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WO2022119380A1 - Nouveau variant d'eca2 et utilisation associée - Google Patents

Nouveau variant d'eca2 et utilisation associée Download PDF

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WO2022119380A1
WO2022119380A1 PCT/KR2021/018229 KR2021018229W WO2022119380A1 WO 2022119380 A1 WO2022119380 A1 WO 2022119380A1 KR 2021018229 W KR2021018229 W KR 2021018229W WO 2022119380 A1 WO2022119380 A1 WO 2022119380A1
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ace2
converting enzyme
angiotensin converting
variant
seq
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권병세
임선우
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Eutilex Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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)

Definitions

  • the present invention relates to an angiotensin converting enzyme II (ACE2) variant having high coronavirus binding affinity and neutralizing ability, a fusion protein comprising the same, and its use for preventing or treating coronavirus.
  • ACE2 angiotensin converting enzyme II
  • Coronavirus is a virus that can infect various animals, including humans, and is a type of RNA virus whose genetic information is composed of ribonucleic acid (RNA). Coronaviruses cause respiratory and digestive system infections in humans and animals.
  • SARS-CoV severe acute respiratory syndrome-coronavirus
  • MERS-CoV Middle East Respiratory Syndrome-coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome-coronavirus-2
  • Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) emerged from Wuhan, China and spread rapidly worldwide, and the WHO named the disease infected with the virus COVID-19.
  • Common symptoms of SARS-CoV-2 infection include loss of taste or smell, cough, sore throat, headache, nausea, fatigue, diarrhea, and muscle pain.
  • SARS-CoV-2 is most easily spread through human-to-human contact through respiratory droplets generated by coughing and sneezing, and it is reported that it can be spread through air in confined spaces or spaces with insufficient ventilation, It has been reported that indirect contact routes to contaminated objects, which are known as the initial main route of infection, are rare. (CDC, C.f.d.c.a.p., How COVID-19 Spreads.
  • Coronavirus Disease 2019 (COVID-19), 2021), mainly spread from symptomatic patients, but it has been reported that asymptomatic infection is possible in some cases (Yu, P ., et al., J Infect Dis, 2020; Hoehl, S., et al., N Engl J Med, 2020; Bendix, A., Science alert, 2020).
  • the World Health Organization declared a 'Public Health Emergency of International Concern' (PHEIC) on January 30, 2020, and declared a pandemic (global pandemic) for the third time in history on March 11, 2020.
  • PHEIC Public Health Emergency of International Concern'
  • pandemic global pandemic
  • Angiotensin-converting enzyme 2 is a cell surface receptor that decomposes angiotensin II, a pro-inflammatory substance, and protects the kidneys, lungs, and heart from inflammation.
  • ACE2 Angiotensin-converting enzyme 2
  • RBD receptor binding domain
  • ACE2 angiotensin converting enzyme 2
  • the spike protein is considered as the most important target in the development of vaccines and therapeutics for coronavirus infection.
  • the SARS-CoV-2 neutralizing protein is the most representative material for the prevention and treatment of coronavirus infection, and the antibody is the most representative.
  • the binding affinity between the RBD of SARS-CoV-2 and the antibody is not necessarily linked to neutralizing ability against the virus, so an antibody that specifically binds to the RBD of SARS-CoV-2 is simply used as a treatment for COVID-19. The risk of failure is high.
  • an angiotensin converting enzyme II mutant library based on the binding structure of ACE2 and RBD was constructed, and from this Wild-type ACE2 protein or ACE2 variants having a significantly higher binding affinity for RBD than previously reported recombinant human ACE2 protein were screened, and the ACE2 variant exhibits significantly higher SARS-CoV-2 virus infection and amplification inhibition than wild-type ACE2. It was confirmed that the present invention was completed.
  • Another object of the present invention is to provide an angiotensin converting enzyme II (ACE2) variant having high coronavirus binding affinity and coronavirus neutralizing ability.
  • ACE2 angiotensin converting enzyme II
  • Another object of the present invention is to provide a fusion protein comprising the angiotensin converting enzyme II (ACE2) variant.
  • ACE2 angiotensin converting enzyme II
  • Another object of the present invention is to provide a nucleic acid encoding the angiotensin converting enzyme II (ACE2) variant or a fusion protein comprising the same.
  • ACE2 angiotensin converting enzyme II
  • Another object of the present invention is to provide a recombinant vector comprising the nucleic acid.
  • Another object of the present invention is to provide a recombinant cell in which the nucleic acid or recombinant vector is introduced into a host cell.
  • Another object of the present invention is to provide a method for producing an angiotensin converting enzyme II (ACE2) mutant or a fusion protein comprising the same.
  • ACE2 angiotensin converting enzyme II
  • Another object of the present invention is to provide the use of the angiotensin converting enzyme II (ACE2) variant or a fusion protein comprising the same to inhibit the proliferation of coronavirus, prevent and treat coronavirus infection.
  • ACE2 angiotensin converting enzyme II
  • the present invention provides an angiotensin converting enzyme II (ACE2) variant having high coronavirus binding affinity and neutralizing ability.
  • ACE2 angiotensin converting enzyme II
  • the present invention also provides a fusion protein comprising the angiotensin converting enzyme II (ACE2) variant.
  • ACE2 angiotensin converting enzyme II
  • the present invention also provides a nucleic acid encoding the angiotensin converting enzyme II (ACE2) variant or a fusion protein comprising the same.
  • ACE2 angiotensin converting enzyme II
  • the present invention also provides a recombinant vector containing the nucleic acid.
  • the present invention also provides a recombinant cell in which the nucleic acid or recombinant vector is introduced into a host cell.
  • the present invention also comprises the steps of culturing the recombinant cell to express the angiotensin converting enzyme II variant or a fusion protein comprising the same; And it provides a method for producing an angiotensin converting enzyme II (ACE2) variant or a fusion protein comprising the same, comprising the step of obtaining the expressed angiotensin converting enzyme II variant or a fusion protein comprising the same.
  • ACE2 an angiotensin converting enzyme II
  • the present invention also provides a pharmaceutical composition for preventing or treating a coronavirus infection comprising the angiotensin converting enzyme II mutant or a fusion protein comprising the same.
  • the present invention also provides a method for preventing and/or treating a coronavirus infection comprising administering to a subject the angiotensin converting enzyme II (ACE2) variant or a fusion protein comprising the same.
  • ACE2 angiotensin converting enzyme II
  • the present invention also provides the use of the angiotensin converting enzyme II (ACE2) mutant or a fusion protein comprising the same for preventing or treating coronavirus infection.
  • ACE2 angiotensin converting enzyme II
  • the present invention also provides the use of the angiotensin converting enzyme II (ACE2) variant or a fusion protein comprising the same for the preparation of a pharmaceutical composition for preventing and/or treating coronavirus infection.
  • ACE2 angiotensin converting enzyme II
  • the angiotensin converting enzyme II (ACE2) mutant of the present invention not only exhibits a higher binding affinity than the wild-type ACE2 protein, but also has significantly high coronavirus neutralizing ability, such as inhibition of coronavirus entry into cells and inhibition of proliferation, and even in variants of coronavirus It exhibits a wide range of neutralizing ability, so it is useful for inhibiting the proliferation of coronavirus infection, preventing infection and treating infectious diseases, and in particular, it can be usefully used as a preventive and therapeutic agent for COVID-19 pandemic.
  • Figure 2 shows a schematic diagram of the pYD5 template vector for the preparation of the ACE2 mutant library.
  • FIG. 3 shows the PCR conditions performed to construct a point mutation library containing a mutation in an amino acid at a specific position of ACE2.
  • Figure 4 schematically shows the transformation method of yeast for yeast surface expression technology (Yeast Surface Display, YSD).
  • FIG. 5 shows the results of FACS analysis of the binding affinity to SARS-CoV-2 S protein RBD of individual clones that have been round-sorted in a point mutation library.
  • FIG. 6 shows a schematic diagram and conditions of the Error prone library.
  • Figure 7a is a Facs analysis result of individual clones of the Q18-L100 error-prone library of the ACE2 H34A template.
  • Figure 7b is a Facs analysis result of individual clones of the Q18-D615 error-prone library of the ACE2 H34A template.
  • 7c is a Facs analysis result of an individual clone of the Q18-L100 error-prone library of the ACE2 8mut ((S19P, T27L, D30V, H34A, E35V, L79V, Q325P, N330Y) template.
