WO2022119380A1 - Novel ace2 variant and use thereof - Google Patents

Novel ace2 variant and use thereof 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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French (fr)
Korean (ko)
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권병세
임선우
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주식회사 유틸렉스
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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

Abstract

The present invention relates to an angiotensin converting enzyme II (ACE2) variant having high binding affinity for and neutralization against coronavirus, a fusion protein including same, and use thereof for prevention or treatment of coronavirus. The angiotensin converting enzyme II (ACE2) variant of the present invention exhibits higher binding affinity than wild-type ACE2 protein as well as having remarkably high neutralization against coronavirus, such as inhibition of intracellular invasion and proliferation of coronavirus, and exhibiting neutralization against a wide spectrum of coronavirus mutants. As such, the variant is useful for inhibiting coronavirus proliferation, preventing coronavirus infection, and treating coronavirus infection symptoms and can be advantageously used as an agent particularly for prevention and treatment of COVID-19 which is an ongoing pandemic.

Description

신규한 ACE2 변이체 및 그의 이용Novel ACE2 variants and uses thereof
본 발명은 높은 코로나바이러스 결합 친화도 및 중화능을 가지는 안지오텐신 변환 효소 II(ACE2) 변이체, 이를 포함하는 융합단백질 및 이의 코로나바이러스의 예방 또는 치료용도에 관한 것이다.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.
코로나바이러스(coronavirus)는 사람을 비롯한 다양한 동물에 감염될 수 있는 바이러스로서, 유전정보가 리보핵산(RNA)으로 구성된 RNA 바이러스의 한 종류이다. 코로나바이러스는 사람과 동물의 호흡기와 소화기계 감염을 유발한다. Coronavirus (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.
코로나바이러스 감염증 중 중증 급성 호흡기 증후군-코로나바이러스(SARS-CoV)는 2003년 4월 중국으로부터 유행하여, 사망률 9.6%를 기록하며 많은 사람이 사망했고, 2015년에는 중동 호흡기 증후군-코로나바이러스(MERS-CoV)는 중동으로부터 유행하여 전세계로 퍼지면서, 사망률 약 36%의 높은 사망률을 나타내었고, 2019년 12월 중국으로부터 유행한 중증 급성 호흡기 증후군-코로나바이러스-2(SARS-CoV-2)는 현재 진행 중이다. Among the coronavirus infections, the severe acute respiratory syndrome-coronavirus (SARS-CoV), which spread from China in April 2003, recorded a mortality rate of 9.6% and killed many people, and in 2015 the Middle East Respiratory Syndrome-coronavirus (MERS-CoV) CoV) originated from the Middle East and spread worldwide, showing a high mortality rate of about 36%, and the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) epidemic from China in December 2019 is currently ongoing. is in progress
중증 급성 호흡기 증후군-코로나바이러스-2(SARS-CoV-2)는 중국 우한에서 출현하여 전세계로 빠르게 확산되었으며 WHO는 해당 바이러스에 감염된 질환을 COVID-19로 명명하였다. SARS-CoV-2 감염의 일반적인 증상으로, 미각 또는 후각의 상실, 기침, 목쓰림, 두통, 구역질, 피로, 설사, 근육통 등이 있으며, 기저질환을 앓는 환자 또는 노약자들에게는 생명에 치명적인 영향을 줄 수 있다. 상기 SARS-CoV-2는 기침과 재채기로 생성된 호흡기 비말을 통한 사람과 사람의 접촉을 통해 가장 쉽게 확산되며, 밀폐된 공간 또는 환기가 부족한 공간의 경우 공기를 통해서도 전파될 수 있는 것으로 보고되며, 초기 주요 감염경로로 알려진 오염된 물체에 간접 접촉 경로는 드문 것으로 보고되었다. (CDC, C.f.d.c.a.p., How COVID-19 Spreads. Coronavirus Disease 2019 (COVID-19), 2021), 주로, 증상이 발현된 환자로부터 전파되나, 일부 사례에서는 무증상 감염이 가능한 것으로 보고된 바 있다(Yu, P., et al., J Infect Dis, 2020; Hoehl, S., et al., N Engl J Med, 2020; Bendix, A., Science alert, 2020). 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. can The 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).
WHO는 2020년 1월 30일 '국제적 공중보건 비상사태'(PHEIC)를 선포했으며, 2020년 3월 11 사상 세 번째로 코로나19에 대해 팬데믹(세계적 대유행)을 선포했다. 2021년 3월 31일을 기준으로 전세계적으로 약 1억 3천만명의 감염 환자가 발생하고, 약 280만명이 사망했으며, 여전히 매일 수만명의 확진환자가 추가되어, 코로나바이러스의 전파가 지속적인 확산세에 있다. 한국의 경우에도, 약103,088명의 환자가 확진되었으며, 약 1700명의 사망자가 보고되고, 매일 400명 이상의 신규 감염자가 확진되고 있어, 코로나바이러스의 기세가 계속되고 있다(COVID-19 Dashboard by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University (JHU) 및 중앙재난안전대책본부). The World Health Organization (WHO) 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. As of March 31, 2021, about 130 million people have been infected worldwide, about 2.8 million people have died, and tens of thousands of confirmed cases are still added every day, so the spread of the coronavirus continues to spread. have. In the case of South Korea, about 103,088 patients have been confirmed, about 1700 deaths are reported, and more than 400 new infections are confirmed every day, so the momentum of the coronavirus continues (COVID-19 Dashboard by the Center for Systems) Science and Engineering (CSSE) at Johns Hopkins University (JHU) and Central Disaster and Safety Countermeasures Headquarters).
이러한 세계적 대유행 및 심각성으로 인해, 많은 연구진 및 기업뿐만아니라 국가적/세계적 차원에서 코로나바이러스 백신 및 치료제의 개발에 많은 노력과 비용을 쏟아붇고 있다. 한국 국가임상시험지원재단에서 공개한 바에 따르면, 2020년 12월 15일 기준 미국 국립보건원(NIH)의 ClinicalTrials.gov에 신규 등록된 코로나19 관련 약물 중재 임상시험은 총 1,636건으로, 치료제 임상시험은 1,509건으로 92.2%, 백신 임상시험은 127건으로 7.8%를 차지하고 있으며, 임상 3상의 비중은 치료제 임상시험이 454건으로 30.1%, 백신 임상시험이 57건이다. 한국의 경우 2021년 3월 13일을 기준으로 코로나 치료제 임상 승인 13건, 허가 심사중 1건, 허가 완료된 치료제 2건(길리어드, 셀트리온)으로 수요에 비해 허가된 치료제가 현저히 부족한 상황이다(식품의약품 안전처).Due to this global pandemic and severity, many researchers and companies, as well as national/global levels, are pouring a lot of effort and money into the development of coronavirus vaccines and therapeutics. According to the Korea National Clinical Trial Support Foundation, as of December 15, 2020, a total of 1,636 drug intervention clinical trials related to COVID-19 newly registered on ClinicalTrials.gov of the National Institutes of Health (NIH) of the United States were 1,509, and 1,509 clinical trials for treatment. 92.2% of cases, and 127 cases of vaccine clinical trials, accounting for 7.8%, and 454 cases of treatment clinical trials, 30.1% of the proportion of phase 3 clinical trials, and 57 cases of vaccine trials. In Korea, as of March 13, 2021, there are 13 cases of clinical approval for corona treatment, 1 case under approval review, and 2 cases with approved treatment (Gilliard, Celltrion). Ministry of Drug Safety).
한편, Angiotensin-converting enzyme 2(ACE2)는 전염증성 물질인 안지오텐신 II를 분해하여, 염증으로부터 신장, 폐, 심장을 보호하는 세포 표면 수용체이다. ACE2를 통한 코로나바이러스의 세포 내 진입 및 증식 경로는 많은 선행 연구의 보고를 통해 밝혀진바 있다. 코로나바이러스는 스파이크 단백질의 수용체 결합 도메인(Receptor Binding Domain, RBD)과 이의 수용체인 안지오텐신 전환효소 2(ACE2)의 결합을 기반으로 i) 내세포 작용 경로 및 ii) 막의 융합에 의한 직접 유입경로를 통해 세포 내로 진입하여 증식 및 방출되는 것으로 보고되었다 (Int. J. Mol. Sci. 2020, 21, 5224). 따라서, 스파이크 단백질은 코로나바이러스감염증의 백신 및 치료제의 개발에 있어서, 가장 주요한 타겟으로 간주된다. SARS-CoV-2 중화 단백질은 코로나바이러스 감염증의 예방 및 치료를 위한 가장 대표적인 물질로, 항체가 가장 대표적이다. 그러나, SARS-CoV-2의 RBD와 항체간의 결합 친화도가 반드시 바이러스에 대한 중화능으로 연결되지 않아, 단순히 SARS-CoV-2의 RBD에 특이적으로 결합하는 항체를 COVID-19의 치료제로 사용하기에는 실패의 위험이 크다. On the other hand, Angiotensin-converting enzyme 2 (ACE2) is a cell surface receptor that decomposes angiotensin II, a pro-inflammatory substance, and protects the kidneys, lungs, and heart from inflammation. The route of entry and proliferation of coronavirus into cells through ACE2 has been revealed through the reports of many previous studies. Coronaviruses are based on the binding of the receptor binding domain (RBD) of the spike protein and its receptor, angiotensin converting enzyme 2 (ACE2), i) through the endocellular pathway and ii) through a direct entry pathway by membrane fusion. It has been reported to enter cells, proliferate and release (Int. J. Mol. Sci. 2020, 21, 5224). Therefore, 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. However, 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.
반면, Monteil 등은 인간 ACE2 재조합 단백질을 인위적으로 주입하는 경우, 코로나바이러스의 인간세포 감염 방지 및 증식 억제능을 나타낸다는 점을 확인하고 보고한 바 있으며(Monteil et al., Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2, Cell 2020), Aperion사는 재조합 ACE2 단백질을 이용하여 코로나바이러스 치료효과에 대한 임상시험을 시작하였다(APN01). 인간 ACE2 재조합 단백질은 인간이 발현하는 ACE2와 동일한 아미노산서열을 갖기 때문에, 대상의 세포 표면에서 자연 발현된 ACE2 수용체와 경쟁적으로 SARS-CoV-2에 결합하며, 이러한 경쟁적 결합능으로 인해 효과가 용량에 크게 의존하므로, ACE2 투여용량 조절의 어려움 및 바이러스 중화효과가 비교적 떨어진다는 한계가 존재한다.On the other hand, Monteil et al. have confirmed and reported that when the human ACE2 recombinant protein is artificially injected, the coronavirus exhibits the ability to prevent human cell infection and inhibit proliferation (Monteil et al., Inhibition of SARS-CoV-2). Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2, Cell 2020), Aperion started a clinical trial for the therapeutic effect of coronavirus using recombinant ACE2 protein (APN01). Since human ACE2 recombinant protein has the same amino acid sequence as human-expressed ACE2, it competitively binds to SARS-CoV-2 with the ACE2 receptor naturally expressed on the cell surface of the subject. Therefore, there are limitations in that it is difficult to adjust the dose of ACE2 and the neutralizing effect of the virus is relatively low.
이러한 배경기술 아래에서, 본 발명자들은 i) 높은 코로나바이러스 포획 및 중화능을 가지며, ii) 중증 감염 환자 및 기저 질환 환자의 폐, 심장 신장을 보호하는 ACE2 전환 효소의 활성이 보충이 가능하면서도, iii) 다양한 코로나바이러스 변이체의 RBD에 야생형 인간 ACE2보다 높은 결합 친화도 및 중화력을 가지는 ACE2 변이체를 개발하고자 예의 노력한 결과, ACE2와 RBD의 결합구조에 기반한 안지오텐신 변환 효소 II 변이체 라이브러리를 구축하고, 이로부터 야생형 ACE2 단백질 또는 기존에 보고된 재조합 인간 ACE2 단백질보다 현저히 높은 RBD에 대한 결합 친화도를 가지는 ACE2 변이체를 스크리닝하였으며, 상기 ACE2 변이체가 야생형 ACE2보다도 현저히 높은 SARS-CoV-2 바이러스 감염 및 증폭 억제능을 나타낸다는 것을 확인하고 본 발명을 완성하였다.Under this background, the present inventors found that i) has high coronavirus trapping and neutralizing ability, ii) the activity of ACE2-converting enzyme that protects lung, heart and kidney of severely infected patients and patients with underlying disease can be supplemented, iii) ) As a result of earnest efforts to develop ACE2 variants having higher binding affinity and neutralizing power than wild-type human ACE2 to RBD of various coronavirus variants, 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.
