored at -80 °C. Experiments were conducted under institutional review and IACUC approval at JAX. Pharmacokinetic parameters were fitted with PKSolver 2.0 using the linear trapezoidal method.
  • ACE2 Catalytic Activity Assay Hydrolysis of a quenched fluorescent peptide substrate was measured on a microplate reader using a Fluorometric ACE2 Activity Assay Kit according to the manufacturer’s directions.
  • Biolayer Interferometry For measuring kinetics of sACE2 2 -IgG1 / RBD interactions, responses were recorded on a biolayer interferometry (BLI) biosensor and analyzed with a 1:1 binding model (global fit) using instrument software.
  • sACE2 2 -IgG1 proteins were immobilized at 100 nM for 60 s to AHC (anti-human IgG Fc) biosensors in assay buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20, 0.5% non-fat dry milk). Loaded sensors were equilibrated in assay buffer, transferred to delta RBD-8h solutions to measure association, and transferred back to buffer to measure dissociation. The purification of delta RBD is previously described (Zhang et al. (2022) Nat Chem Biol 18: 342-351).
  • Biosensors were then transferred to sACE2 2 -IgG1 solutions in the same buffer for association kinetics and back to buffer for dissociation kinetics.
  • 19 derivatives of sACE2 2 .v2.4-IgG1 15 were secreted at high levels in transiently transfected Expi293F culture and were purified. While the designs were all catalytically active, as shown in FIGs.6A, 6B, and 6C, those with cavity-filling mutations tended to have reduced proteolytic activity.
  • Glycomics analysis indicated that sACE2 2 .S19-IgG1, expressed and purified from Expi293F cell culture, was highly sialylated and similar to parental sACE2 2 .v2.4-IgG1 54 IPTS/125367707.1 Attorney Docket No.: CYR-004WO protein (FIG.8A, 8B, 9A, 9B, and 9C).
  • Glycopeptidomics analysis of sACE2 2 .S19-IgG1 produced in Expi293F cell culture was compared to the parental sACE2 2 .v2.4-IgG1 protein produced in Expi293F cells and ExpiCHO-S cells.
  • glycoforms present at each site are highly heterogenous (TABLES 9-18).
  • ⁇ 20-60% of the glycoforms contained at least one sialic acid, with the exceptions of N322 and N810 (equivalent to N297 of IgG1 Fc using EU numbering) where sialylation was absent (FIG.10B).
  • Sialylated glycans had very low relative abundance ( ⁇ 2%) at all positions in ExpiCHO-S-produced sACE2 2 .v2.4-IgG1, although it was moderately higher (8.7%) at position N546.
  • the O-glycosylation site at T730 was poorly glycosylated in the proteins produced in Expi293F cells (2.3-5.0% glycosylated) and approximately half the glycoforms were sialylated (TABLE 19).
  • sACE2 2 .v2.4-IgG1 expressed in ExpiCHO-S cells was more highly O-glycosylated (11.1%) and almost exclusively with a single N- acetylhexosamine (TABLE 19).
  • T730 was found to be 97% glycosylated (Shajahan et al. (2021) Glycobiology 31:410-424) and may 68 IPTS/125367707.1 Attorney Docket No.: CYR-004WO reflect differences in expression systems, construct length, and fusion to IgG1 (here, sACE2 2 .v2.4-IgG1 and sACE2 2 .S19-IgG1 are fused at residue G732 to IgG1 Fc, thus changing the environment around T730).
  • YTE mutations enhanced the affinities of sACE2 2 .v2.4-IgG1 and sACE2 2 .S19-IgG1 for FcRn using biolayer interferometry (BLI).
  • FcRn binding at pH 6.0 was tighter than at pH 7.4, reflecting the biological role of FcRn to capture internalized IgG in the acidic endosome and recycle it to the cell surface for release.
  • Dissociation constants of modified ACE2 fusion protein in the absence of YTE mutations were estimated to be > 100 nM (accurate fitting of kinetic rate constants was not possible for these samples due to incomplete dissociation of the analyte).
  • sACE2 2 .v2.4- IgG1(YTE) and sACE2 2 .S19-IgG1(YTE) had dissociation constants for FcRn at pH 6.0 of 21 nM and 39 nM, respectively, with slow off rates of 5.9 x 10 -4 s -1 and 1.2 x 10 -3 s -1 .
  • IPTS/125367707.1 Attorney Docket No.: CYR-004WO [00184] 10 mg/kg of sACE2 2 .v2.4-IgG1 with or without YTE mutations, or 10 mg/kg sACE2 2 .S19-IgG1 with and without YTE mutations were administered to human FcRn transgenic mice intravenously and plasma protein concentrations were measured by ELISA (FIG.12A).