  • 7d is a Facs analysis result of individual clones of the Q18-D615 error-prone library of the ACE2 8mut ((S19P, T27L, D30V, H34A, E35V, L79V, Q325P, N330Y) template.
  • FIG. 8 shows a schematic diagram of the vector used for the production of Fc domain fusion ACE2 variants.
  • Figure 9 is an SDS-PAGE result of the produced ACE2 mutant.
  • Figure 9a is a result of SDS-PAGE reduction by adding a reducing agent
  • Figure 9b is a result of SDS_PAGE without the addition of a reducing agent.
  • FIG. 10 shows the results of size exclusion chromatography of the produced binding affinity-enhancing ACE2 candidates (a: standard, b: wt ACE2, c: ACE2.V.43, d: ACE2.V.44, e: ACE2.V .45, f: ACE2.V.46, g: ACE2.V.47, h: ACE2.V.48)
  • Figure 13 is a comparison of the neutralizing ability of ACE2 wild type, ACE2.V.46 against the SARS-CoV-2 RBD mutant from the UK.
  • Figure 21 shows the result of SARS-CoV-2 protein reduction by the ACE2 mutant ACE2.V.06 treatment of the present invention.
  • FIG. 22 shows the results of SARS-CoV-2 CPE reduction by treatment with ACE2-WT or ACE2.V.06 of the present invention.
  • FIG. 23 shows the results of changes in the amount of SARS-CoV-2 protein and RNA by treatment with ACE2-WT or ACE2.V.06 of the present invention.
  • 24 shows the CPE reduction results of SARS-CoV-2 in culture for 24 hours after treatment with ACE2-WT or ACE2.V.06 of the present invention.
  • 25 shows the protein reduction results of SARS-CoV-2 in culture for 24 hours after treatment with ACE2-WT or ACE2.V.06 of the present invention.
  • 26 shows the RNA reduction results of SARS-CoV-2 in culture for 24 hours after treatment with ACE2-WT or ACE2.V.06 of the present invention.
  • FIG. 27 shows the CPE reduction results of SARS-CoV-2 in culture for 24 hours after treatment with ACE2-WT or ACE2.V.41 of the present invention.
  • RNA reduction results of SARS-CoV-2 after treatment with ACE2-WT or ACE2.V.41 of the present invention shows RNA reduction results of SARS-CoV-2 after treatment with ACE2-WT or ACE2.V.41 of the present invention.
  • FIG. 30 shows the CPE reduction results of SARS-CoV-2 after treatment with ACE2-WT or ACE2.V.41 of the present invention.
  • Figure 31 shows the result of SARS-CoV-2 protein reduction after treatment with ACE2-WT or ACE2.V.41 of the present invention.
  • Figure 32 shows the result of SARS-CoV-2 RNA reduction after treatment with ACE2-WT or ACE2.V.41 of the present invention.
  • nucleotide sequences described in the present specification were written from the 5' end to the 3' end, and all amino acid sequences were written from the N' end to the C' end.
  • COVID-19 caused by the SARS-CoV-2 virus is a global pandemic, and despite the fact that there have been more than 100 million confirmed cases and millions of deaths worldwide, delay in development and supply of vaccines and therapeutics The shortage is still not reducing the spread.
  • RBD of the spike protein and its receptor ACE2 a key molecule involved in cell infection and proliferation of coronavirus, are major targets for the prevention or treatment of coronavirus infection, and most currently developed therapeutic agents aim to neutralize RBD.
  • Antibodies which are representative RBD neutralizing proteins, are screened based on binding affinity, but binding affinity does not necessarily lead to neutralizing ability, so the success rate for therapeutic efficacy is low. Although reported, it binds to RBD competitively with ACE2 expressed in the target cells, and has a disadvantage in that the concentration-dependent and neutralizing effect is low.
  • the present inventors prepared an angiotensin converting enzyme II mutant library having a high coronavirus binding affinity by screening candidate amino acids for mutation that can exhibit a higher coronavirus binding affinity than wild-type ACE2.
  • angiotensin converting enzyme II variants having coronavirus neutralizing ability with high coronavirus binding affinity were screened from the prepared angiotensin converting enzyme II (ACE2) mutant library, and the screened angiotensin converting enzyme II It was confirmed that the mutant exhibited significantly superior coronavirus binding affinity and neutralizing ability compared to wild-type angiotensin converting enzyme II.
  • the ACE2 variant of the present invention binds to coronavirus with higher affinity than wild-type ACE2 upon administration, lowers the binding rate of coronavirus and cell surface ACE2, and exhibits high coronavirus neutralizing ability, prevention of coronavirus infections including COVID-19 And it can be usefully used for treatment.
  • the present invention relates to an angiotensin converting enzyme II (ACE2) variant having high coronavirus binding affinity and coronavirus neutralizing ability.
  • ACE2 angiotensin converting enzyme II
  • angiotensin converting enzyme II is one of metallo-carboxypeptidase, a type 1 transmembrane protein homologous to angiotensin converting enzyme, heart, lung, kidney, vascular endothelium It is mainly expressed in the digestive system and catalyzes the hydrolysis of angiotensin II and is known to play a role in preventing cardiovascular diseases.
  • ACE2 angiotensin converting enzyme II
  • angiotensin converting enzyme II is used interchangeably in the same meaning as "ACE2".
  • the sequence of SEQ ID NO: 1 disclosed in the present invention is a human wild-type ACE2 (hACE2) sequence, reported to UniProtKB as seq ID: Q9BYF1, and many wild-type ACE2 sequences substantially identical thereto have been reported.
  • the binding structure of wild-type ACE2 and coronavirus has been reported in detail in the art, and the sequence is conserved between species of coronavirus.
  • the angiotensin converting enzyme II may be one represented by the amino acid sequence of SEQ ID NO: 1, but is not limited thereto, and includes a sequence that is considered to be biologically and functionally substantially identical thereto. For example, it may be a sequence having 80% or more, 90% or more, preferably 95% or more, and most preferably 99% or more homology to SEQ ID NO: 1, but is not limited thereto.
  • the sequence of SEQ ID NO: 2 disclosed in the present invention is a fragment of angiotensin converting enzyme II (ACE2) including a signal sequence of angiotensin converting enzyme II (ACE2) of SEQ ID NO: 1 and a part of the extracellular domain.
  • ACE2 angiotensin converting enzyme II
  • ACE2 variant is used to mean including full-length protein and fragments thereof, so that the variant of SEQ ID NO: 2 is included in the range of angiotensin converting enzyme II (ACE2) variants of the present invention should be interpreted
  • the angiotensin converting enzyme II is interpreted to include variants or fragments thereof in which amino acid residues are conservatively substituted at specific amino acid residue positions.
  • conservative substitution refers to a modification of a receptor binding domain comprising substituting one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function of angiotensin converting enzyme II. .
  • Constant amino acid substitution means a substitution of an amino acid residue with an amino acid residue having a similar side chain.
  • classes of amino acid residues with similar side chains are well known in the art. These classes have basic side chains Amino acids (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg glycine, asparagine, glutamine, serine) , threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (eg, threonine, valine, isoleucine) and amino acids with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).
  • angiotensin converting enzyme II variant includes mutations of some amino acid residues in wild-type angiotensin converting enzyme II, preferably substitutions, deletions, insertions, etc. of amino acid residues, as well as N-terminal or C- It is used as a concept including all the cleavage of some amino acid residues at the terminal.
  • the angiotensin converting enzyme II (ACE2) variant has a mutation in any one or more amino acids selected from the group consisting of S19, T27, D30, H34, E35, L79, Q325 and N330 in SEQ ID NO: 2 can be done with
  • the angiotensin converting enzyme II (ACE2) variant may be characterized in that it has a mutation in the H34 amino acid in SEQ ID NO: 2.
  • the angiotensin converting enzyme II (ACE2) mutant is a mutation in the H34 amino acid in SEQ ID NO: 2;
  • It may be characterized by having a mutation in any one or more amino acids selected from the group consisting of N49, L79, L91, S280 and S602.
  • the angiotensin converting enzyme II (ACE2) variant may be characterized in that it has mutations in S19, T27, D30, H34, E35, L79, Q325 and N330 amino acids in SEQ ID NO: 2.
  • the angiotensin converting enzyme II (ACE2) variant is S19, T27, D30, H34, E35, L79, Q325 and N330 amino acids in SEQ ID NO: 2;
  • It may be characterized as having a mutation in any one or more amino acids selected from the group consisting of K26, K31, K74, L91, V185, N250 and G448.