본 배경기술 부분에 기재된 상기 정보는 오직 본 발명의 배경에 대한 이해를 향상시키기 위한 것이며, 이에 본 발명이 속하는 기술분야에서 통상의 지식을 가지는 자에게 있어 이미 알려진 선행기술을 형성하는 정보를 포함하지 않을 수 있다.The information described in the background section is only for improving the understanding of the background of the present invention, and it does not include information forming prior art known to those of ordinary skill in the art to which the present invention pertains. it may not be
본 발명의 목적은 높은 코로나바이러스 결합 친화도 및 중화능을 가지는 안지오텐신 변환 효소 II 변이체를의 스크리닝 방법을 제공하는 데 있다.It is an object of the present invention to provide a screening method for an angiotensin converting enzyme II variant having high coronavirus binding affinity and neutralizing ability.
본 발명의 다른 목적은 높은 코로나바이러스 결합 친화도 및 코로나바이러스 중화능을 가지는 안지오텐신 변환효소 II(ACE2) 변이체를 제공하는 데 있다.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.
본 발명의 또 다른 목적은 상기 안지오텐신 변환효소 II(ACE2) 변이체를 포함하는 융합단백질을 제공하는 데 있다.Another object of the present invention is to provide a fusion protein comprising the angiotensin converting enzyme II (ACE2) variant.
본 발명의 또 다른 목적은 상기 안지오텐신 변환효소 II(ACE2) 변이체 또는 이를 포함하는 융합단백질을 코딩하는 핵산을 제공하는데 있다.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.
본 발명의 또 다른 목적은 상기 핵산을 포함하는 재조합 벡터를 제공하는 데 있다.Another object of the present invention is to provide a recombinant vector comprising the nucleic acid.
본 발명의 또 다른 목적은 상기 핵산 또는 재조합 벡터가 숙주세포에 도입된 재조합세포를 제공하는 데 있다.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.
본 발명의 또 다른 목적은 상기 안지오텐신 변환효소 II(ACE2) 변이체 또는 이를 포함하는 융합단백질의 제조방법을 제공하는 데 있다.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.
본 발명의 또 다른 목적은 상기 안지오텐신 변환 효소 II(ACE2) 변이체또는 이를 포함하는 융합단백질의 코로나바이러스의 증식억제, 코로나바이러스감염증의 예방 및 치료 용도를 제공하는 데 있다.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.
상기 목적을 달성하기 위하여, 본 발명은 높은 코로나바이러스 결합 친화도 및 중화능을 가지는 안지오텐신 변환 효소 II(ACE2) 변이체를 제공한다.In order to achieve the above object, the present invention provides an angiotensin converting enzyme II (ACE2) variant having high coronavirus binding affinity and neutralizing ability.
본 발명은 또한, 상기 안지오텐신 변환 효소 II(ACE2) 변이체를 포함하는 융합단백질을 제공한다.The present invention also provides a fusion protein comprising the angiotensin converting enzyme II (ACE2) variant.
본 발명은 또한, 상기 안지오텐신 변환 효소 II(ACE2) 변이체또는 이를 포함하는 융합단백질을 코딩하는 핵산을 제공한다.The present invention also provides a nucleic acid encoding the angiotensin converting enzyme II (ACE2) variant or a fusion protein comprising the same.
본 발명은 또한, 상기 핵산을 함유하는 재조합 벡터를 제공한다.The present invention also provides a recombinant vector containing the nucleic acid.
본 발명은 또한, 상기 핵산 또는 재조합 벡터가 숙주세포에 도입된 재조합세포를 제공한다.The present invention also provides a recombinant cell in which the nucleic acid or recombinant vector is introduced into a host cell.
본 발명은 또한, 상기 재조합세포를 배양하여 상기 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 발현하는 단계; 및 상기 발현된 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 수득하는 단계를 포함하는 안지오텐신 변환 효소 II(ACE2) 변이체또는 이를 포함하는 융합단백질의 제조방법을 제공한다.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.
본 발명은 또한, 상기 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 포함하는 코로나바이러스 감염증의 예방 또는 치료용 약학 조성물을 제공한다.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.
본 발명은 또한, 대상에게 상기 안지오텐신 변환효소 II(ACE2) 변이체 또는 이를 포함하는 융합단백질을 투여하는 단계를 포함하는 코로나바이러스감염증의 예방 및/또는 치료 방법을 제공한다.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.
본 발명은 또한, 상기 안지오텐신 변환 효소 II(ACE2) 변이체 또는 이를 포함하는 융합단백질의 코로나바이러스 감염증의 예방 또는 치료용도를 제공한다.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.
본 발명은 또한 상기 안지오텐신 변환 효소 II(ACE2) 변이체 또는 이를 포함하는 융합단백질의 코로나바이러스 감염증의 예방 및/또는 치료용 약학 조성물의 제조를 위한 용도를 제공한다.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.
본 발명의 안지오텐신 변환 효소 II(ACE2) 변이체는 야생형 ACE2 단백질보다 높은 결합 친화도를 나타낼뿐만 아니라, 코로나바이러스의 세포 내 진입 억제 및 증식 억제와 같은 코로나바이러스 중화능이 현저히 높으며, 코로나바이러스의 변이형에도 광범위한 중화능을 나타내어, 코로나바이러스 감염증의 증식억제, 감염 예방 및 감염증의 치료에 유용하며, 특히, 대유행 중인 COVID-19의 예방 및 치료제로 유용하게 사용될 수 있다.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.
도 1은 인간 ACE2 단백질의 전장 서열을 나타낸 것이다(UniProtKB seq ID: Q9BYF1).1 shows the full-length sequence of human ACE2 protein (UniProtKB seq ID: Q9BYF1).
도 2는 ACE2 변이체 라이브러리 제조를 위한 pYD5 템플릿벡터의 모식도를 나타낸 것이다.Figure 2 shows a schematic diagram of the pYD5 template vector for the preparation of the ACE2 mutant library.
도 3은 ACE2 특정 위치의 아미노산에 변이를 포함하는 point mutation library 제작에 수행된 PCR 조건을 나타낸 것이다.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.
도 4는 효모표면발현기술 (Yeast Surface Display, YSD)을 위한 효모의 형질전환방법을 개략적으로 나타낸 것이다.Figure 4 schematically shows the transformation method of yeast for yeast surface expression technology (Yeast Surface Display, YSD).
도 5는 점 돌연변이(point mutation) 라이브러리에서 round sorting 완료된 개별클론의 SARS-CoV-2 S protein RBD에 대한 결합 친화도를 FACS 분석한 결과를 나타낸 것이다.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.
도 6은 Error prone 라이브러리의 모식도 및 조건을 나타낸 것이다. 6 shows a schematic diagram and conditions of the Error prone library.
도 7은 각 Error prone 라이브러리로부터 4 round sorting된 개별 클론의 SARS-CoV-2 S protein RBD에 대한 결합 친화도를 FACS 분석한 결과를 나타낸 것이다.7 shows the results of FACS analysis of the binding affinity to SARS-CoV-2 S protein RBD of individual clones sorted 4 rounds from each error prone library.
도 7a는 ACE2 H34A 템플릿의 Q18-L100 error-prone 라이브러리의 개별 clone의 Facs 분석결과이다. Figure 7a is a Facs analysis result of individual clones of the Q18-L100 error-prone library of the ACE2 H34A template.
도 7b는 ACE2 H34A 템플릿의 Q18-D615 error-prone 라이브러리의 개별 clone의 Facs 분석결과이다. Figure 7b is a Facs analysis result of individual clones of the Q18-D615 error-prone library of the ACE2 H34A template.
도 7c는 ACE2 8mut((S19P, T27L, D30V, H34A, E35V, L79V, Q325P, N330Y) 템플릿의 Q18-L100 error-prone 라이브러리의 개별 clone의 Facs 분석결과이다. 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.
도 7d는 ACE2 8mut((S19P, T27L, D30V, H34A, E35V, L79V, Q325P, N330Y) 템플릿의 Q18-D615 error-prone 라이브러리의 개별 clone의 Facs 분석결과이다.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.
도 8은 Fc 도메인 융합 ACE2 변이체의 생산을 위해 사용된 벡터의 모식도를 나타낸 것이다.8 shows a schematic diagram of the vector used for the production of Fc domain fusion ACE2 variants.
도 9는 생산된 ACE2 변이체의 SDS-PAGE 결과이다. 도 9a는 환원제를 첨가하여 환원한 SDS-PAGE 결과이며, 도 9b는 환원제를 첨가하지 않은 SDS_PAGE 결과이다.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.
도 10은 생산된 결합 친화도 향상 ACE2 후보군의 Size exclusion chromatography 결과를 나타낸 것이다(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)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)
도 11은 ACE2.V.43 내지 ACE2.V.48의 SARS-CoV-2 중화능을 ACE2.wt와 비교한 것이다. 11 is a comparison of the SARS-CoV-2 neutralizing ability of ACE2.V.43 to ACE2.V.48 with ACE2.wt.
도 12는 ACE2.V.46 및 ACE2.V.48의 SARS-CoV-2 중화능을 ACE2.wt와 비교한 것이다. 12 is a comparison of the SARS-CoV-2 neutralizing ability of ACE2.V.46 and ACE2.V.48 with ACE2.wt.
도 13은 영국발 SARS-CoV-2 RBD 변이체에 대한 ACE2 wild type, ACE2.V.46의 중화능을 비교한 것이다. 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.
도 14는 남아공발 SARS-CoV-2 RBD 변이체에 대한 ACE2 wild type, ACE2.V.46의 중화능을 비교한 것이다. 14 is a comparison of the neutralizing ability of ACE2 wild type, ACE2.V.46 against the SARS-CoV-2 RBD mutant from South Africa.
도 15는 ACE2.V.40 내지 V.42 변이체의 촉매 활성을 확인한 결과이다.15 is a result confirming the catalytic activity of ACE2.V.40 to V.42 variants.
도 16은 ACE2.V.43 내지 V.48 변이체의 촉매 활성을 확인한 결과이다.16 is a result confirming the catalytic activity of ACE2.V.43 to V.48 variants.
도 17은 ACE2.V.46 변이체와 야생형 ACE2의 생체 내 안정성을 확인한 결과이다. 좌측은 생체 내 안정성 테스트를 모식적으로 나타낸 것이다.17 is a result confirming the in vivo stability of ACE2.V.46 mutant and wild-type ACE2. The left side schematically shows the in vivo stability test.
도 18은 ACE2 변이체의 serum 내 촉매 활성을 확인한 결과이다.18 is a result confirming the catalytic activity in serum of the ACE2 mutant.
도 19는 본 발명의 ACE2 변이체 ACE2.V.06 처리에 의한 SARS-CoV-2 (MOI=1)의 CPE 감소 결과를 보여준다.19 shows the CPE reduction result of SARS-CoV-2 (MOI=1) by treatment with the ACE2 mutant ACE2.V.06 of the present invention.
도 20은 본 발명의 ACE2 변이체 ACE2.V.06 처리에 의한 SARS-CoV-2 (MOI=0.1)의 CPE 감소 결과를 보여준다.20 shows the CPE reduction result of SARS-CoV-2 (MOI=0.1) by treatment with the ACE2 mutant ACE2.V.06 of the present invention.