  • SC subcutaneously
  • substrate protein (5 mg/ml) was incubated with PNGase F (New England Biolabs; 125 units/ ⁇ l) in 1x Glycobuffer (New England Biolabs) prepared in PBS.
  • PNGase F New England Biolabs
  • 1x Glycobuffer New England Biolabs
  • substrate protein (6.8 mg/ml) was incubated with Arthrobacter ureafaciens neuraminidase (Roche; 1.7 units/ml) in PBS. Mixtures were separated on a gel filtration column to purify the treated sACE2 2 .S19-IgG1(YTE) proteins.
  • PNGase F cleaves the amide linkage between asparagine and the innermost N- acetylglucosamine, releasing the entire glycan.
  • sACE2 2 .S19-IgG1(YTE) produced in Expi293F cells
  • PNGase F under native conditions
  • the modified ACE2 fusion protein had increased electrophoretic mobility consistent with removal of large glycans (FIG.13A).
  • Multiple charged species of the protein were still observed on an isoelectric focusing (IEF) gel (FIG.13B), suggesting heterogenous sialylated glycans remained and deglycosylation was incomplete.
  • IEF isoelectric focusing
  • the elimination phase t 1/2 ⁇ values were 73 and 60 h for sACE2 2 .S19-IgG1(YTE) and sACE2 2 .v2.4-IgG1(YTE), respectively, and exposures (AUC 0-120h ) were 510 and 330 ⁇ g/mL ⁇ h. Exposure was decreased following PNGase F treatment of sACE2 2 .S19-IgG1(YTE), but the most dramatic effect was seen following treatment with neuraminidase. Desialylated sACE2 2 .S19- IgG1(YTE) was rapidly cleared and its exposure was decreased by two orders of magnitude. Sialylation of the decoy receptor is thus necessary for optimal pharmacokinetics.
  • EXAMPLE 4 Decoy receptors from stable pooled expression in CHOK1SV GS-KO cells
  • This example describes representative manufacture from stable CHOK1SV GS-KO pools of modified ACE2 fusion proteins derived from sACE2 2 .v2.4-IgG1 and their pharmacokinetics.
  • Biologic drugs are generally purified from CHO expression systems that have well described safety characteristics and for which there is extensive clinical experience.
  • CHOK1SV GS-KO stable pools were generated for the production of sACE2 2 .v2.4-IgG1(YTE) and sACE2 2 .S19-IgG1 (YTE).
  • sACE2 2 .v2.4-IgG1(YTE) and sACE2 2 .S19-IgG1(YTE) were designed with a consensus Kozak sequence and codon optimized for Cricetulus griseus. Genes were synthesized and subcloned into the HindIII and EcoRI sites of pPV-A.
  • the pPV- 71 IPTS/125367707.1 Attorney Docket No.: CYR-004WO A parts vectors were assembled into piggyBac destination vectors in an assembly reaction containing a 2:1 ratio of part vector to destination vector, T4 DNA ligase, and Esp3I. The reaction was cycled between 37 oC and 16 oC for the digestion and ligation phases of the assembly reaction, respectively.
  • CHOK1SV GS-KO cells were cultured in CD-CHO media supplemented with 6 mM L-glutamine. Cells were grown at 36.5 °C, 5% CO 2 , 85% humidity, 140 rpm. Cells were transfected via electroporation.
  • Bolus feeds were administered on days 4 and 8 consisting of a mixture of feeds. Production cultures were harvested on day 10 and sterile filtered. Fusion proteins were purified using affinity chromatography. The column was equilibrated with 50 mM sodium phosphate pH 7.0, 125 mM NaCl, and washed with 50 mM sodium phosphate pH 7.0, 1 M NaCl, followed by a second wash with equilibration buffer. Proteins were eluted with 10 mM NaHCOO pH 3.5 and eluents were neutralized with 10x PBS, pH 7.4, and 1 M Tris-HCl pH 8.0. to pH ⁇ 7.3.
  • Eluted protein fractions were concentrated and separated on a dextran-agarose matrix gel filtration column.
  • Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS) analysis was performed on an automated electrophoresis bioanalyzer instrument. Protein solutions were mixed 1:3 with denaturing solution with and without ⁇ -mercaptoethanol for reduced and non- reduced samples, respectively. Samples were heated to 95 °C for five minutes, diluted 1:14 with water, and loaded onto a primed microfluidic chip for electrophoresis.