  • the mutant in which the amino acid of the derived mutation site was substituted showed high binding affinity for SARS-CoV-2 S protein RBD.
  • the mutation is used as the broadest concept including all amino acid mutations such as substitution, deletion, insertion, glycosylation of amino acids, substitution of side chains, and the like.
  • the mutation may be characterized in that the substitution of amino acids.
  • the angiotensin converting enzyme II (ACE2) variant is S19W or S19P in SEQ ID NO: 2; K26N; T27W, T27D, T27A, T27Y, T27L, T27M or T27C; D30V; K31R; H34A or H34V; E35V; N49D; K74R; L79I, L79Y or L79V; L91P; V185A; N250K; S280R; Q325P; N330Y, N330H or N330F; G448V; R514Q; And any one or more selected from the group consisting of S602T, it may be characterized in that it has three or more mutations.
  • the angiotensin converting enzyme II (ACE2) variant is S19W or S19P in SEQ ID NO: 2; T27W, T27D, T27A, T27Y, T27L, T27M or T27C; D30V; H34A or H34V; E35V; L79Y or L79V; Q325P; and N330Y, N330H, or N330F may be characterized as having one or more mutations selected from the group consisting of.
  • the angiotensin converting enzyme II variant is S19W or S19P, T27L, D30V, H34A, E35V, L79V, Q325P and N330Y in SEQ ID NO: 2 It can be characterized in that it has any one or more mutations selected from the group consisting of have.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of H34A in SEQ ID NO: 2.
  • the angiotensin converting enzyme II (ACE2) mutant is a mutation of H34A in SEQ ID NO: 2;
  • It may be characterized as having one or more mutations selected from the group consisting of N49D, L79I, L91P, S280R and S602T.
  • the angiotensin converting enzyme II variant may be characterized in that it has mutations of S19W or S19P, T27L, D30V, H34A, E35V, L79V, Q325P and N330Y in SEQ ID NO: 2.
  • the angiotensin converting enzyme II variant is S19W or S19P, T27L, D30V, H34A, E35V, L79V, Q325P and N330Y in SEQ ID NO: 2;
  • It may be characterized as having one or more mutations selected from the group consisting of K26N, K31R, K74R, L91P, V185A, N250K and G448V.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of H34V in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of S19W in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of S19P in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of T27D in SEQ ID NO:2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of T27Y in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of T27L in SEQ ID NO:2.
  • the angiotensin converting enzyme II variant may be characterized in that it has a mutation of T27C in SEQ ID NO:2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of D30V in SEQ ID NO:2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of E35V in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of L79Y in SEQ ID NO: 2.
  • the angiotensin converting enzyme II variant may be characterized in that it has a mutation of L79V in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of Q325P in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of N330Y in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of N330F in SEQ ID NO: 2.
  • the angiotensin converting enzyme II variant may be characterized in that it has any one or more mutations selected from the group consisting of S19P, T27L, H34A, L79V and N330Y in SEQ ID NO: 2, preferably S19P, T27L , may be characterized as having mutations of H34A, L79V and N330Y.
  • the angiotensin converting enzyme II variant is selected from the group consisting of S19P, T27L, D30V, H34A, E35V, L79V, Q325P and N330Y in SEQ ID NO: 2 It may be characterized in that it has one or more mutations, Preferably, it may be characterized as having mutations of S19P, T27L, D30V, H34A, E35V, L79V, Q325P and N330Y.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of N330H in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has mutations of H34A and N330Y in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has mutations of H34V and N330H in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has mutations of T27Y and N330Y in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has mutations in T27Y and H34A in SEQ ID NO: 2.
  • the angiotensin converting enzyme II variant may be characterized in that it has one or more mutations selected from the group consisting of T27Y, H34A and N330Y in SEQ ID NO: 2, preferably T27Y, H34A and N330Y mutations It can be characterized as having.
  • the angiotensin converting enzyme II mutant may be characterized in that it has mutations of H34V and N330Y in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of K26N in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of K31R in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of L91P in SEQ ID NO: 2.
  • the angiotensin converting enzyme II variant may be characterized in that it has any one or more mutations selected from the group consisting of K26N, K31R and L91P in SEQ ID NO: 2, preferably K26N, K31R and L91P mutations It may be characterized as having
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of R514Q in SEQ ID NO: 2.
  • the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of L79I in SEQ ID NO: 2.
  • the angiotensin converting enzyme II variant may be characterized in that it has any one or more mutations selected from the group consisting of T27Y, H34A and L79Y in SEQ ID NO: 2, preferably T27Y, H34A and L79Y mutations It may be characterized as having
  • the angiotensin converting enzyme II variant may be characterized in that it has any one or more mutations selected from the group consisting of T27Y, H34A, L79Y and N330Y in SEQ ID NO: 2, preferably T27Y, H34A, L79Y and N330Y mutation.
  • the angiotensin converting enzyme II variant may be characterized in that it has any one or more mutations selected from the group consisting of T27Y, H34A, L79V and N330Y in SEQ ID NO: 2, preferably T27Y, H34A, L79V and N330Y mutation.
  • the angiotensin converting enzyme II variant may be characterized in that it has any one or more mutations selected from the group consisting of T27D, H34A, L79V and N330Y in SEQ ID NO: 2, preferably T27D, H34A, L79V and N330Y mutation.
  • the angiotensin converting enzyme II variant may be characterized in that it has any one or more mutations selected from the group consisting of T27Y, H34A, L79Y, L91P and N330Y in SEQ ID NO: 2, preferably T27Y, H34A , L79Y, L91P and N330Y may be characterized as having mutations.
  • the angiotensin converting enzyme II variant may be characterized in that it has any one or more mutations selected from the group consisting of T27Y, H34A, L79V, L91P and N330Y in SEQ ID NO: 2, preferably T27Y, H34A , L79V, L91P and N330Y may be characterized as having mutations.
  • the angiotensin converting enzyme II variant may be characterized in that it has any one or more mutations selected from the group consisting of T27D, H34A, L79V, L91P and N330Y in SEQ ID NO: 2, preferably T27D, H34A , L79V, L91P and N330Y may be characterized as having mutations.
  • the angiotensin converting enzyme II variant may be characterized in that it has any one or more mutations selected from the group consisting of S19P, T27D, H34A, L79V and N330Y in SEQ ID NO: 2, preferably S19P, T27D , may be characterized as having mutations of H34A, L79V and N330Y.
  • the angiotensin converting enzyme II variant may have any one or more mutations selected from the group consisting of S19P, T27D, H34A, L79V, L91P and N330Y in SEQ ID NO: 2, preferably S19P , T27D, H34A, L79V, L91P and N330Y may be characterized as having mutations.
  • the angiotensin converting enzyme II variant may be characterized in that it has any one or more mutations selected from the group consisting of S19P, T27Y, H34A, L79Y and N330Y in SEQ ID NO: 2, preferably S19P, T27Y , may be characterized as having mutations of H34A, L79Y and N330Y.
  • the angiotensin converting enzyme II variant may be characterized in that it has any one or more mutations selected from the group consisting of S19P, T27Y, H34A, L79V and N330Y in SEQ ID NO: 2, preferably S19P, T27Y , may be characterized as having mutations of H34A, L79V and N330Y.
  • the angiotensin converting enzyme II variant has any one or more mutations selected from the group consisting of S19P, T27Y, H34A, L79V, L91P and N330Y in SEQ ID NO: 2, preferably S19P, T27Y, H34A, It can be characterized as having three or more mutations selected from the group consisting of L79V, L91P and N330Y, and most preferably having mutations of S19P, T27Y, H34A, L79V, L91P and N330Y.
  • the angiotensin converting enzyme II variant has any one or more mutations selected from the group consisting of S19P, T27Y, H34A, L79Y, L91P and N330Y in SEQ ID NO: 2, preferably S19P, T27Y, H34A, It can be characterized as having three or more mutations selected from the group consisting of L79Y, L91P and N330Y, and most preferably having mutations of S19P, T27Y, H34A, L79Y, L91P and N330Y.
  • the "angiotensin converting enzyme II variant characterized in that it has a mutation” is used in the sense of including the angiotensin converting enzyme II variant having an additional mutation in addition to the mutation disclosed in the corresponding context.
  • the angiotensin converting enzyme II variant may be characterized as a variant or fragment thereof comprising a sequence selected from the group consisting of SEQ ID NOs: 3 to 45.