도 21은 본 발명의 ACE2 변이체 ACE2.V.06 처리에 의한 SARS-CoV-2 단백질 감소 결과를 보여준다.Figure 21 shows the result of SARS-CoV-2 protein reduction by the ACE2 mutant ACE2.V.06 treatment of the present invention.
도 22는 ACE2-WT 또는 본 발명의 ACE2 변이체 ACE2.V.06 처리에 의한 SARS-CoV-2 CPE 감소 결과를 보여준다.22 shows the results of SARS-CoV-2 CPE reduction by treatment with ACE2-WT or ACE2.V.06 of the present invention.
도 23은 ACE2-WT 또는 본 발명의 ACE2 변이체 ACE2.V.06 처리에 의한 SARS-CoV-2 단백질과 RNA양의 변화 결과를 보여준다.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.
도 24는 ACE2-WT 또는 본 발명의 ACE2 변이체 ACE2.V.06 처리 후 24시간 배양에서 SARS-CoV-2의 CPE 감소 결과를 보여준다.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.
도 25는 ACE2-WT 또는 본 발명의 ACE2 변이체 ACE2.V.06 처리 후 24시간 배양에서 SARS-CoV-2의 단백질 감소 결과를 보여준다.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.
도 26은 ACE2-WT 또는 본 발명의 ACE2 변이체 ACE2.V.06 처리 후 24시간 배양에서 SARS-CoV-2의 RNA 감소 결과를 보여준다.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.
도 27는 ACE2-WT 또는 본 발명의 ACE2 변이체 ACE2.V.41 처리 후 24시간 배양에서 SARS-CoV-2의 CPE 감소 결과를 보여준다.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.
도 28은 ACE2-WT 또는 본 발명의 ACE2 변이체 ACE2.V.41 처리 후 SARS-CoV-2의 단백질 감소 결과를 보여준다.28 shows the protein reduction results of SARS-CoV-2 after treatment with ACE2-WT or ACE2.V.41 of the present invention.
도 29은 ACE2-WT 또는 본 발명의 ACE2 변이체 ACE2.V.41 처리 후 SARS-CoV-2의 RNA 감소 결과를 보여준다.29 shows RNA reduction results of SARS-CoV-2 after treatment with ACE2-WT or ACE2.V.41 of the present invention.
도 30은 ACE2-WT 또는 본 발명의 ACE2 변이체 ACE2.V.41 처리 후 SARS-CoV-2의 CPE 감소 결과를 보여준다.30 shows the CPE reduction results of SARS-CoV-2 after treatment with ACE2-WT or ACE2.V.41 of the present invention.
도 31은 ACE2-WT 또는 본 발명의 ACE2 변이체 ACE2.V.41 처리 후 SARS-CoV-2 단백질 감소 결과를 보여준다.Figure 31 shows the result of SARS-CoV-2 protein reduction after treatment with ACE2-WT or ACE2.V.41 of the present invention.
도 32는 ACE2-WT 또는 본 발명의 ACE2 변이체 ACE2.V.41 처리 후 SARS-CoV-2 RNA 감소 결과를 보여준다.Figure 32 shows the result of SARS-CoV-2 RNA reduction after treatment with ACE2-WT or ACE2.V.41 of the present invention.
도 33은 SARS-CoV-2 중화(neutralization)에 필요한 ACE2-WT 또는 본 발명의 ACE2 변이체 ACE2.V.41의 IC50 값 산출 결과를 보여준다.33 shows the result of calculating the IC 50 value of ACE2-WT or ACE2 mutant ACE2.V.41 of the present invention required for SARS-CoV-2 neutralization.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 가진다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.
특별한 언급이 없는 경우, 본 명세서에 기재된 모든 염기서열은 5'말단에서 3'말단으로, 모든 아미노산 서열은 N'말단에서 C'말단 방향으로 작성하였다.Unless otherwise specified, all 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.
현재 코로나바이러스 중 하나인 SARS-CoV-2 바이러스에 의한 COVID-19가 전세계적으로 대유행 중에 있으며, 전세계적으로 1억명이 넘는 확진자 및 수백만명의 사망자가 발생하였음에도, 백신 및 치료제의 개발 지연 및 공급부족으로 여전히 확산세가 감소되지 않고 있다.Currently, COVID-19 caused by the SARS-CoV-2 virus, one of the coronaviruses, 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.
코로나바이러스의 세포 감염 및 증식에 관여하는 핵심 분자인 스파이크 단백질의 RBD와 이의 수용체인 ACE2는 코로나바이러스 감염증의 예방 또는 치료를 위한 주요 타겟으로, 현재 개발되는 대부분의 치료제는 RBD의 중화를 목적으로 한다. 대표적인 RBD 중화 단백질인 항체는 결합 친화도를 기반으로 스크리닝하는데 반해 결합 친화도가 중화능으로 반드시 이어지지 않아, 치료 효능에 대한 성공률이 낮으며, 인간 재조합 ACE2 단백질의 투여가 코로나바이러스 중화효과를 나타내는 것으로 보고되었으나, 대상의 세포에서 발현하는 ACE2와 경쟁적으로 RBD에 결합하여, 농도의존성 및 중화효과가 낮다는 단점이 있다.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.
본 발명의 일 실시예에서, 본 발명자들은 야생형 ACE2보다 높은 코로나바이러스 결합 친화도를 나타낼 수 있는 변이 후보 아미노산을 선별하여 높은 코로나바이러스 결합 친화도를 가지는 안지오텐신 변환 효소 II 변이체 라이브러리를 제조하였다. In one embodiment of the present invention, 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.
또한, 본 발명의 다른 실시예에서, 제조된 안지오텐신 변환 효소 II(ACE2) 변이체 라이브러리로부터 높은 코로나바이러스 결합 친화도와 함께 코로나바이러스 중화능을 가지는 안지오텐신 변환 효소 II 변이체를 스크리닝 하였으며, 스크리닝된 안지오텐신 변환 효소 II 변이체가 야생형 안지오텐신 변환 효소 II에 비해 현저히 뛰어난 코로나바이러스 결합 친화도 및 중화능을 나타내는 것을 확인하였다.In addition, in another embodiment of the present invention, 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.
본 발명의 ACE2 변이체는 투여시에 야생형 ACE2보다 높은 친화력으로 코로나바이러스에 결합하여, 코로나바이러스와 세포표면 ACE2 결합률을 낮추고, 높은 코로나바이러스 중화능을 나타내므로, COVID-19를 비롯한 코로나바이러스 감염증의 예방 및 치료에 유용하게 사용될 수 있다.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.
따라서, 본 발명은 일 관점에서, 높은 코로나바이러스 결합 친화도 및 코로나바이러스 중화능을 가지는 안지오텐신 변환 효소 II(ACE2) 변이체에 관한 것이다.Accordingly, in one aspect, the present invention relates to an angiotensin converting enzyme II (ACE2) variant having high coronavirus binding affinity and coronavirus neutralizing ability.
본 발명의 용어, "안지오텐신 변환 효소 II(ACE2)"는 메탈로카복시펩티다제 (metallo-carboxypeptidase)의 하나로, 안지오텐신 전환효소와 상동하는 1형 막관통 단백질이며, 심장, 폐, 콩팥, 혈관내피와 소화계통에서 주로 발현되어, 안지오텐신 II의 가수분해를 촉매하고, 심혈관계 질환을 방지하는 역할을 수행하는 것으로 알려져 있다. 또한, SARS-CoV와 SARS-CoV-2를 포함한 여러 코로나바이러스가 가지는 스파이크 단백질의 수용체 결합 도메인(RBD)의 수용체로서, 코로나바이러스의 세포 내 진입 및 증식에 필수적인 역할을 담당하는 것으로 보고되었다(W. Li, at al., Nature. Vol. 426, No. 6965, November 2003, S. 450-454; Yushun Wan, at al., doi 10.1128/JVI.00127-20; H. Hofmann, at al., Proceedings of the National Academy of Sciences. Vol. 102, No. 22, May 2005, S. 79808-7993 등). 본 발명에서, 안지오텐신 변환 효소 II는"ACE2"와 동일한 의미에서 상호호환적으로 사용된다.As used herein, the term "angiotensin converting enzyme II (ACE2)" 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. In addition, as a receptor of the receptor binding domain (RBD) of the spike protein of several coronaviruses including SARS-CoV and SARS-CoV-2, it has been reported that it plays an essential role in the entry and proliferation of the coronavirus into cells (W Li, at al., Nature. Vol. 426, No. 6965, November 2003, S. 450-454; Yushun Wan, at al., doi 10.1128/JVI.00127-20; Proceedings of the National Academy of Sciences. Vol. 102, No. 22, May 2005, S. 79808-7993 et al.). In the present invention, angiotensin converting enzyme II is used interchangeably in the same meaning as "ACE2".
본 발명에서 개시하는 서열번호 1의 서열은 인간 야생형 ACE2(hACE2) 서열로, UniProtKB에 seq ID: Q9BYF1으로 보고되었으며, 이와 실질적으로 동일한 많은 야생형 ACE2 서열이 보고되어 있다. 야생형 ACE2와 코로나바이러스의 결합 구조는 본 기술 분야에 상세히 보고되어 있으며, 코로나바이러스의 종 간에 그 서열이 보존되어 있다. 본 발명에 있어서, 안지오텐신 변환 효소 II는 서열번호 1의 아미노산 서열로 표시되는 것일 수 있으나, 이에 제한되는 것은 아니며, 이와 생물학적, 기능적으로 실질적으로 동일한 것으로 간주되는 서열을 포함한다. 예를 들어, 서열번호 1과 80% 이상, 90% 이상, 바람직하게는 95% 이상, 가장 바람직하게는 99% 이상의 상동성을 가지는 서열일 수 있으나, 이에 제한되는 것은 아니다. 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. In the present invention, 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.
본 발명에서 개시하는 서열번호 2의 서열은 서열번호 1의 안지오텐신 변환 효소 II(ACE2)의 신호서열 및 세포 외 도메인의 일부를 포함하는 안지오텐신 변환 효소 II(ACE2)의 절편이다. 본 발명의 실시예에서, 상기 서열번호 2를 주형으로 변이체를 제작하였다. 본 발명에 있어서, 안지오텐신 변환 효소 II(ACE2) 변이체는 전장 단백질 및 이의 절편을 포함하는 의미로 사용되므로, 서열번호 2의 변이체는 본 발명의 안지오텐신 변환 효소 II(ACE2) 변이체의 범위에 포함되는 것으로 해석되어야 한다.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. In an example of the present invention, a variant was prepared using SEQ ID NO: 2 as a template. In the present invention, 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
본 발명에 있어서, 상기 안지오텐신 변환 효소 II는 특정 아미노산 잔기 위치에서, 아미노산 잔기가 보존적으로 치환된 변이체 또는 이의 절편들도 포함하는 의미로 해석된다. 본 명세서에서 "보존적 치환"이란 1개 이상의 아미노산을 안지오텐신 변환 효소 II의 생물학적 또는 생화학적 기능의 손실을 야기하지 않는 유사한 생화학적 특성을 가지는 아미노산으로 치환하는 것을 포함하는 수용체 결합 도메인의 변형을 의미한다.In the present invention, 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. As used herein, "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. .
“보존적 아미노산 치환"은 아미노산 잔기를 유사한 측쇄를 가지는 아미노산 잔기로의 치환을 의미한다. 예를 들어, 유사한 측쇄를 가지는 아미노산 잔기 부류는 해당 기술분야에 잘 알려져 있다. 이들 부류는 염기성 측쇄를 가지는 아미노산(예를 들어, 라이신, 아르기닌, 히스티딘), 산성 측쇄를 가지는 아미노산(예를 들어, 아스파르트산, 글루탐산), 대전되지 않은 극성 측쇄를 가지는 아미노산(예를 들어, 글리신, 아스파라진, 글루타민, 세린, 트레오닌, 티로신, 시스테인), 비-극성 측쇄를 가지는 아미노산(예를 들어, 알라닌, 발린, 류신, 이소류신, 프롤린, 페닐알라닌, 메티오닌, 트립토판), 베타-분지된 측쇄를 가지는 아미노산(예를 들어, 트레오닌, 발린, 이소류신) 및 방향족 측쇄를 가지는 아미노산(예를 들어, 티로신, 페닐알라닌, 트립토판, 히스티딘)을 포함한다."Conservative amino acid substitution" means a substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, 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).