  • Capillary isoelectric focusing electrophoresis cIEF was performed on a capillary electrophoresis system.
  • Samples were prepared at 0.2 mg/ml in sample buffer (0.35% methyl cellulose, 4% pharmalyte 3-10, 10 mM arginine) containing 4.05 and 9.99 pI markers.
  • 72 IPTS/125367707.1 Attorney Docket No.: CYR-004WO [00195]
  • Sialic acids were released by acid hydrolysis (2 M acetic acid) of the test samples under heat and the ⁇ -keto functionality of the free sialic acids underwent a condensation reaction with 1,2-diamino-4,5-methylenedioxybenzene (DMB) to form fluorescent reaction products.
  • DMB 1,2-diamino-4,5-methylenedioxybenzene
  • N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) were quantified.
  • N-acetylneuraminic acid (Neu5Ac)
  • N-glycolylneuraminic acid (Neu5Gc) were quantified.
  • decoy receptors purified from stable CHOK1SV GS-KO culture had equivalent purity to those from transiently transfected Expi293F cells (FIG.14A) but had sharper peaks with lower isoelectric points (pI) under isoelectric focusing (FIGs.14B-14E), suggesting reduced glycoform heterogeneity with high levels of negatively charged sugars.
  • Sialic acids were released by acid hydrolysis and reacted to form a fluorescent product that was quantified by reverse phase high performance liquid chromatography (RP-HPLC).
  • Decoy receptors from CHOK1SV GS-KO stable pools had twice the molar content of N-acetylneuraminic acid (Neu5Ac) than proteins transiently expressed by Expi293F (TABLE 21).
  • decoy receptors from CHOK1SV GS-KO stable pools tightly bound the RBDs of gamma and delta SARS-CoV-2 variants with monovalent binding affinities equivalent to proteins with less sialylation from Expi293F culture (FIG.15 and TABLE 22).
  • the data demonstrate that a stable CHO expression system is suitable for producing decoy receptors with high levels of sialylated N-glycans and potent affinity for virus spikes.
  • the highly sialylated decoy receptors had superior PK, with plasma concentrations remaining >10-fold higher than the EC90 for virus neutralization for at least 8 days (FIG 16A and TABLE 22).
  • EXAMPLE 5 Modified ACE2 Fusion Proteins Have Low Probability of Presenting Immunogenic Epitopes on HLA Class II
  • This example describes the predicted immunogenicity of modified ACE2 fusion proteins of the present disclosure.
  • sequences of modified ACE2 fusion proteins were scanned for peptides predicted to be displayed on a set of 14 common HLA-II allotypes.
  • sACE2 2 .v2.4-IgG1 has 3 mutations: there are no high affinity antigenic peptides predicted to encompass the T27Y mutation; the L79T mutation may be found at the end positions of a small number of presented peptides; and the N330Y mutation is predicted to remove an HLA-II epitope (FIG. 17).
  • V491I falls within a predicted epitope but is a highly conservative change of a single methyl group; while the mutations that add N-glycans at residues 660 and 718 were not analyzed as the computational algorithm is not trained on glycopeptides, although glycosylation may reduce display and T Cell receptor (TCR) recognition.
  • TCR T Cell receptor
  • Expi293F cells were transfected with 500 ng pcDNA3-myc-S plasmid per mL of culture using Expifectamine. Cells were harvested (600 ⁇ g, 1 min) 24 h post- transfection without the addition of transfection enhancers. Cells were washed with cold PBS supplemented with 0.2% bovine serum albumin (PBS-BSA) to reduce non-specific binding. Cells were resuspended in PBS-BSA and incubated with serial dilutions of sACE2 2 -IgG1 proteins on ice for 30 minutes.
  • PBS-BSA bovine serum albumin

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Abstract

La présente divulgation concerne de manière générale des polypeptides de l'enzyme de conversion de l'angiotensine humaine 2 (ACE2) et des protéines de fusion de ACE2 qui présentent une activité et/ou une pharmacocinétique améliorées, qui peuvent être utilisés en tant qu'agents thérapeutiques pour la prophylaxie (prophylaxie pré- ou post-exposition), ou le traitement de la COVID-19, ou d'une maladie provoquée par un quelconque coronavirus qui utilise l'ACE2 en tant que récepteur cellulaire.
EP23901407.9A 2022-12-05 2023-12-04 Protéines solubles de l'enzyme de conversion de l'angiotensine 2 (ace2) et protéines de fusion d'ace2 Pending EP4646222A1 (fr)

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