  • the angiotensin converting enzyme II variant is preferably a sequence selected from the group consisting of SEQ ID NOs: 33 (ACE2.V.36) to 45 (ACE2.V.48), more preferably SEQ ID NO: 43 ( ACE2.V.46) or 45 (ACE2.V.48) may be characterized as a variant or fragment thereof comprising the sequence represented by, most preferably, SEQ ID NO: 43 (ACE2.V46). .
  • the angiotensin converting enzyme II mutant may be characterized in that it exhibits significantly higher coronavirus binding affinity than wild-type angiotensin converting enzyme II.
  • a library was prepared by inducing mutations in the extracellular domain fragment of wild-type ACE2 (SEQ ID NO: 2).
  • the angiotensin converting enzyme II variant is, for example, "angiotensin converting enzyme II full-length protein, as well as variants of each domain or part of the full-length protein contained therein (eg, SEQ ID NO: 2)" It should be understood as a broad concept encompassing "fragments of enzyme II variants”.
  • the angiotensin converting enzyme II mutant discloses an amino acid containing a mutation based on SEQ ID NO: 2, which is the full-length sequence reported as a wild type of human angiotensin converting enzyme II.
  • SEQ ID NO: 2 is a fragment (M1-D615) including a signal sequence of the full-length human angiotensin converting enzyme II of SEQ ID NO: 1 and some fragments of the extracellular domain, and includes a SARS-CoV-2 binding site. .
  • SEQ ID NO: 1 is the full-length sequence of angiotensin converting enzyme II. Even when analyzing a mutant amino acid site or substitution of an amino acid based on SEQ ID NO: 1, it has the same amino acid number as SEQ ID NO: 2.
  • any fragment including the amino acid mutation site of the present invention other than SEQ ID NO: 2 is included in the scope of the present invention.
  • it may be an extracellular domain fragment (Q18 to 740) or a part thereof (Q18 to D615) excluding the signal sequence, but is not limited thereto.
  • angiotensin converting enzyme II derived from the same species or different species has an amino acid sequence different from SEQ ID NO: 1 or SEQ ID NO: 2 (eg, ACE2 in which some amino acids are conservatively substituted, human and ACE2, etc. derived from other species), or a fragment thereof, it will be apparent that those skilled in the art can easily derive the amino acid corresponding to the amino acid mutation site described above through sequence alignment and analysis. In this case, it is obvious that the angiotensin converting enzyme II of the present invention may be characterized by having a mutation in the amino acid corresponding to the amino acid described above.
  • an expression in which an amino acid residue name of one letter and a number (n) are written together, such as "S19”, means an amino acid residue and type at the corresponding nth position in each amino acid sequence.
  • S19 in the amino acid sequence of SEQ ID NO: 2 means that the amino acid residue at the 19th position of SEQ ID NO: 2 (or SEQ ID NO: 1) is serine.
  • amino acid residue name after the number means the substitution of amino acids, and the "S19W” is serine (Ser, S) at position 137 of SEQ ID NO: 2 (or SEQ ID NO: 1) to tryptophan (Try, W) means that it is substituted.
  • the angiotensin converting enzyme II variant is used in fusion with other polypeptides, proteins or structures such as sugars and PEGs for the modification of biological properties or physical/chemical properties. .
  • a fusion protein was prepared by fusing an angiotensin converting enzyme II mutant and an Fc domain derived from human IgG1. Even when fused to the Fc domain, it was confirmed that the same level of SARS-CoV-2 (including variant) binding affinity was exhibited, and the neutralization ability was maintained at the same level.
  • the fusion protein bound to the Fc domain may exhibit improvement in biological/functional properties, such as an increase in half-life and an increase in expression level, while minimizing loss of biological activity.
  • the present invention relates to a fusion protein comprising an angiotensin converting enzyme II variant.
  • the fusion protein may be characterized in that it further comprises an Fc domain.
  • the term “Fc domain” refers to a tail region of an antibody that interacts with a receptor on the cell surface of immunoglobulin and a protein of the complement system, and includes heavy chain constant domains, CH2 and CH3, and the hinge of the heavy chain constant region. It may further include a region.
  • the Fc domain may be cleaved, amino acid substitution, etc. may be performed for the modification (modulation) of its properties. Therefore, in the present invention, the Fc domain is used as a concept including all of the Fc domain of immunoglobulin, fragments thereof, and variants thereof.
  • the Fc domain may be a mammalian immunoglobulin Fc domain, preferably, a human immunoglobulin Fc domain, but is not limited thereto.
  • the Fc domain may be an IgA, IgM, IgE, IgD, or an IgG Fc domain, a fragment thereof, or a modification thereof, and preferably, the Fc domain is an IgG Fc domain (eg, IgG1, IgG2a). , an Fc domain of IgG2b, IgG3, or IgG4), but is not limited thereto.
  • the Fc domain may be a human IgG1 Fc domain.
  • the Fc domain is used to include a variant of the Fc domain derived from immunoglobulin.
  • the Fc domain may be characterized as an Fc domain variant with reduced Fc ⁇ R binding affinity, for example, L234A, L235A, and/or K322A variants of a human IgG1 Fc domain (SEQ ID NO: 46). may be, but is not limited thereto.
  • the Fc domain may be characterized in that it comprises a sequence represented by SEQ ID NO: 46 or 47.
  • the angiotensin converting enzyme II variant may be characterized in that it is linked to the N'-terminus or the C'-terminus of the Fc domain, preferably it may be characterized in that it is linked to the N'-terminus.
  • the angiotensin converting enzyme II variant and the Fc domain may be characterized in that they are linked by a linker.
  • the linker may be a glycine-serine linker (Glycin-Serine Linker, GS linker), more preferably a GS linker as in the examples of the present invention, but is not limited thereto.
  • the Fc domain may include sugar chains, and may include increased or decreased sugar chains compared to the wild type, or may be in a form in which sugar chains are removed.
  • the increase, decrease or removal of sugar chains of the immunoglobulin Fc domain may be performed by conventional methods known in the art, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removal of sugar chains from the Fc domain sharply reduces the binding affinity of the primary complement component C1 to C1q, resulting in reduction or elimination of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), As a result, it can exhibit a characteristic that does not induce an unnecessary immune response in the living body.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • angiotensin converting enzyme II (ACE2) variant and the Fc domain fusion protein may be in the form of a monomer, but is not limited thereto.
  • the angiotensin converting enzyme II (ACE2) variant and the Fc domain fusion protein may be a homodimer or a heterodimer, and may be in the form of a trimer or more of a multimer.
  • the multimeric form of the fusion protein including the Fc domain may be formed through a disulfide bond at the hinge portion of the Fc domain, but is not limited thereto.
  • the present invention relates to a nucleic acid encoding an angiotensin converting enzyme II variant or a fusion protein comprising the same of the present invention.
  • Nucleic acids as used herein may be present in cells, cell lysates, or may exist in partially purified or substantially pure form. Nucleic acids can be removed from other cellular components or other contaminants, e.g., by standard techniques including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. "Isolated” or “substantially pure” when purified from the nucleic acid or protein of another cell.
  • the nucleic acid of the invention may be, for example, DNA or RNA.
  • the present invention relates to a recombinant vector containing a nucleic acid encoding an angiotensin converting enzyme II variant of the present invention or a fusion protein comprising the same.
  • the recombinant vector is a vector capable of inducing the expression of a nucleic acid encoding an angiotensin converting enzyme II mutant or a fusion protein comprising the same of the present invention
  • a person skilled in the art can appropriately select a vector known in the art without limitation. can be used for example, when E.
  • coli is used as a host, a vector containing a T7 family (T7A1, T7A2, T7A3, etc.), lac, lacUV5, temperature-dependent ( ⁇ phoA, phoB, rmB, tac, trc, trp or 1PL promoter can be used) , when yeast is used as a host, a vector containing an ADH1, AOX1, GAL1, GAL10, PGK or TDH3 promoter can be used, and in the case of Bacillus, a vector containing a P2 promoter can be used, but this is a listing of some embodiments, In addition to the vector containing the promoter, as a vector containing a promoter for inducing the expression of the angiotensin converting enzyme II mutant or fusion protein containing the same according to the present invention, as long as it is suitable for the host, various vectors known in the art can be used without limitation. can be appropriately selected and used.
  • the term “vector” refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host.