본 명세서에서 사용된 "안지오텐신 변환 효소 II 변이체"는 야생형 안지오텐신 변환 효소 II에서 일부 아미노산 잔기의 변이, 바람직하게는 아미노산 잔기의 치환, 결실, 삽입 등을 포함하는 것뿐만 아니라, N-말단 또는 C-말단에서의 일부 아미노산 잔기의 절단 등을 모두 포함하는 개념으로 사용된다.As used herein, "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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II(ACE2) 변이체는 서열번호 2에서, S19, K26, T27, D30, K31, H34, E35, N49, K74, L79, L91, V185, N250, S280, Q325, N330, G448, R514 및 S602로 구성된 군에서 선택되는 어느 하나 이상의 아미노산에 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II (ACE2) variant in SEQ ID NO: 2, S19, K26, T27, D30, K31, H34, E35, N49, K74, L79, L91, V185, N250, S280, Q325, It may be characterized by having a mutation in any one or more amino acids selected from the group consisting of N330, G448, R514 and S602.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II(ACE2) 변이체는 서열번호 2에서 S19, T27, D30, H34, E35, L79, Q325 및 N330로 구성된 군에서 선택되는 어느 하나 이상의 아미노산에 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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
본 발명에 있어서, 상기 안지오텐신 변환 효소 II(ACE2) 변이체는 서열번호 2에서 H34 아미노산에 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II(ACE2) 변이체는 서열번호 2에서 H34 아미노산에 변이; 및In the present invention, the angiotensin converting enzyme II (ACE2) mutant is a mutation in the H34 amino acid in SEQ ID NO: 2; and
N49, L79, L91, S280 및 S602로 구성된 군에서 선택되는 어느 하나 이상의 아미노산에 변이를 가지는 것을 특징으로 할 수 있다.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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II(ACE2) 변이체는 서열번호 2에서 S19, T27, D30, H34, E35, L79, Q325 및 N330 아미노산에 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II(ACE2) 변이체는 서열번호 2에서 S19, T27, D30, H34, E35, L79, Q325 및 N330 아미노산에 변이; 및In the present invention, the angiotensin converting enzyme II (ACE2) variant is S19, T27, D30, H34, E35, L79, Q325 and N330 amino acids in SEQ ID NO: 2; and
K26, K31, K74, L91, V185, N250 및 G448로 구성된 군에서 선택되는 어느 하나 이상의 아미노산에 변이를 가지는 것을 특징으로 할 수 있다.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.
본 발명의 일 실시예에서는 상기 도출된 변이 부위의 아미노산이 치환된 변이체에서, 높은 SARS-CoV-2 S protein RBD에 대한 결합친화도를 나타내는 것을 확인하였다.In an embodiment of the present invention, it was confirmed that 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.
본 발명에 있어서, 상기 변이는 치환, 결실, 삽입 등의 아미노산 돌연변이, 아미노산의 당쇄화, 측쇄의 치환 등을 모두 포함하는 최광의의 개념으로 사용된다. In the present invention, 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.
본 발명에 있어서, 상기 변이는 아미노산의 치환인 것을 특징으로 할 수 있다.In the present invention, the mutation may be characterized in that the substitution of amino acids.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II(ACE2) 변이체는 서열번호 2에서 S19W 또는 S19P; K26N; T27W, T27D, T27A, T27Y, T27L, T27M 또는 T27C; D30V; K31R; H34A 또는 H34V; E35V; N49D; K74R; L79I, L79Y 또는 L79V; L91P; V185A; N250K; S280R; Q325P; N330Y, N330H 또는 N330F; G448V; R514Q; 및 S602T로 구성된 군에서 선택되는 어느 하나 이상, 바람직하게는 셋 이상의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II(ACE2) 변이체는 서열번호 2에서 S19W 또는 S19P; T27W, T27D, T27A, T27Y, T27L, T27M 또는 T27C; D30V; H34A 또는 H34V; E35V; L79Y 또는 L79V; Q325P; 및 N330Y, N330H 또는 N330F로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 S19W 또는 S19P, T27L, D30V, H34A, E35V, L79V, Q325P 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 H34A의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of H34A in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II(ACE2) 변이체는 서열번호 2에서 H34A의 변이; 및In the present invention, the angiotensin converting enzyme II (ACE2) mutant is a mutation of H34A in SEQ ID NO: 2; and
N49D, L79I, L91P, S280R 및 S602T로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있다.It may be characterized as having one or more mutations selected from the group consisting of N49D, L79I, L91P, S280R and S602T.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 S19W 또는 S19P, T27L, D30V, H34A, E35V, L79V, Q325P 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 S19W 또는 S19P, T27L, D30V, H34A, E35V, L79V, Q325P 및 N330Y의 변이; 및In the present invention, the angiotensin converting enzyme II variant is S19W or S19P, T27L, D30V, H34A, E35V, L79V, Q325P and N330Y in SEQ ID NO: 2; and
K26N, K31R, K74R, L91P, V185A, N250K 및 G448V로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있다.It may be characterized as having one or more mutations selected from the group consisting of K26N, K31R, K74R, L91P, V185A, N250K and G448V.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 H34V의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of H34V in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 S19W의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of S19W in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 S19P의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of S19P in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27D의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of T27D in SEQ ID NO:2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of T27Y in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27L의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of T27L in SEQ ID NO:2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27C의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II variant may be characterized in that it has a mutation of T27C in SEQ ID NO:2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 D30V의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of D30V in SEQ ID NO:2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 E35V의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of E35V in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 L79Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of L79Y in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 L79V의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II variant may be characterized in that it has a mutation of L79V in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 Q325P의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of Q325P in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of N330Y in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 N330F의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of N330F in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 S19P, T27L, H34A, L79V 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 S19P, T27L, H34A, L79V 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 S19P, T27L, D30V, H34A, E35V, L79V, Q325P 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 S19P, T27L, D30V, H34A, E35V, L79V, Q325P 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 N330H의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of N330H in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 H34A 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has mutations of H34A and N330Y in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 H34V 및 N330H의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has mutations of H34V and N330H in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27Y 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has mutations of T27Y and N330Y in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27Y 및 H34A의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has mutations in T27Y and H34A in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27Y, H34A 및 N330Y로 구성된 군에서 선택된 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 T27Y, H34A 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 H34V 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has mutations of H34V and N330Y in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 K26N의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of K26N in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 K31R의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of K31R in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 L91P의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of L91P in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 K26N, K31R 및 L91P로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 K26N, K31R 및 L91P의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 R514Q의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of R514Q in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 L79I의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, the angiotensin converting enzyme II mutant may be characterized in that it has a mutation of L79I in SEQ ID NO: 2.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27Y, H34A 및 L79Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 T27Y, H34A 및 L79Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27Y, H34A, L79Y 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 T27Y, H34A, L79Y 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27Y, H34A, L79V 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 T27Y, H34A, L79V 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27D, H34A, L79V 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 T27D, H34A, L79V 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27Y, H34A, L79Y, L91P 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 T27Y, H34A, L79Y, L91P 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27Y, H34A, L79V, L91P 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 T27Y, H34A, L79V, L91P 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 T27D, H34A, L79V, L91P 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 T27D, H34A, L79V, L91P 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 S19P, T27D, H34A, L79V 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 S19P, T27D, H34A, L79V 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 S19P, T27D, H34A, L79V, L91P 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 S19P, T27D, H34A, L79V, L91P 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 S19P, T27Y, H34A, L79Y 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 S19P, T27Y, H34A, L79Y 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 S19P, T27Y, H34A, L79V 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것을 특징으로 할 수 있으며, 바람직하게는 S19P, T27Y, H34A, L79V 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다. In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환효소 II 변이체는 서열번호 2에서 S19P, T27Y, H34A, L79V, L91P 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것, 바람직하게는 S19P, T27Y, H34A, L79V, L91P 및 N330Y로 구성된 군에서 선택되는 셋 이상의 변이를 가지는 것, 가장 바람직하게는 S19P, T27Y, H34A, L79V, L91P 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 2에서 S19P, T27Y, H34A, L79Y, L91P 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 것, 바람직하게는 S19P, T27Y, H34A, L79Y, L91P 및 N330Y로 구성된 군에서 선택되는 셋 이상의 변이를 가지는 것, 가장 바람직하게는 S19P, T27Y, H34A, L79Y, L91P 및 N330Y의 변이를 가지는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 "변이를 가지는 것을 특징으로 하는 안지오텐신 변환 효소 II 변이체"는 해당 문맥에서 개시된 변이뿐만 아니라, 이 외에 추가적인 변이를 갖는 안지오텐신 변환 효소 II 변이체를 포함하는 의미로 사용된다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 서열번호 3 내지 45로 구성된 군에서 선택되는 서열을 포함하는 변이체 또는 이의 절편인 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 바람직하게는 서열번호 33(ACE2.V.36) 내지 45(ACE2.V.48)로 구성된 군에서 선택되는 서열, 더욱 바람직하게는 서열번호 43(ACE2.V.46) 또는 45(ACE2.V.48)로 표시되는 서열, 가장 바람직하게는 서열번호 43(ACE2.V46)로 표시되는 서열을 포함하는 변이체 또는 이의 절편인 것을 특징으로 할 수 있다.In the present invention, 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). .
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 야생형 안지오텐신 변환 효소 II에 비해 현저히 높은 코로나바이러스 결합 친화도를 나타내는 것을 특징으로 할 수 잇다.In the present invention, 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.
본 발명의 실시예에서, 안지오텐신 변환 효소 II 변이체를 스크리닝하기 위해, 야생형 ACE2의 세포외 도메인 절편(서열번호 2)에 돌연변이를 유도하여 라이브러리를 제조하였다. In an example of the present invention, in order to screen for angiotensin converting enzyme II variants, a library was prepared by inducing mutations in the extracellular domain fragment of wild-type ACE2 (SEQ ID NO: 2).
따라서, 본 발명에 있어서, 안지오텐신 변환 효소 II 변이체는 예를 들어, 안지오텐신 변환 효소 II 전장 단백질뿐만 아니라, 이에 포함된 각 도메인 또는 전장 단백질의 일부(예, 서열번호 2)의 변이체와 같은 "안지오텐신 변환 효소 II 변이체의 절편"을 포함하는 광의의 개념으로 이해되어야 한다.Therefore, in the present invention, 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".
본 명세서에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 인간 안지오텐신 변환 효소 II의 야생형으로 보고된 전장 서열인 서열번호 2을 기준으로 변이를 포함하는 아미노산을 개시하였다. 앞서 설명한 바와 같이, 서열번호 2는 서열번호 1의 전장 인간 안지오텐신 변환 효소 II의 신호서열 및 세포 외 도메인의 일부 단편을 포함하는 절편(M1~D615)으로서, SARS-CoV-2 결합 부위를 포함한다.In the present specification, 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. As described above, 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. .
따라서, 본 발명은 안지오텐신 변환 효소 II의 전장 서열인 서열번호 1을 기준으로 해석될 수 있다. 서열번호 1을 기준으로 변이 아미노산 부위 또는 아미노산의 치환을 해석하는 경우에도, 서열번호 2와 동일한 아미노산 번호를 갖는다.Accordingly, the present invention can be interpreted based on SEQ ID NO: 1, which 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.
본 발명에 있어서, 상기 서열번호 2 이외에도, 본 발명의 아미노산 변이 부위를 포함하는 절편이라면, 모두 본 발명의 범위에 포함된다. 예를 들어, 신호서열을 제외한 세포 외 도메인 절편(Q18~740) 또는 이의 일부(Q18~D615) 등일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, 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. For example, 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.