  • a vector may be a plasmid, a phage particle, or simply a potential genomic insert. Upon transformation into an appropriate host, the vector may replicate and function independently of the host genome, or in some cases may be integrated into the genome itself. Since a plasmid is currently the most commonly used form of vector, "plasmid” and “vector” are sometimes used interchangeably in the context of the present invention. However, the present invention includes other forms of vectors that have an equivalent function as known or coming to be known in the art. Examples of protein expression vectors used in E.
  • coli include the pET family of Novagen (USA); pBAD family of Invitrogen (USA); pHCE or pCOLD from Takara (Japan); pACE family of Xenofocus (Korea USA); etc. can be used.
  • Bacillus subtilis protein expression can be realized by inserting a target gene into a specific part of the genome, or a pHT-based vector from MoBiTech (Germany) can be used.
  • mold or yeast protein expression is possible using genome insertion or self-replicating vectors.
  • a plant protein expression vector can be used using a T-DNA system such as Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • Typical expression vectors for expression in mammalian cell culture are based, for example, on pRK5 (EP 307,247), pSV16B (WO 91/08291) and pVL1392 (Pharmingen) and the like.
  • expression control sequence refers to a DNA sequence essential for the expression of an operably linked coding sequence in a particular host organism.
  • regulatory sequences include promoters for effecting transcription, optional operator sequences for regulating such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences regulating the termination of transcription and translation.
  • regulatory sequences suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells include promoters, polyadenylation signals and enhancers. The factor most affecting the expression amount of a gene in a plasmid is a promoter.
  • the promoter for high expression the SR ⁇ promoter, the cytomegalovirus-derived promoter, etc. are preferably used.
  • any of a wide variety of expression control sequences can be used in the vector.
  • useful expression control sequences include, in addition to the promoters described above, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, phage lambda major operator and promoter regions, regulatory regions of the fd coding protein, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of said phosphatases such as Pho5, promoters of yeast alpha-crossing systems and prokaryotes or Other sequences of construction and induction known to regulate the expression of genes in eukaryotic cells or viruses thereof, and various combinations thereof are included.
  • the T7 RNA polymerase promoter ⁇ can be usefully used to express proteins in E. coli.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. It can be a gene and regulatory sequence(s) linked in such a way that an appropriate molecule (eg, a transcriptional activation protein) allows gene expression when bound to the regulatory sequence(s).
  • an appropriate molecule eg, a transcriptional activation protein
  • DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a preprotein that participates in secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or the ribosome binding site is operably linked to a coding sequence if it affects transcription of the sequence; or the ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation.
  • "operably linked” means that the linked DNA sequences are in contact and, in the case of a secretory leader, in contact and in reading frame. However, the enhancer does not need to be in contact. Linking of these sequences is accomplished by ligation (conjugation) at convenient restriction enzyme sites. If such a site does not exist, a synthetic oligonucleotide adapter or linker according to a conventional method is used.
  • expression vector generally refers to a fragment of double-stranded DNA as a recombinant carrier into which a heterologous DNA fragment is inserted.
  • heterologous DNA refers to heterologous DNA that is not naturally found in host cells.
  • the expression vector once in the host cell, can replicate independently of the host chromosomal DNA and several copies of the vector and its inserted (heterologous) DNA can be produced.
  • the gene in order to increase the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host.
  • the expression control sequence and the corresponding gene are included in one expression vector including the bacterial selection marker and the replication origin.
  • the expression vector may further comprise an expression marker useful in the eukaryotic expression host.
  • the present invention provides a nucleic acid encoding an angiotensin converting enzyme II variant or a fusion protein comprising the same of the present invention; and/or to a recombinant cell into which the recombinant vector is introduced into a host cell.
  • the host cell means an expression cell into which a gene or a recombinant vector can be introduced to produce a protein or the like.
  • the host cell may be used without limitation as long as it is a cell capable of expressing the angiotensin converting enzyme II mutant of the present invention or a fusion protein comprising the same, preferably a eukaryotic cell, more preferably a yeast, insect cell, animal cell, most Preferably, it may be an animal cell.
  • a CHO cell line or a HEK cell line mainly used for protein expression may be used, but is not limited thereto.
  • Expression vectors suitable for eukaryotic hosts include, for example, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus.
  • Expression vectors that can be used for bacterial hosts include pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and derivatives thereof, such as bacterial plasmids exemplified from E. coli, and a broader host range such as RP4.
  • phage DNA exemplified by a wide variety of phage lambda derivatives such as ⁇ and ⁇ NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages.
  • Useful expression vectors for yeast cells are the 2 ⁇ plasmid and derivatives thereof.
  • a useful vector for insect cells is pVL 941.
  • the recombinant vector may be introduced into a host cell by a method such as transformation or transfection.
  • transformation refers to the introduction of DNA into a host such that the DNA becomes replicable either as an extrachromosomal factor or by chromosomal integrity.
  • transfection means that an expression vector is accepted by a host cell, whether or not any coding sequence is actually expressed.
  • the single-celled host is capable of transferring the product encoded by the DNA sequence of the invention from the host to the selected vector, toxicity, secretion characteristics, ability to correctly fold the protein, culture and fermentation requirements, and the product encoded by the DNA sequence of the invention. It should be selected in consideration of factors such as ease of purification. Within the scope of these parameters, one of ordinary skill in the art can select a variety of vector/expression control sequence/host combinations capable of expressing the DNA sequences of the present invention in fermentation or large-scale animal culture. As a screening method for cloning cDNA by expression cloning, a binding method, a panning method, a film emulsion method, etc. may be applied.
  • the gene and recombinant vector may be introduced into a host cell through a variety of methods known in the prior art.
  • the gene encoding the angiotensin converting enzyme II mutant or a fusion protein comprising the same of the present invention may be directly introduced into the genome of a host cell and present as a chromosomal factor.
  • a chromosomal factor For those skilled in the art to which the present invention pertains, it will be apparent that even when the gene is inserted into the genomic chromosome of the host cell, it will have the same effect as when the recombinant vector is introduced into the host cell.
  • the present invention relates to a method for producing an angiotensin converting enzyme II mutant or a fusion protein comprising the same of the present invention comprising the step of culturing the recombinant cell.
  • angiotensin converting enzyme II variant of the present invention or a fusion protein comprising the same When a recombinant expression vector capable of expressing an angiotensin converting enzyme II variant of the present invention or a fusion protein comprising the same is introduced into a mammalian host cell, the angiotensin converting enzyme II variant or a fusion protein comprising the same is expressed in recombinant cells. It can be prepared by culturing the host cells for a sufficient period of time or for a period sufficient to secrete the angiotensin converting enzyme II variant or a fusion protein comprising the same into the culture medium in which the recombinant cells are cultured.
  • the expressed angiotensin converting enzyme II mutant or a fusion protein comprising the same can be isolated from recombinant cells and purified to be uniform. Separation or purification of the angiotensin converting enzyme II mutant or a fusion protein comprising the same may be performed by a separation or purification method used in conventional proteins, for example, chromatography.
  • the chromatography may be, for example, affinity chromatography, ion exchange chromatography, or a combination of one or more selected from hydrophobic chromatography, but is not limited thereto.
  • filtration, ultrafiltration, salting out, dialysis, etc. may be used in combination.
  • the angiotensin converting enzyme II mutant of the present invention can effectively inhibit the entry and proliferation of SARS-CoV-2 into cells through its excellent SARS-CoV-2 neutralizing ability
  • a recent report Various strains of SARS-CoV-2 (SARS-CoV-2 RBD_N501Y from the UK, strains from South Africa (SARS-CoV-2 RBD_N501Y, K417N, E484K)) were also confirmed to exhibit high neutralizing ability.
  • the angiotensin converting enzyme II mutant of the present invention exhibits excellent pharmacokinetic properties, thereby demonstrating that it can be usefully used for the prevention or treatment of coronavirus infections including COVID-19.
  • the present invention relates to a pharmaceutical composition for preventing or treating a coronavirus infection comprising the angiotensin converting enzyme II mutant or a fusion protein comprising the same.
  • the present invention also relates to a method for preventing and/or treating a coronavirus infection comprising administering to a subject the angiotensin converting enzyme II (ACE2) variant or a fusion protein comprising the same.
  • ACE2 angiotensin converting enzyme II
  • the present invention also relates to the preventive or therapeutic use of the angiotensin converting enzyme II (ACE2) variant or a fusion protein comprising the same for the prevention or treatment of coronavirus infection.