또한, 통상의 기술자들은 동일 종 또는 상이한 종으로부터 유래한 안지오텐신 변환 효소 II가 서열번호 1 또는 서열번호 2와 상이한 아미노산 서열을 가지는 경우(예를 들어, 일부 아미노산이 보존적으로 치환된 ACE2, 인간과 다른 종 유래의 ACE2 등), 또는 이의 절편인 경우 통상의 기술자는 서열의 정렬(alignment) 및 분석 등을 통해 상기 기재된 아미노산 변이 부위에 상응하는 아미노산을 용이하게 도출할 수 있음은 자명하다 할 것이다. 이 경우, 본 발명의 안지오텐신 변환 효소 II는 상기 기재된 아미노산에 상응하는 아미노산에서 변이를 갖는 것을 특징으로 할 수 있음은 자명하다.In addition, those skilled in the art know that when 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.
본 발명에 있어서, "S19"와 같이 1 글자(one letter)의 아미노산 잔기명과 숫자(n)가 함께 기재된 표현은 각 아미노산 서열에서의 해당 n번째 위치에서의 아미노산 잔기 및 종류를 의미한다.In the present invention, 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.
예를 들어 서열번호 2(또는 서열번호 1)의 아미노산 서열에서 "S19"은 서열번호 2(또는 서열번호 1)의 19번째 위치에서의 아미노산 잔기가 세린임을 의미한다. "S19W"와 같이, 숫자 뒤의 아미노산 잔기명은 아미노산의 치환을 의미하며, 상기 "S19W"는 서열번호 2(또는 서열번호 1)의 137번째 위치에서의 세린(Ser, S)이 트립토판(Try, W)으로 치환된 것을 의미한다.For example, "S19" in the amino acid sequence of SEQ ID NO: 2 (or SEQ ID NO: 1) means that the amino acid residue at the 19th position of SEQ ID NO: 2 (or SEQ ID NO: 1) is serine. Like "S19W", the 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체는 생물학적 특성 또는 물리/화학적 특성의 변형(modulation)을 위해, 다른 폴리펩타이드, 단백질 또는 당, PEG와 같은 구조체와 융합되어 사용되는 것을 특징으로 할 수 있다.In the present invention, 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. .
본 발명의 일 실시예에서, 안지오텐신 변환 효소 II 변이체와 인간 IgG1 유래의 Fc 도메인을 융합하여 융합단백질을 제조하였다. Fc 도메인과 융합하는 경우에도, 동일한 수준의 SARS-CoV-2(변종 포함) 결합 친화도를 나타냈으며, 중화능도 동일한 수준으로 유지됨을 확인하였다. Fc 도메인을 결합한 융합 단백질은 생물학적 활성 손실이 최소화되면서도, 반감기의 증가, 발현 수준의 증가 등의 생물학적/기능적 특성의 향상을 나타낼 수 있다.In an embodiment of the present invention, 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.
본 발명은 또 다른 관점에서, 안지오텐신 변환 효소 II 변이체를 포함하는 융합 단백질에 관한 것이다.In another aspect, the present invention relates to a fusion protein comprising an angiotensin converting enzyme II variant.
본 발명에 있어서, 상기 융합단백질은 Fc 도메인을 추가로 포함하는 것을 특징으로 할 수 있다. In the present invention, the fusion protein may be characterized in that it further comprises an Fc domain.
본 발명의 용어 “Fc 도메인”은 면역글로불린(immunoglobulin)의 세포 표면의 수용체 및 보체 시스템의 단백질 등과 상호작용하는 항체의 꼬리영역으로, 중쇄 불변 도메인, CH2 및 CH3를 포함하며, 중쇄 불변 영역의 힌지 영역을 추가로 포함할 수 있다. 본 발명에 있어서, 상기 Fc 도메인은 그 특성의 변형(modulation)을 위해, 절단, 아미노산의 치환 등이 수행될 수 있다. 따라서, 본 발명에 있어서, 상기 Fc 도메인은 면역글로불린의 Fc 도메인, 이의 단편 및 이의 변이체를 모두 포함하는 개념으로 사용된다.As used herein, 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. In the present invention, 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.
본 발명에 있어서, 상기 Fc 도메인은 포유 동물의 면역글로불린의 Fc 도메인일 수 있으며, 바람직하게는, 인간 면역글로불린의 Fc 도메인일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the Fc domain may be a mammalian immunoglobulin Fc domain, preferably, a human immunoglobulin Fc domain, but is not limited thereto.
본 발명에 있어서, 상기 Fc 도메인은 IgA, IgM, IgE, IgD, 또는 IgG의 Fc 도메인, 이의 단편 혹은 이들의 변형일 수 있고, 바람직하게는 상기 Fc 도메인은 IgG의 Fc 도메인(예, IgG1, IgG2a, IgG2b, IgG3, 또는 IgG4의 Fc 도메인)일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, 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.
본 발명에 있어서, 상기 Fc 도메인은 인간 IgG1 Fc 도메인일 수 있다. In the present invention, the Fc domain may be a human IgG1 Fc domain.
본 발명에 있어서, 상기 Fc 도메인은 면역글로불린으로부터 유래한 Fc 도메인의 변이체를 포함하는 의미로 사용된다.In the present invention, the Fc domain is used to include a variant of the Fc domain derived from immunoglobulin.
본 발명에 있어서, 상기 Fc 도메인은 FcγR 결합 친화도가 감소된 Fc 도메인 변이체인 것을 특징으로 할 수 있으며, 예를 들어, 인간 IgG1 Fc 도메인(서열번호 46)의 L234A, L235A, 및/또는 K322A 변이체일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the Fc domain may be characterized as an Fc domain variant with reduced FcγR 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.
본 발명에 있어서, 상기 Fc 도메인은 서열번호 46 또는 47로 표시되는 서열을 포함하는 것을 특징으로 할 수 있다.In the present invention, the Fc domain may be characterized in that it comprises a sequence represented by SEQ ID NO: 46 or 47.
본 발명에 있어서, 안지오텐신 변환 효소 II 변이체는 Fc 도메인의 N'-말단 또는 C'-말단에 연결되는 것을 특징으로 할 수 있으며, 바람직하게는 N'-말단에 연결되는 것을 특징으로 할 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II 변이체 및 Fc 도메인은 링커에 의해 연결된 것을 특징으로 할 수 있다. 예를 들어, 상기 링커는 글리신-세린 링커(Glycin-Serine Linker, GS 링커), 더욱 바람직하게는 본 발명의 실시예와 같이, GS 링커일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the angiotensin converting enzyme II variant and the Fc domain may be characterized in that they are linked by a linker. For example, 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.
본 발명에 있어서, 상기 Fc 도메인은 당쇄를 포함할 수 있으며, 야생형에 비해 증가 또는 감소된 당쇄를 포함하거나 당쇄가 제거된 형태일 수 있다. 화학적 방법, 효소적 방법 및 미생물을 사용한 유전공학적 엔지니어링 방법 등과 같은 당업계에 알려진 통상적인 방법으로 면역글로불린 Fc 도메인의 당쇄의 증가, 감소 또는 제거가 수행될 수 있다. Fc 도메인에서 당쇄의 제거는 1차 보체 구성요소 C1의 C1q에의 결합 친화력을 급격하게 감소시키고, ADCC(antibody-dependent cell-mediated cytotoxicity) 또는 CDC(complement-dependent cytotoxicity)의 감소 또는 소실을 가져오며, 그로 인하여 생체 내의 불필요한 면역반응을 유도하지 않는 특징을 나타낼 수 있다.In the present invention, 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.
본 발명에 있어서, 안지오텐신 변환 효소 II(ACE2) 변이체 및 Fc 도메인 융합단백질은 단량체 형태일 수 있으며 이에 제한되는 것은 아니다.In the present invention, the 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.
본 발명에 있어서, 상기 안지오텐신 변환 효소 II(ACE2) 변이체 및 Fc 도메인 융합단백질은 동종이량체 또는 이종이량체일 수 있으며, 3량체 이상의 다량체 형태일 수 있다. 예를 들어, Fc 도메인을 포함하는 융합단백질의 다량체 형태는 Fc 도메인의 힌지 부분에서 이황화결합을 통해 형성될 수 있으나, 이에 제한되는 것은 아니다.In the present invention, 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. For example, 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.
본 발명은 또 다른 관점에서, 본 발명의 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 코딩하는 핵산에 관한 것이다.In another aspect, 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.
본 명세서에서 사용되는 핵산은 세포, 세포 용해물(lysate) 중에 존재하거나, 또는 부분적으로 정제된 형태 또는 실질적으로 순수한 형태로 존재할 수도 있다. 핵산은 알칼리/SDS 처리, CsCl 밴드화(banding), 컬럼 크로마토그래피, 아가로스 겔 전기 영동 및 해당 기술분야에 잘 알려진 기타의 것을 포함하는 표준 기술에 의해 다른 세포 성분 또는 기타 오염 물질, 예를 들어 다른 세포의 핵산 또는 단백질로부터 정제되어 나올 경우 "단리"되거나 "실질적으로 순수하게 된" 것이다. 본 발명의 핵산은 예를 들어 DNA 또는 RNA일 수 있다.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.
본 발명은 또 다른 관점에서, 본 발명의 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 코딩하는 핵산을 함유하는 재조합 벡터에 관한 것이다.In another aspect, 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.
본 발명에 있어서, 상기 재조합 벡터는 본 발명의 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 코딩하는 핵산의 발현을 유도할 수 있는 벡터라면 당업자가 당업계에 공지된 벡터를 적절히 선택하여 제한없이 사용할 수 있다. 예를 들어, 대장균을 숙주로 사용할 경우 T7계열 (T7A1, T7A2, T7A3 등), lac, lacUV5, 온도의존형 (λλphoA, phoB, rmB, tac, trc, trp 또는 1PL 프로모터를 포함하는 벡터를 사용할 수 있고, 효모를 숙주로 사용할 경우 ADH1, AOX1, GAL1, GAL10, PGK 또는 TDH3 프로모터를 포함하는 벡터를 사용할 수 있으며, 바실러스의 경우 P2 프로모터를 포함하는 벡터를 사용할 수 있으나, 이는 일부 실시 양태를 나열한 것으로, 상기 프로모터를 포함하는 벡터 이외에도 본 발명에 따른 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질의 발현을 유도하기 위한 프로모터를 포함하는 벡터로써 숙주에 적합한 것이라면 제한없이, 당업계에 공지된 다양한 벡터를 당업자가 적절히 선택하여 사용할 수 있다.In the present invention, if 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 (λλphoA, 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.
본 발명에서 용어 "벡터 (vector)"는 적합한 숙주 내에서 DNA를 발현시킬 수 있는 적합한 조절 서열에 작동가능하게 연결된 DNA 서열을 함유하는 DNA 제조물을 의미한다. 벡터는 플라스미드, 파지 입자, 또는 간단하게 잠재적 게놈 삽입물일 수 있다. 적당한 숙주로 형질전환되면, 벡터는 숙주 게놈과 무관하게 복제하고 기능할 수 있거나, 또는 일부 경우에 게놈 그 자체에 통합될 수 있다. 플라스미드가 현재 벡터의 가장 통상적으로 사용되는 형태이므로, 본 발명의 명세서에서 "플라스미드 (plasmid)" 및 "벡터 (vector)"는 때로 상호 교환적으로 사용된다. 그러나, 본 발명은 당업계에 알려진 또는 알려지게 되는 바와 동등한 기능을 가지는 벡터의 다른 형태를 포함한다. 대장균에서 사용되는 단백질 발현 벡터로는 Novagen (미국) 사의 pET 계열; Invitrogen (미국)의 pBAD 계열; Takara (일본)의 pHCE 나 pCOLD; 제노포커스 (대한미국)의 pACE 계열; 등을 사용할 수 있다. 고초균에서는 게놈의 특정부분에 목적 유전자를 삽입하여 단백질 발현을 구현하거나, MoBiTech (독일)의 pHT 계열의 벡터, 등을 사용할 수 있다. 곰팡이나 효모에서도 게놈 삽입이나 자가복제 벡터들 이용하여 단백질 발현이 가능하다. Agrobacterium tumefaciens나 Agrobacterium rhizogenes 등의 T-DNA 시스템을 이용하여 식물용 단백질 발현 벡터를 사용할 수 있다. 포유동물 세포 배양물 발현을 위한 전형적인 발현 벡터는 예를 들면 pRK5 (EP 307,247호), pSV16B (WO 91/08291호) 및 pVL1392 (Pharmingen) 등을 기초로 한다.As used herein, 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. In 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. In 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.
"발현 조절 서열 (expression control sequence)"이라는 표현은 특정한 숙주 생물에서 작동가능하게 연결된 코딩 서열의 발현에 필수적인 DNA 서열을 의미한다. 그러한 조절 서열은 전사를 실시하기 위한 프로모터, 그러한 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 코딩하는 서열 및 전사 및 해독의 종결을 조절하는 서열을 포함한다. 예를 들면, 원핵생물에 적합한 조절 서열은 프로모터, 임의로 오퍼레이터 서열 및 리보좀 결합 부위를 포함한다. 진핵세포는 프로모터, 폴리아데닐화 시그날 및 인핸서가 이에 포함된다. 플라스미드에서 유전자의 발현 양에 가장 영향을 미치는 인자는 프로모터이다. 고 발현용의 프로모터로서 SRα 프로모터와 사이토메갈로바이러스 (cytomegalovirus) 유래 프로모터 등이 바람직하게 사용된다.The expression "expression control sequence" refers to a DNA sequence essential for the expression of an operably linked coding sequence in a particular host organism. Such 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. For example, 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. As the promoter for high expression, the SRα promoter, the cytomegalovirus-derived promoter, etc. are preferably used.
본 발명의 핵산을 발현시키기 위하여, 매우 다양한 발현 조절 서열 중 어느 것이라도 벡터에 사용될 수 있다. 유용한 발현 조절서열의 예에는, 상기 기술한 프로모터 이외에도, 예를 들어, SV40 또는 아데노바이러스의 초기 및 후기 프로모터들, lac 시스템, trp 시스템, TAC 또는 TRC 시스템, T3 및 T7 프로모터들, 파지 람다의 주요 오퍼레이터 및 프로모터 영역, fd 코드 단백질의 조절 영역, 3-포스포글리세레이트 키나제 또는 다른 글리콜분해 효소에 대한 프로모터, 상기 포스파타제의 프로모터들, 예를 들어 Pho5, 효모 알파-교배 시스템의 프로모터 및 원핵세포 또는 진핵세포 또는 이들의 바이러스의 유전자의 발현을 조절하는 것으로 알려진 구성과 유도의 기타 다른 서열 및 이들의 여러 조합이 포함된다. T7 RNA 폴리메라아제 프로모터 Φ은 E.coli에서 단백질을 발현시키는데 유용하게 사용될 수 있다.To express the nucleic acids of the invention, any of a wide variety of expression control sequences can be used in the vector. Examples of 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 Φ can be usefully used to express proteins in E. coli.
핵산은 다른 핵산 서열과 기능적 관계로 배치될 때 "작동가능하게 연결 (operably linked)"된다. 이것은 적절한 분자 (예를 들면, 전사 활성화 단백질)은 조절 서열(들)에 결합될 때 유전자 발현을 가능하게 하는 방식으로 연결된 유전자 및 조절 서열(들)일 수 있다. 예를 들면, 전서열(pre-sequence) 또는 분비 리더 (leader)에 대한 DNA는 폴리펩타이드의 분비에 참여하는 전단백질로서 발현되는 경우 폴리펩타이드에 대한 DNA에 작동가능하게 연결되고; 프로모터 또는 인핸서는 서열의 전사에 영향을 끼치는 경우 코딩서열에 작동가능하게 연결되거나; 또는 리보좀 결합 부위는 서열의 전사에 영향을 끼치는 경우 코딩 서열에 작동가능하게 연결되거나; 또는 리보좀 결합 부위는 번역을 용이하게 하도록 배치되는 경우 코딩 서열에 작동가능하게 연결된다. 일반적으로, "작동가능하게 연결된"은 연결된 DNA 서열이 접촉하고, 또한 분비 리더의 경우 접촉하고 리딩 프레임 내에 존재하는 것을 의미한다. 그러나, 인핸서 (enhancer)는 접촉할 필요가 없다. 이들 서열의 연결은 편리한 제한 효소 부위에서 라이게이션(접합)에 의해 수행된다. 그러한 부위가 존재하지 않는 경우, 통상의 방법에 따른 합성 올리고뉴클레오티드 어댑터 (oligonucleotide adaptor) 또는 링커(linker)를 사용한다.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). For example, 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. In general, "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.
본원 명세서에 사용된 용어 "발현 벡터"는 통상 이종의 DNA의 단편이 삽입된 재조합 캐리어 (recombinant carrier)로서 일반적으로 이중 가닥의 DNA의 단편을 의미한다. 여기서, 이종 DNA는 숙주세포에서 천연적으로 발견되지 않는 DNA인 이형 DNA를 의미한다. 발현 벡터는 일단 숙주 세포 내에 있으면 숙주 염색체 DNA와 무관하게 복제할 수 있으며 벡터의 수 개의 카피 및 그의 삽입된 (이종) DNA가 생성될 수 있다.As used herein, the term "expression vector" generally refers to a fragment of double-stranded DNA as a recombinant carrier into which a heterologous DNA fragment is inserted. Here, 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.
당업계에 주지된 바와 같이, 숙주세포에서 형질감염 유전자의 발현 수준을 높이기 위해서는, 해당 유전자가, 선택된 발현 숙주 내에서 기능을 발휘하는 전사 및 해독 발현 조절 서열에 작동가능하도록 연결되어야만 한다. 바람직하게는 발현 조절서열 및 해당 유전자는 세균 선택 마커 및 복제 개시점 (replication origin)을 같이 포함하고 있는 하나의 발현 벡터 내에 포함되게 된다. 발현 숙주가 진핵세포인 경우에는, 발현 벡터는 진핵 발현 숙주 내에서 유용한 발현 마커를 더 포함할 수 있다.As is well known in the art, 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. Preferably, the expression control sequence and the corresponding gene are included in one expression vector including the bacterial selection marker and the replication origin. When the expression host is a eukaryotic cell, the expression vector may further comprise an expression marker useful in the eukaryotic expression host.
본 발명은 또 다른 관점에서, 본 발명의 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 코딩하는 핵산; 및/또는 상기 재조합 벡터가 숙주세포에 도입된 재조합세포에 관한 것이다.In another aspect, 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.
본 발명에 있어서, 상기 숙주세포는 단백질 등을 생산하기 위해, 유전자 또는 재조합 벡터 등이 도입될 수 있는 발현용 세포를 의미한다. 상기 숙주세포는 본 발명의 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 발현할 수 있는 세포라면 제한없이 사용될 수 있으며, 바람직하게는 진핵세포, 더욱 바람직하게는 효모, 곤충세포, 동물세포, 가장 바람직하게는 동물세포일 수 있다. 예를 들어 단백질의 발현에 주로 사용되는 CHO 세포주 또는 HEK 세포주 등이 사용될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, 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. For example, a CHO cell line or a HEK cell line mainly used for protein expression may be used, but is not limited thereto.
본 발명의 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 발현시키기 위해 매우 다양한 발현 숙주/벡터 조합이 이용될 수 있다. 진핵 숙주에 적합한 발현 벡터에는, 예를 들어 SV40, 소 유두종바이러스, 아네노바이러스, 아데노-연관 바이러스(adeno-associated virus), 사이토메갈로바이러스 및 레트로바이러스로부터 유래된 발현 조절 서열을 포함한다. 세균 숙주에 사용할 수 있는 발현 벡터에는 pBluescript, pGEX2T, pUC벡터, col E1, pCR1, pBR322, pMB9 및 이들의 유도체와 같이 E. coli에서 얻는 것을 예시할 수 있는 세균성 플라스미드, RP4와 같이 보다 넓은 숙주 범위를 가지는 플라스미드, λ과 λNM989와 같은 매우 다양한 파지 람다(phage lambda) 유도체로 예시될 수 있는 파지 DNA, 및 M13과 필라멘트성 단일가닥의 DNA 파지와 같은 기타 다른 DNA 파지가 포함된다. 효모 세포에 유용한 발현 벡터는 2μ 플라스미드 및 그의 유도체이다. 곤충 세포에 유용한 벡터는 pVL 941이다.A wide variety of expression host/vector combinations may be used to express the angiotensin converting enzyme II mutant of the present invention or a fusion protein comprising the same. 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 λ and λNM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages. Useful expression vectors for yeast cells are the 2μ plasmid and derivatives thereof. A useful vector for insect cells is pVL 941.
상기 재조합 벡터는 형질전환 또는 형질감염 등의 방법으로 숙주세포에 도입될 수 있다. 본원 명세서에 사용된 용어 "형질전환"은 DNA를 숙주로 도입하여 DNA가 염색체외 인자로서 또는 염색체 통합완성에 의해 복제가능하게 되는 것을 의미한다. 본원 명세서에 사용된 용어 "형질감염"은 임의의 코딩 서열이 실제로 발현되든 아니든 발현 벡터가 숙주 세포에 의해 수용되는 것을 의미한다. The recombinant vector may be introduced into a host cell by a method such as transformation or transfection. As used herein, the term “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. As used herein, the term “transfection” means that an expression vector is accepted by a host cell, whether or not any coding sequence is actually expressed.
물론 모든 벡터와 발현 조절 서열이 본 발명의 DNA 서열을 발현하는데 모두 동등하게 기능을 발휘하지는 않는다는 것을 이해하여야만 한다. 마찬가지로 모든 숙주가 동일한 발현 시스템에 대해 동일하게 기능을 발휘하지는 않는다. 그러나, 당업자라면 과도한 실험적 부담없이 본 발명의 범위를 벗어나지 않는 채로 여러 벡터, 발현 조절 서열 및 숙주 중에서 적절한 선택을 할 수 있다. 예를 들어, 벡터를 선택함에 있어서는 숙주를 고려하여야 하는데, 이는 벡터가 그 안에서 복제되어야만 하기 때문이다. 벡터의 복제 수, 복제 수를 조절할 수 있는 능력 및 당해 벡터에 의해 코딩되는 다른 단백질, 예를 들어 항생제 마커의 발현도 또한 고려되어야만 한다. 발현 조절 서열을 선정함에 있어서도, 여러 가지 인자들을 고려하여야만 한다. 예를 들어, 서열의 상대적 강도, 조절 가능성 및 본 발명의 DNA 서열과의 상용성 등, 특히 가능성 있는 이차 구조와 관련하여 고려하여야 한다. 단세포 숙주는 선정된 벡터, 본 발명의 DNA 서열에 의해 코딩되는 산물의 독성, 분비 특성, 단백질을 정확하게 폴딩시킬 수 있는 능력, 배양 및 발효 요건들, 본 발명 DNA 서열에 의해 코딩되는 산물을 숙주로부터 정제하는 것의 용이성 등의 인자를 고려하여 선정되어야만 한다. 이들 변수의 범위내에서, 당업자는 본 발명의 DNA 서열을 발효 또는 대규모 동물 배양에서 발현시킬 수 있는 각종 벡터/발현 조절 서열/숙주 조합을 선정할 수 있다. 발현클로닝에 의해 cDNA를 클로닝 하려고 할 때의 스크리닝법으로서 바인딩법(binding법), 페닝법(panning법), 필름 에멀션법(film emulsion 법)등이 적용될 수 있다.Of course, it should be understood that not all vectors and expression control sequences function equally in expressing the DNA sequences of the present invention. Likewise, not all hosts function equally against the same expression system. However, a person skilled in the art can make an appropriate selection among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The copy number of the vector, its ability to control the copy number and the expression of other proteins encoded by the vector, for example antibiotic markers, must also be considered. In selecting the expression control sequence, various factors must be considered. For example, the relative strength of the sequences, their controllability and compatibility with the DNA sequences of the invention, etc., should be taken into account, particularly with regard to possible secondary structures. 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.