  • ACE2 angiotensin converting enzyme II
  • the present invention also relates to the use of the angiotensin converting enzyme II (ACE2) variant or a fusion protein comprising the same for the preparation of a pharmaceutical composition for preventing and/or treating coronavirus infection.
  • ACE2 angiotensin converting enzyme II
  • the binding affinity and neutralizing ability of the angiotensin converting enzyme II (ACE2) variant and the Fc domain fusion protein to SARS-CoV-2 and its variants were confirmed.
  • MERS, and other coronaviruses such as SARS-CoV-1 also enter, infect, and proliferate into the cells of the host centering on the binding of the receptor binding domain of the spike protein and its receptor ACE2, particularly in the S1 subunit of each species.
  • the included receptor binding domain is highly conserved, so the angiotensin converting enzyme II (ACE2) variant of the present invention is not limited to use only for the prevention and treatment of COVID-19, but it is self-evident that it can be used for all coronavirus infections. .
  • ACE2 angiotensin converting enzyme II
  • the coronavirus refers to an RNA virus belonging to the genus Coronavirinae (Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier, Oxford. pages 806-828).
  • the coronavirus subfamily can be divided into four genera of alpha / beta / gamma / delta coronavirus, for example, SARS-CoV, MERS-CoV, SARS-CoV-2, SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-229E, HCoV-OC43, HKU1, HCoV-NL63, and the like, but are not limited thereto.
  • prevention refers to any action of suppressing or delaying the onset of a desired disease by administration of the pharmaceutical composition according to the present invention.
  • treatment refers to any action in which symptoms for a target disease are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.
  • the pharmaceutical composition may further include suitable carriers, excipients and diluents commonly used in pharmaceutical compositions in addition to the angiotensin converting enzyme II (ACE2) variant or fusion protein comprising the same as the active ingredient.
  • suitable carriers, excipients and diluents commonly used in pharmaceutical compositions in addition to the angiotensin converting enzyme II (ACE2) variant or fusion protein comprising the same as the active ingredient.
  • ACE2 angiotensin converting enzyme II
  • compositions must be compatible with the active ingredient of the present invention, and include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or two or more of these components. They can be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats can be added as needed.
  • diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, and the like.
  • inhalation formulations can be prepared in various forms such as dry powder, liquid, and aerosol, and dry powder inhalers (DPIs), nebulizers, and metered dose inhalers. It can be administered using a metered-dose inhaler (MDI) or a spacer.
  • DPI dry powder inhalers
  • nebulizers nebulizers
  • metered dose inhalers metered dose inhalers
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.
  • a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant.
  • the pharmaceutical composition according to the present invention may be formulated and used in various forms according to conventional methods. Suitable formulations include tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, oral formulations such as aerosols, external preparations, suppositories, and sterile injection solutions,
  • Suitable formulations include tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, oral formulations such as aerosols, external preparations, suppositories, and sterile injection solutions,
  • the present invention is not limited thereto.
  • the pharmaceutical composition according to the present invention can be prepared in a suitable dosage form using a pharmaceutically inert organic or inorganic carrier. That is, when the formulation is a tablet, a coated tablet, a dragee, and a hard capsule, it may contain lactose, sucrose, starch or a derivative thereof, talc, calcium carbonate, gelatin, stearic acid or a salt thereof. In addition, when the formulation is a soft capsule, it may contain vegetable oils, waxes, fats, semi-solid and liquid polyols. In addition, when the formulation is in the form of a solution or syrup, water, polyol, glycerol, and vegetable oil may be included.
  • the pharmaceutical composition according to the present invention may further include a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizing agent, a sweetener, a colorant, an osmotic pressure regulator, an antioxidant, and the like, in addition to the carrier described above.
  • the pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the pharmaceutical composition of the present invention may be administered to an individual by various routes.
  • the pharmaceutical composition may be administered orally or parenterally.
  • parenteral administration intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc. can be administered.
  • oral compositions may be formulated to coat the active agent or to protect it from degradation in the stomach.
  • the composition may be administered by any device capable of transporting the active agent to a target cell.
  • the administration method of the pharmaceutical composition according to the present invention can be easily selected according to the dosage form, and can be administered orally or parenterally.
  • the dosage may vary depending on the patient's age, sex, weight, severity of disease, and route of administration.
  • amino acid sequence substantially identical to the enzyme to be practiced in the present invention and a nucleotide sequence encoding the same fall within the scope of the present invention.
  • Substantially identical means a protein that shares structural features or has the same function as used in the present invention, including cases in which homology of amino acid or nucleotide sequence is very high, and in addition, regardless of sequence homology.
  • a fragment of a protein or a nucleotide sequence encoding the same may also be included in the present invention, in which other sequences except for the sequence constituting the core of the present invention are partially deleted. All amino acids or nucleotide sequences having the same function are included.
  • Example 1 Construction of an ACE2 mutant library using the ACE2 protein as a template
  • Example 1-1 Template vector construction for ACE2 mutant library preparation
  • Q18-N720 which is part of the extracellular domain of wild-type human ACE2 (SEQ ID NO: 1, UniProtKB seq ID: Q9BYF1, FIG. 1), was cloned into pYD5 vector to create an ACE2 mutant library.
  • a V5 tag was spliced after the c-terminal EcoR1 site of ACE2, and sequences homologous to both ends of the pYD5 vector cut with Nhe1 and EcoR1 restriction enzymes were synthesized at both ends of the synthetic gene (FIG. 2).
  • the synthesized insert gene and linearized vector were cloned using In-Fusion® HD Cloning Kit (Takara).
  • the cloned plasmid was transformed into StellarTM Competent Cells (Takara), and then midi-prep with ZymoPUREII Plasmid Midiprep kit (Zymo research, Cat# D4201) to secure more than 15ug of DNA.
  • Example 1-2 Preparation of a point mutation library containing mutations in amino acids at specific positions of ACE2
  • Example 1-3 Yeast transformation
  • yeast competent cells For yeast competent cells, EBY100 yeast strain was streaked on a YPD plate and cultured at 30°C for 2 days, then one colony was picked and cultured overnight at 30°C, 250rpm in 5ml YPD medium. The cultured yeast cells were subcultured on a 100ml scale and grown under the same conditions for about 6 hours until the O.D value was about 1.0-1.5, and then 1.0 ml of sterilized Tris-DTT solution (0.39g 1,4-dithiothreitol in 1ml). 1M Tris pH8.0 buffer) was added and incubated for 15 minutes.
  • Tris-DTT solution (0.39g 1,4-dithiothreitol in 1ml). 1M Tris pH8.0 buffer
  • yeast cells After the cultured yeast cells were centrifuged, they were washed with cold E buffer (1.2g Tris base, 92.4g sucrose, 1M MgCl2 in distilled water) and resuspensioned with E buffer to make a total volume of 450ul to prepare competent cells. 50ul of the prepared competent cells, 5ug of concentrated insert DNA, and 1ug of vector were placed in an electrocuvette, electroporated under 0.54kV, 25uF conditions, and then 2ml of YPD medium was immediately added, followed by 1 hour at 30°C, 250rpm. incubated for a while.
  • E buffer 1.2g Tris base, 92.4g sucrose, 1M MgCl2 in distilled water
  • yeast cells cultured in YPD were cultured in 10 ml SDCAA medium (20 g glucose, 14.7 g sodium citrate, 4.3 g citric acid monohydrate, 6.7 g yeast nitrogen base, 5 g bacto casamino acid in 100 ⁇ g/ml kanamycin antibiotic). After resuspension with 1L distilled water), dilute with 1/100 SDCAA medium and add 100ul of SDCAA::Km plate (100ug/ml kanamycin, 20g glucose, 14.7g sodium citrate, 4.3g citric acid monohydrate, 6.7g to 10 4 cells). It was spread on yeast nitrogen base, 5g bacto casamino acid, 16g bacto agar/1L distilled water).
  • the transformed yeast cells were resuspensioned in SDCAA medium and cultured in 100 ml SDCAA medium for 24 hours so that the initial O.D was 0.2. After subculture in the same way, 25ml SGCAA medium containing 100ug/ml kanamycin so that the initial O.D is 1 (20g galactose, 14.7g sodium citrate, 4.3g citric acid monohydrate, 6.7g yeast nitrogen base, 5g bacto casamino acid in 1L After resuspension with distilled water), induction was performed at 30°C and 250rpm for 16-20 hours.
  • Example 2 Sorting and enrichment using the ACE2 variant library
  • 26 point mutation libraries were stained with ACE2 ECD (Q18-N720) expressed on the yeast cell surface to improve antigen binding affinity through FACS sorting.
  • the primary staining is 200-500 nM biotinylated SARS-CoV-2 spike protein RBD-his, which can confirm antigen-binding affinity, and 1:500 anti-V5 antibody (Invitrogen, cat#R960-25) that can confirm the expression of the ACE2 library ) was diluted with FACS buffer (0.1% BSA in pH7.4 PBS buffer) and incubated at room temperature for 30 minutes in a rotational mixer.
  • Example 3 Antigen-binding affinity and sequence analysis of individual clones of ACE2 variants with improved binding affinity
  • Example 2 The cells finally selected in Example 2 were cultured in some SDCAA::Km plates. After picking colonies, growing them in SDCAA media, induction with SGCAA media, FACS analysis was performed in the same manner as above. By comparing wild type ACE2 (Q18-N720) and PE histogram, clones with improved binding affinity were selected, stained together, and analyzed by FACS (FIG. 5).
  • Yeast cells grown with SDCAA::Km media after colony picking on SDCAA::Km plate were DNA prep with ZymoPrep Yeast Plasmid MiniPrep II Kit (zymo research, Cat#D2004-50).
  • the extracted DNA was transformed into E. coli StellarTM Competent Cells and sequence analysis was performed. Amino acid mutation information of ACE2 variants having improved binding affinity derived from each library is shown in Table 4 below.
  • point mutation library (variant residue number) clone name mutation information 19 19_203 S19W 19_204 S19P 27 27_102 T27W 27_106 T27D 27_107 T27A 27_114 T27Y 27_202 T27L 27_205 T27M 27_207 T27C 30 30_101 D30V 34 34_101 H34A 34_110 H34V 35 35_202 E35V 79 79_101 L79Y 79_105 L79V 325 325_102 Q325P 330 330_101 N330Y 330_105 N330F
  • Example 4 ACE2 error prone library production and variant derivation based on ACE2 variants with improved binding affinity
  • Example 4-1 ACE2 error prone library production
  • a primer specific to each template was prepared, and an error rate of 0.23%, 0.48%, and 0.81% libraries were prepared using PCR ramdom mutagenesis kit (TAKARA, cat no. 630703), respectively. Transformation and culture were performed in the same manner as above.
  • Example 4-2 sorting and using the ACE2 error prone library enrichment
  • Each error prone library with a different template was stained with ACE2 (Q18-D615) expressed on the yeast cell surface to improve antigen binding affinity through FACS sorting.
  • the staining method is the same as in Example 2, and round sorting was performed 4 times by lowering the antigen concentration to 0.5 nM.
  • No. library type clone number mutation information One 8mut_err 100 A.A 2 K26N, K31R 2 4 K26N, L91P 3 8mut_err Full length 2 N250K 4 5 N250K, G448V 5 6 G448V 6 9 K74,V185A 7 H34A_err 100A.A R4-2 N49D 8 H34A_err Full length R4-1 L79I, L91P, S280R, S602T 9 R4-3 L79I, L91P
  • An ACE2-GS linker-Fc_pcDNA3.3 plasmid was constructed for the production of ACE2 variants fused with the Fc domain.
  • an animal cell expression vector, EcoRI at the C' end of the wild type ACE2 sequence, and BamHI restriction enzyme sites at the N' end were inserted, and a sequence homologous to pcDNA3.3 vector was added to both ends of the sequence in IDT.
  • the signal peptide (signal peptide) of human ACE2 (seq ID: Q9BYF1) was used.
  • Each individual clone was synthesized and cloned by varying the restriction enzyme site according to the mutation site. Mutations within the 100th amino acid were synthesized by synthesizing BspEI at both ends and cloned with the BspEI cut vector, and the mutations after the 100th amino acid were synthesized by dividing them into fragments 1 and 2 for assembly, and cloned using EcoRI and BamHI cut vectors. In addition, mutants consisting of a combination of derived mutations were additionally synthesized and produced by fusion of Fc or Fc-his tag depending on efficacy.
  • the synthesized insert gene was cloned by putting each linearized vector using the In-Fusion® HD Cloning Kit (Clontech), and the sequencing primer was confirmed using CmV Forward and pcDNA3.3 reverse primer (Table 15) (Fig. 8). ).
  • the mutation information of the finally selected and cloned ACE2 mutant is shown in Table 6 below.
  • ACE2 Variants name Mutation Sites SEQ ID NO: ACE2 wild type (M1-D615) - 2 ACE2.V.06 H34A 3 ACE2.V.07 H34V 4 ACE2.V.08 S19W 5 ACE2.V.09 S19P 6 ACE2.V.10 T27D 7 ACE2.V.11 T27Y 8 ACE2.V.12 T27L 9 ACE2.V.13 T27C 10 ACE2.V.14 D30V 11 ACE2.V.15 E35V 12 ACE2.V.16 L79Y 13 ACE2.V.17 L79V 14 ACE2.V.18 Q325P 15 ACE2.V.19 N330Y 16 ACE2.V.20 N330F 17 ACE2.V.21 S19P, T27L, H34A, L79V, N330Y 18 ACE2.V.22 S19P, T27L, D30V, H34A, E35V, L79V, Q325P, N330Y 19 ACE2.V.23 N330
  • the plasmid into which the ACE2 variant designed to improve binding affinity was introduced was expressed using the Expi293 expression system (Invitrogen), which was then expressed in AktaPure (GE healthcare), AktaPrime purifier (GE healthcare) and MabselectSURE column (GE healthcare, Cat#11). -0034-95) was used for purification.
  • the purified antibody was buffer-changed with PBS through a desalting column (GE healthcare, Cat #17-1408-01), and the concentration was measured through Multiskan GO (Thermo).
  • the production of each ACE2 variant is shown in Table 7 below.
  • Example 7-2 Binding affinity analysis of ACE2 mutant and SARS-CoV-2 spike protein RBD
  • CM5 chip GE Healthcare, Cat#BR-1005
  • a human capture kit GE healthcare, Cat#BR-1008-39
  • the ligand was immobilized.
  • the analyte SARS-CoV-2_His (Sino) was serially diluted to 100, 50, 25, 12.5, 6.25, 3.125 nM, and the association time was 150 seconds and the dissociation time was 240 seconds. Binding affinity was analyzed by fitting the sensorgram measured through the Biacore T200 (GE healthcare) device to a 1:1 binding model (Table 8 and Table 9).
  • ACE2.V.46 was prepared in the form of a fusion of Fc mutants (L234A, L235A, K322A) with lower Fc ⁇ R binding affinity, and similarly, binding to SARS-CoV-2 RBD was confirmed (Table 10).
  • Example 7-3 Binding affinity analysis of ACE2 mutant and SARS-CoV-2 spike protein RBD mutant (variant from UK and strain from South Africa)
  • each RBD mutant was used as an analyte and analyzed under the same conditions as above (Table 11 and Table 12). ).
  • Example 7-4 Size exclusion chromatography analysis
  • the produced ACE2 variant was analyzed using AKTA pure connected to Superdex200 Increase 10/300 GL (GE healthcare, Cat#.28-9909-44).
  • column equilibration is performed by flowing PBS at a flow rate of 0.3 mL/min.
  • 0.5 mL standard and sample were injected into the FPLC device using a 1 mL syringe, and the sample was allowed to pass through the column. It was analyzed using the UNICORN (GE healthcare) program (FIGS. 10a to 10h).
  • Example 7-5 SARS-CoV-2 neutralizing ability analysis of ACE2 variants
  • IVnAT In Vitro Neutralizing Antibody Test
  • SARS-CoV-2 RBD SARS-CoV-2 RBD
  • the ACE2 mutant was serially diluted and reacted with RBD-HRP 1:1, followed by pre-incubation at 37° C. for 15 minutes, and then treated on a hACE2-coated plate to confirm the neutralizing ability according to the concentration ( FIGS. 11 and 12 ).
  • Example 7-6 Analysis of neutralizing ability of SARS-CoV-2 mutant (from UK, South Africa) of ACE2 mutant
  • SARS-CoV-2 inhibitor screening kit (Acro, cat no.EP-) to confirm the neutralizing ability against two SARS-CoV-2 RBD variants (from England (N501Y), from South Africa (N501Y, K417N, E484K)) 105) was used.