상기 유전자 및 재조합 벡터는 종래에 공지된 다양한 방법을 통해, 숙주세포에 도입될 수 있다. 본 발명의 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 코딩하는 유전자가 직접적으로 숙주세포의 게놈에 도입되어 염색체 상 인자로서 존재할 수 있다. 본 발명이 속하는 기술분야의 당업자에게 있어 상기 유전자를 숙주세포의 게놈 염색체에 삽입하여서도 재조합 벡터를 숙주세포에 도입한 경우와 동일한 효과를 가질 것은 자명하다 할 것이다.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. 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.
본 발명은 또 다른 관점에서, 상기 재조합세포를 배양하는 단계를 포함하는 본 발명의 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질의 제조방법에 관한 것이다.In another aspect, 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.
본 발명의 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 발현할 수 있는 재조합 발현 벡터가 포유류 숙주세포 내로 도입될 경우, 상기 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질은 재조합세포에서 발현되기에 충분한 기간 동안, 또는 재조합세포가 배양되는 배양 배지 내로 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 분비하기에 충분한 기간 동안 숙주세포를 배양함으로써 제조될 수 있다.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.
경우에 따라서, 발현된 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질은 재조합세포로부터 분리하여 균일하도록 정제할 수 있다. 상기 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질의 분리 또는 정제는 통상의 단백질에서 사용되고 있는 분리, 정제 방법, 예를 들어 크로마토그래피에 의해 수행될 수 있다. 상기 크로마토그래피는 예를 들어, 친화성 크로마토그래피, 이온교환 크로마토그래피 또는 소수성 크로마토그래피에서 선택된 하나 이상의 조합일 수 있지만, 이에 한정되지는 않는다. 상기 크로마토그래피 이외에, 추가로 여과, 초여과, 염석, 투석 등을 조합되어 사용될 수 있다.In some cases, 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. In addition to the above chromatography, filtration, ultrafiltration, salting out, dialysis, etc. may be used in combination.
본 발명의 일 실시예에서, 본 발명의 안지오텐신 변환 효소 II 변이체가 뛰어난 SARS-CoV-2 중화 능력을 통해 SARS-CoV-2의 세포 내 진입 및 증식을 효과적으로 억제할 수 있음을 확인하였으며, 최근 보고되는 SARS-CoV-2의 다양한 변종(영국발(SARS-CoV-2 RBD_N501Y), 남아프리카공화국발 변종 (SARS-CoV-2 RBD_N501Y, K417N, E484K))에도 높은 중화능을 나타내는 것을 확인하였다. 나아가, 본 발명의 안지오텐신 변환 효소 II 변이체는 약동학적으로도 뛰어난 특성을 나타내어, COVID-19를 포함하는 코로나바이러스 감염증의 예방 또는 치료에 유용하게 사용될 수 있음을 입증하였다. In one embodiment of the present invention, it was confirmed that 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. Furthermore, 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.
따라서, 본 발명은 또 다른 관점에서, 상기 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 포함하는 코로나바이러스 감염증의 예방 또는 치료용 약학 조성물에 관한 것이다.Accordingly, in another aspect, 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.
본 발명은 또한, 대상에게 상기 안지오텐신 변환효소 II(ACE2) 변이체 또는 이를 포함하는 융합단백질을 투여하는 단계를 포함하는 코로나바이러스감염증의 예방 및/또는 치료 방법에 관한 것이다.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.
본 발명은 또한, 상기 안지오텐신 변환 효소 II(ACE2) 변이체 또는 이를 포함하는 융합단백질의 코로나바이러스 감염증의 예방 또는 치료용도에 관한 것이다.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.
본 발명은 또한 상기 안지오텐신 변환 효소 II(ACE2) 변이체 또는 이를 포함하는 융합단백질의 코로나바이러스 감염증의 예방 및/또는 치료용 약학 조성물의 제조를 위한 용도에 관한 것이다.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.
본 발명의 일 실시예에서는 안지오텐신 변환 효소 II(ACE2) 변이체 및 Fc 도메인 융합단백질의 SARS-CoV-2 및 이의 변종(영국발 변종, 남아프리카공화국발 변종)에 대한 결합 친화도 및 중화능력을 확인하였으나, MERS, SARS-CoV-1과 같은 다른 코로나바이러스또한, 스파이크 단백질의 수용체 결합 도메인과 이의 수용체인 ACE2의 결합을 중심으로 숙주의 세포 내로 진입 및 감염시키고 증식되며, 특히 각 종간의 S1 서브유닛에 포함된 수용체 결합도메인은 고도로 보존되어 있는 바, 본 발명의 안지오텐신 변환효소 II(ACE2) 변이체는 COVID-19의 예방 및 치료에만 사용에만 국한되지 않고 모든 코로나바이러스 감염증에 사용될 수 있음은 자명하다 할 것이다.In one embodiment of the present invention, 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 (variants from England, variants from South Africa) 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. .
따라서, 본 발명에 있어서, 상기 코로나바이러스는 코로나바이러스아과(Coronavirinae) 속에 속하는 RNA 바이러스를 의미한다(Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier, Oxford. 806-828쪽). 상기 코로나바이러스아과에는 알파/베타/감마/델타 코로나바이러스의 4개의 속으로 구분될 수 있으며, 예를 들어, 인간에게 감염 가능한 코로나바이러스로는 SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-229E, HCoV-OC43, HKU1, HCoV-NL63 등이 있으나, 이에 제한되는 것은 아니다.Therefore, in the present invention, 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.
본 발명에서 사용되는 용어, "예방"은 본 발명에 따른 약학 조성물의 투여에 의해 목적하는 질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "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.
본 발명에서 사용되는 용어, "치료"는 본 발명에 따른 약학 조성물의 투여에 의해 목적하는 질환에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "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.
상기 약학 조성물은 유효성분인 안지오텐신 변환효소 II(ACE2) 변이체 또는 이를 포함하는 융합단백질 이외에도 통상적으로 약학 조성물에 사용되는 적절한 담체, 부형제 및 희석제를 추가로 포함할 수 있다.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.
약제학적으로 허용 가능한 부형제는 본 발명의 유효성분과 양립 가능하여야 하며, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 한 성분 또는 둘 이상의 성분을 혼합하여 사용할 수 있고, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형으로 제제화 할 수 있다. 특히, 동결건조(lyophilized)된 형태의 제형 또는 흡입형으로 제제화하여 제공하는 것이 바람직하다. 동결건조 제형 제조를 위해서 본 발명이 속하는 기술분야에서 통상적으로 알려져 있는 방법이 사용될 수 있으며, 동결건조를 위한 안정화제가 추가될 수도 있다. 특히, 흡입형 제제의 경우, 흡입형 제제는 건조분말형태, 액체형태, 에어로졸 형태 등 다양한 형태로 제조될 수 있으며, 건조분말 흡입기(Dry powder inhalers: DPIs), 네뷸라이저(Nebulizer), 정량식 흡입기(metered-dose inhaler: MDI), 스페이서(spacer)등을 사용하여 투여될 수 있다. Pharmaceutically acceptable excipients 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. In addition, 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. In particular, it is preferable to provide a formulation in a lyophilized form or an inhalation form. For the preparation of the freeze-dried formulation, a method commonly known in the art to which the present invention pertains may be used, and a stabilizer for freeze-drying may be added. In particular, in the case of inhalation formulations, 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.
상기 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등이 있다. 상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.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. When formulating the composition, it is usually prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant.
본 발명에 따른 약학 조성물은 통상의 방법에 따라 다양한 형태로 제형화하여 사용될 수 있다. 적합한 제형으로는 정제, 환제, 산제, 과립제, 당의정, 경질 또는 연질의 캡슐제, 용액제, 현탁제 또는 유화액제, 주사제, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액 등이 있으나, 이에 한정되는 것은 아니다.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, The present invention is not limited thereto.
본 발명에 따른 약학 조성물은 약학적으로 불활성인 유기 또는 무기 담체를 이용하여 적합한 제형으로 제조할 수 있다. 즉, 제형이 정제, 코팅된 정제, 당의정 및 경질 캡슐제인 경우 락토스, 수크로스, 전분 또는 그 유도체, 탈크, 칼슘 카보네이트, 젤라틴, 스테아르산 또는 그 염을 포함할 수 있다. 또한, 제형이 연질 캡슐제인 경우에는 식물성 오일, 왁스, 지방, 반고체 및 액체의 폴리올을 포함할 수 있다. 또한, 제형이 용액 또는 시럽 형태인 경우, 물, 폴리올, 글리세롤, 및 식물성 오일 등을 포함할 수 있다.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.
본 발명에 따른 약학 조성물은 상기의 담체 외에도 보존제, 안정화제, 습윤제, 유화제, 용해제, 감미제, 착색제, 삼투압 조절제, 산화방지제 등을 더 포함할 수 있다.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.
본 발명에 따른 약학 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 따른 약학 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "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.
본 발명의 약학 조성물은 개체에 다양한 경로로 투여될 수 있다. 상기 약학 조성물은 경구 또는 비경구로 투여할 수 있다. 비경구 투여인 경우에는 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 내피 투여, 국소 투여, 비내 투여, 폐내 투여 및 직장내 투여 등으로 투여할 수 있다. 경구 투여 시, 단백질 또는 펩타이드는 소화가 되기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화될 수 있다. 또한, 상기 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.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. In the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc. can be administered. Since the protein or peptide is digested upon oral administration, oral compositions may be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition may be administered by any device capable of transporting the active agent to a target cell.
본 발명에 따른 약학 조성물의 투여방법은 제형에 따라 용이하게 선택될 수 있으며, 경구 또는 비경구 투여될 수 있다. 투여량은 환자의 나이, 성별, 체중, 병증의 정도, 투여경로에 따라 달라질 수 있다.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.
본 발명에서는 특정 아미노산 서열 및 염기 서열을 기재하였으나, 본 발명에서 실시하고자 하는 효소와 실질적으로 동일한 아미노산 서열 및 이를 코딩하는 염기 서열이 본 발명의 권리범위에 속하는 것은 당업자에게 자명할 것이다. 실질적으로 동일하다는 것은, 아미노산 또는 염기서열의 상동성이 매우 높은 경우를 포함하고, 그 밖에도 서열의 상동성과는 무관하게 구조적 특징을 공유하거나 본 발명에서 사용된 것과 동일한 기능을 가지는 단백질을 의미한다. 본 발명의 핵심을 구성하는 서열을 제외한 다른 서열이 일부 결실된 단백질 또는 이를 코딩하는 염기서열의 단편도 본 발명에 포함될 수 있으며, 따라서 본 발명은 단편의 길이와는 무관하게 본 발명에서 사용된 것과 동일한 기능을 가지는 아미노산 또는 염기 서열을 모두 포함한다.Although a specific amino acid sequence and base sequence have been described in the present invention, it will be apparent to those skilled in the art that an 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.
실시예Example
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예에서 사용된 장비 및 재료Equipment and materials used in the examples
본 발명의 실시예에서 사용된 장비 및 재료는 아래 표 1 및 표 2와 같다.Equipment and materials used in the embodiment of the present invention are shown in Tables 1 and 2 below.
장비equipment
No.No. 모델Model 제조사manufacturing company Cat. No.Cat. No. Serial No.Serial No.