  • a 1:1 mixture of biotinylated hACE2 and serial diluted ACE2 variant was reacted on a microplate coated with the RBD variant, and incubated at 37°C for 1 hour. Thereafter, streptavidin HRP was treated at 37° C. for 1 hour, and color was developed with TMB to confirm the SARS-CoV-2 RBD neutralizing ability according to the ACE2 concentration ( FIGS. 13 and 14 ).
  • the ACE2 mutant of the present invention exhibited significantly superior neutralizing ability compared to wild-type ACE2 even for two SARS-CoV-2 RBD mutants.
  • Example 7-7 Analysis of catalytic activity of ACE2 variants
  • Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric) (Biovision, cat no. K897) was used to confirm the catalytic activity of the angiotensin converting enzyme II (ACE2) variant.
  • the present invention including ACE2.V.46 as well as individual clones sorted from the library (Tables 4 and 5) and ACE2.V.06 to ACE2.V.48
  • the ACE2 mutant of the present invention has a significantly higher binding affinity to the RBD of the coronavirus spike protein compared to wild-type ACE2, and exhibits excellent neutralizing ability for coronavirus, thereby effectively inhibiting viral invasion and amplification.
  • Example 8-1 In vivo stability test of ACE2.V.46 (EU129)
  • Anti-Human IgG (Fc specific) antibody (Sigma, Cat# I2136) was coated on a 96-wells immune plate at a concentration of 2ug/ml at 100ul/well overnight at 4°C to prepare. After blocking at RT for 1 hr with 5% skim milk, prepare a serum sample of ACE2.V.46 (EU129) by diluting it in 1X PBS to a concentration that matches the rage in the standard curve, and ACE2 serum sample at 100ul/well, 1 hr, Treated with RT.
  • ACE2.V.46 EU129
  • ACE2 binding was performed with 20 nM biotinylated RBD-his at 1 hr and RT, and 1:5000 (v/v) HRP-Conjugated Streptavidin was reacted in the same manner.
  • TMB substrate was used, and after 5-10 min of development, a stop solution was added and analyzed at 450 nm (FIG. 17).
  • the ACE2 mutant of the present invention exhibits a significantly improved level of stability in vivo at the same level as that of wild-type ACE2 or over a long period of time.
  • Example 8-2 Evaluation of catalytic activity of ACE2 variants
  • Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric) (Biovision, cat no. K897) was used to confirm the catalytic activity of the angiotensin converting enzyme II (ACE2) variant in the serum sample of the in vivo experiment. Based on the residual amount of ACE2 confirmed through the ACE2 pk test, a sample was prepared by diluting 24 hr of serum with 5 nM of ACE2 in 1X PBS.
  • Example 9 Inhibition of infection and amplification of actual coronavirus at the cellular level
  • ACE2.V.06-Fc hereinafter, ACE2.V .06
  • ACE2.V.41-Fc hereinafter, ACE2.V.46
  • ACE2-WT ACE2 WT-Fc
  • SARS-CoV-2 Real virus was mixed with ACE2 Wt-Fc or mutated proteins ACE2.V.06-Fc and ACE2.V.41-Fc at various MOIs to induce virus neutralization, and then, monkey kidney cells, Vero Infection was induced in the E6 cell line. After culturing at room temperature for 1 hour, the virus is removed, the cells are washed with OPTI-MEM medium to remove the virus that has not been bound to the cells, then a new medium is added and cultured for 24 hours, and the amount of virus protein and RNA is measured after collecting the cells. did
  • Cytopathic effect assay was used to observe the cytopathic effect in VeroE6 cell line infected with SARS-CoV-2 and the decrease in CPE by ACE2 protein treatment under a light microscope.
  • lysis buffer Cell culture lysis reagent, Promega
  • VeroE6 cell line infected with SARS-CoV-2 to lyse the cells
  • Western using an antibody that detects SARS-CoV-2 nucleoprotein Sino Biological, China
  • the amount of expressed viral protein was determined through blotting.
  • VeroE6 cells were lysed using NucleoZol (MN), total RNA was isolated, cDNA was synthesized using a cDNA synthesis kit (Toyobo, Japan), and primers (SEQ ID NO: 107; sense, 5'-GTG AAA TGG TCA TGT GTG GCG) qRT-PCR (SYBR Green Master Mix, Bio-Rad Laboratories) was performed using G-3' and SEQ ID NO: 108; antisense, 5'-CAA ATG TTA AAA ACA CTA TTA GCA TA-3').
  • the present inventors confirmed the antiviral effect of ACE2-V.06 on SARS-CoV-2.
  • the present inventors confirmed the antiviral effect of ACE2-V.41 on SARS-CoV-2.
  • nucleoprotein increased by SARS-CoV-2 infection did not decrease in all the experimental groups treated with ACE2-WT.
  • nucleoprotein was not detected in the experimental group treated with ACE2-V.41 at least 1ug/ml, so it can be seen that the antiviral efficacy is higher than that of WT.
  • CPE caused by SARS-CoV-2 infection in the experimental group cultured for 48 hours decreased at a concentration of 10ug/ml of ACE2-WT.
  • ACE2-V.41 a phenomenon in which CPE was significantly reduced or disappeared was observed at a concentration of 0.5ug/ml or more.
  • ACE2-WT a concentration-dependent decrease in nucleoprotein was observed by treatment with ACE2-WT in 24-hour culture, and was not detected in the experimental group treated with 5ug/ml.
  • nucleoprotein was not detected in the experimental group treated with ACE2-V.41 at a concentration of 0.5ug/ml or higher, and thus, higher antiviral efficacy was observed compared to WT.
  • nucleoprotein increased by SARS-CoV-2 infection did not decrease in all the experimental groups treated with ACE2-WT.
  • nucleoprotein was not detected, so it showed higher antiviral efficacy than WT.
  • IC 50 values of ACE2-WT or V.41 required for SARS-CoV-2 neutralization were calculated as follows:
  • ACE2-WT, ACE2-V.06 and ACE2-V.41 proteins were mixed with SARS-CoV-2 virus to induce virus-protein binding, and then treated with VeroE6 cell line.
  • the neutralizing effect was shown dependently, and when the concentration of all proteins was increased, a decrease in CPE was observed, and a decrease in the amount of SARS-CoV-2 nucleoprotein and RNA was induced.
  • ACE2-V.06 had a greater neutralizing effect at a low concentration than WT.
  • ACE2-V.41 had a greater neutralizing effect at a low concentration than WT.
  • the amount of viral protein was measured in VeroE6 cells infected with SARS-CoV-2 at 48 hours, in the case of ACE2-WT, the viral protein was detected even in the group treated with 10 ug/ml, but in the case of ACE2-V.41, 0.5 ug/ml Considering that it was not detected in the abnormally treated experimental group, ACE2-V.41 showed a neutralizing effect with high efficiency against SARS-CoV-2 in the in vitro cell model.
  • ACE2-WT IC 50 of ACE2-WT was measured to be 1.56 ug/ml and 0.23 ug/ml in two experiments, and the IC 50 of ACE-V.41 was measured to be 0.17 ug/ml and 0.079 ug/ml, respectively.
  • WT WT
  • ACE2 variant of the present invention can be utilized as an effective therapeutic agent for SARS-CoV-2.
  • ACE2 wild type full sequence
  • Q9BYF1 One ACE2 wild type (M1 ⁇ D615) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTK

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Abstract

La présente invention concerne une variante de l'enzyme de conversion de l'angiotensine II (ECA2) ayant une affinité de liaison élevée pour et une neutralisation contre le coronavirus, une protéine de fusion la comprenant et une utilisation associée pour la prévention ou le traitement du coronavirus. Le variant de l'enzyme de conversion de l'angiotensine II (ECA2) de la présente invention présente une affinité de liaison plus élevée que la protéine ECA2 de type sauvage ainsi qu'une neutralisation remarquablement élevée contre le coronavirus, tel que l'inhibition de l'invasion et de la prolifération intracellulaires du coronavirus, et présente une neutralisation contre un large spectre de mutants de coronavirus. En tant que tel, le variant est utile pour inhiber la prolifération du coronavirus, prévenir une infection à coronavirus et traiter des symptômes d'une infection à coronavirus et peut être avantageusement utilisée en tant qu'agent, en particulier pour la prévention et le traitement de la COVID-19 qui est une pandémie en cours.
PCT/KR2021/018229 2020-12-03 2021-12-03 Nouveau variant d'eca2 et utilisation associée Ceased WO2022119380A1 (fr)

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