1One SH800SH800 SonySony -- 03010050301005
22 PCR Thermal Cycler DicePCR Thermal Cycler Dice TAKARATAKARA -- AC2009-06AC2009-06
33 Gene pulserGene pulser Bio-radBio-rad 16526601652660 617BR11690 617BR11690
44 Multiskan GoMultiskan Go ThermoThermo 5111930051119300 1510-04454C1510-04454C
55 AKTA PrimeAKTA Prime GE healthcareGE healthcare 1100131311001313 22827122282712
66 AKTA PureAKTA Pure GE healthcareGE healthcare 2904666529046665 20932132093213
77 PowerPac power supplyPowerPac power supply Bio-radBio-rad 16450521645052 043BR59008 043BR59008
88 ChemiDocChemiDoc Bio-radBio-rad -- 721BR14182 721BR14182
99 Mupid ElectrophoresisMupid Electrophoresis AdvanceAdvance -- 077661077661
1010 Biacore T200Biacore T200 GE healthcareGE healthcare 2897500128975001 043BR59008043BR59008
재료ingredient
No.No. 제품명product name 제조사manufacturing company Cat. No.Cat. No. Lot. No.Lot. No.
1One Stellar competent cellStellar competent cells TAKARATAKARA 636763636763 1807636A1807636A
22 Phusion polymerasePhusion polymerase Thermo scientificThermo scientific F-520LF-520L 00625150062515
33 5x HF buffer5x HF buffer Thermo scientificThermo scientific F-518F-518 100605129100605129
44 DMSODMSO Thermo scientificThermo scientific F-515F-515 0069165300691653
55 dNTP mixdNTP mix invitrogeninvitrogen 18270881827088 --
66 Intron 100bp ladderIntron 100bp ladder IntronIntron 2407324073 401110951401110951
77 Seakem LE agaroseSeakem LE agarose LonzaLonza 5000450004 00004328780000432878
88 RedSafe staining solutionRedSafe staining solution IntronIntron O008-090150.49O008-090150.49 2114121141
99 6x puple loading dye6x puple loading dye NEBNEB B7024SB7024S 1001841910018419
1010 Pellet paintPellet paint NovagenNovagen 69049-469049-4 31521523152152
1111 GenepHlow DNA cleanup kitGenepHlow DNA cleanup kit GeneaidGeneaid DFM025DFM025 FF05201FF05201
1212 SAPESAPE invitrogeninvitrogen S866S866 19735011973501
1313 Anti mouse IgG FITCAnti mouse IgG FITC invitrogeninvitrogen 11-4011-8511-4011-85 19701601970160
1414 Anti V5 antibodyAnti V5 antibody invitrogeninvitrogen 46-070546-0705 20742802074280
1515 SOC mediaSOC media TAKARATAKARA 636763636763 1807636A1807636A
1616 In-fusioninfusion TAKARATAKARA ST0345ST0345 1805235A1805235A
1717 PBS pH7.4PBS pH7.4 gibcogibco 10010-03110010-031 20467692046769
1818 UltraPure D.I waterUltraPure D.I water invitrogeninvitrogen 10977-01510977-015 20520862052086
1919 Expi293F cellExpi293F cell InvitrogenInvitrogen A14527A14527 --
2020 Expi293 MediaExpi293 Media InvitrogenInvitrogen A14351-01A14351-01 21188952118895
2121 FectoPROFectoPRO PolyplusPolyplus 116-001116-001 08W0205E408W0205E4
2222 Percision unstained standardPrecision unstained standard Bio-radBio-rad 161-0363161-0363 6416705764167057
2323 Stain-free GelStain-free Gel Bio-radBio-rad 456-8096456-8096 6423097164230971
2424 MabselectSUREMabselectSURE GE healthcareGE healthcare 11-0034-9511-0034-95 1027099810270998
2525 Amine compling KitAmine compiling kit GE healthcareGE healthcare BR-1000-50BR-1000-50 20861592086159
2626 Sensor CM5 ChipSensor CM5 Chip GE healthcareGE healthcare BR-1005-30BR-1005-30 1026607410266074
2727 10X HBS-EP10X HBS-EP GE healthcareGE healthcare BR-1006BR-1006 BCBX0006BCBX0006
2828 SARS-CoV2 RBDSARS-CoV2 RBD Genscript Genscript Z03483-100Z03483-100 --
2929 HRP-streptavidinHRP-streptavidin Thermo scientificThermo scientific N100N100 RJ236399RJ236399
3030 TMB substrateTMB substrate Thermo scientificThermo scientific N301N301 UA274904UA274904
3131 Skim milkSkim milk biowordbioword 30620074-130620074-1 V17042602V17042602
3232 Anti human IgG Fc-HRPAnti human IgG Fc-HRP Jackson LabJackson Lab 109-035-008109-035-008 --
3333 Tween20Tween20 SIGMASIGMA P1379P1379 SLBV3799SLBV3799
3434 PCR random mutagenesis kitPCR random mutagenesis kit TAKARATAKARA 630703630703 --
3535 SARS CoV2 RBD_N501YSARS CoV2 RBD_N501Y SinobiologicalSinobiological 40592-V08H8240592-V08H82 --
3636 SARS CoV2 RBD_N501Y, K417N, E484KSARS CoV2 RBD_N501Y, K417N, E484K SinobiologicalSinobiological 40592-V08H8540592-V08H85 --
실시예 1: ACE2 단백질을 템플릿으로 하는 ACE2 변이체 라이브러리의 제작Example 1: Construction of an ACE2 mutant library using the ACE2 protein as a template
실시예 1-1: ACE2 변이체 라이브러리 제조를 위한 템플릿 벡터 제작Example 1-1: Template vector construction for ACE2 mutant library preparation
ACE2 변이체 라이브러리를 만들기 위해 야생형 인간 ACE2(서열번호 1, UniProtKB seq ID: Q9BYF1, 도 1)의 세포외 도메인의 일부인 Q18-N720를 pYD5 vector에 클로닝하였다.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.
야생형 인간 ACE2(서열번호 1, UniProtKB seq ID: Q9BYF1) 서열의 토폴로지는 아래 표 3과 같다.The topology of wild-type human ACE2 (SEQ ID NO: 1, UniProtKB seq ID: Q9BYF1) sequence is shown in Table 3 below.
Position (S)Position (S) DescriptionDescription
1-171-17 Signal peptide signal peptide
18-74018-740 Extracellularextracellular
741-761741-761 HelicalHelical
762-805762-805 CytoplasmicCytoplasmic
ACE2의 c-terminal EcoR1 site 뒤에 V5 tag을 접합하였고 Nhe1, EcoR1 제한효소로 자른 pYD5 vector의 양 말단과 homologous 한 서열을 합성 유전자 양 말단에 합성하였다(도 2). 합성이 완료된 insert gene과 linearized vector를 In-Fusion® HD Cloning Kit (Takara)을 사용하여 클로닝하였다. 클로닝이 완료된 플라스미드를 Stellar™ Competent Cells(Takara)에 형질전환(transformation)한 후 ZymoPUREⅡ Plasmid Midiprep kit(Zymo research, Cat# D4201) 로 midi-prep 하여 DNA를 15ug 이상 확보하였다.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 Stellar™ Competent Cells (Takara), and then midi-prep with ZymoPUREⅡ Plasmid Midiprep kit (Zymo research, Cat# D4201) to secure more than 15ug of DNA.
실시예 1-2: ACE2 특정 위치의 아미노산에 변이를 포함하는 point mutation library 제작Example 1-2: Preparation of a point mutation library containing mutations in amino acids at specific positions of ACE2
ACE2의 서열을 분석하여 SARS-CoV-2 스파이크 단백질(M1~N720)의 수용체 결합 도메인(receptor binding domain; RBD)와 결합하거나 결합표면 근처에 위치하는 ACE2의 아미노산 26개 (S19, Q24, A25, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L45, L79, M82, Y83, T92, Q325, E329, N330, K353, D354, G355, R357, A386)를 선정하여 NNK codon 으로 random library를 제작하였다.By analyzing the sequence of ACE2, 26 amino acids of ACE2 (S19, Q24, A25, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L45, L79, M82, Y83, T92, Q325, E329, N330, K353, D354, G355, R357, A386) and NNK A random library was created with codon.
선정된 아미노산을 표적으로 하여 NNK randomization시키는 프라이머를 디자인하여, IDT technology (www.IDTDNA.com)에 주문하였다(표 14). Primers for NNK randomization targeting the selected amino acids were designed and ordered from IDT technology (www.IDTDNA.com) (Table 14).
Random 프라이머를 포함한 insert 양 말단 2쌍의 프라이머(표 14)를 이용해 PCR로 증폭시켜 gel-extraction을 한 후(1차 PCR), 2개의 fragment를 overlap extension PCR(2차 PCR)로 1개의 아미노산에 NNK random sequence가 있는 library를 제작하였다. 아래와 같은 방법으로 각 PCR을 진행하였고 42번 변이를 제외한 22개의 라이브러리를 완성하였다. 상기 1차 PCR 및 2차 PCR의 조건은 도 3과 같다.Amplify by PCR using two pairs of primers (Table 14) at both ends of the insert, including random primers, and gel-extraction (1st PCR), and 2 fragments to 1 amino acid by overlap extension PCR (2nd PCR) A library with NNK random sequence was prepared. Each PCR was performed in the following manner, and 22 libraries were completed except for mutation No. 42. The conditions of the first PCR and the second PCR are as shown in FIG. 3 .
실시예 1-3: 효모 형질전환Example 1-3: Yeast transformation
Yeast competent cell는 EBY100 효모 균주를 YPD 플레이트에 streaking하여 30℃에서 2일 배양한 후 1개의 콜로니를 picking 하여 5ml YPD 배지에서 30℃, 250rpm으로 밤새 배양하였다. 배양한 효모세포를 100ml scale로 계대배양하여 약 6시간 동안 동일한 조건에서 O.D 측정값이 약 1.0-1.5가 될 때까지 키운 후 1.0 ml의 sterilized Tris-DTT 용액(0.39g 1,4-dithiothreitol in 1ml 1M Tris pH8.0 buffer)을 넣어주고 15분동안 인큐베이션하였다. 배양이 끝난 효모세포를 원심분리 후 cold E buffer(1.2g Tris base, 92.4g sucrose, 1M MgCl2 in distilled water)로 washing 하고 total volume이 450ul 가 되도록 E buffer로 resuspension 하여 competent cell을 만들었다. 준비된 competent cell 50ul 와 농축된 insert DNA 5ug, vector 1ug을 전기큐벳(electrocuvette)에 넣고 0.54kV, 25uF 조건으로 전기천공(electroporation)을 수행한 후 YPD 배지 2ml을 즉시 넣어주었고 30℃, 250rpm 에서 1시간동안 인큐베이션하였다. 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. 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℃, 250rpm. incubated for a while.
실시예 1-4: 라이브러리의 배양Example 1-4: Culture of the library
형질전환 후 YPD 에서 배양한 효모 세포를 100ug/ml의 카나마이신(kanamycin) 항생제를 넣은 10ml SDCAA 배지(20g glucose, 14.7g sodium citrate, 4.3g citric acid monohydrate, 6.7g yeast nitrogen base, 5g bacto casamino acid in 1L distilled water) 로 resuspension 후 1/100 SDCAA 배지로 희석하여 104개의 세포가 되도록 100ul를 SDCAA::Km plate(100ug/ml kanamycin, 20g glucose, 14.7g sodium citrate, 4.3g citric acid monohydrate, 6.7g yeast nitrogen base, 5g bacto casamino acid, 16g bacto agar / 1L 증류수) 에 spreading 하였다. After transformation, 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 μg/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).
형질전환된 효모세포를 SDCAA 배지에 resuspension 하여 initial O.D 가 0.2이 되도록 100ml SDCAA 배지에서 24시간 배양하였다. 동일한 방법으로 계대배양 한 후에 initial O.D 가 1이 되도록 100ug/ml 카나마이신을 함유하는 25ml SGCAA 배지(20g galactose, 14.7g sodium citrate, 4.3g citric acid monohydrate, 6.7g yeast nitrogen base, 5g bacto casamino acid in 1L distilled water)로 resuspension 후 30℃, 250rpm으로 16-20시간 induction 하였다.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.
실시예 2: ACE2 변이체 라이브러리를 사용한 sorting 및 enrichmentExample 2: Sorting and enrichment using the ACE2 variant library
26개의 점 돌연변이 라이브러리(point mutation library)는 FACS sorting을 통해 항원 결합 친화도을 향상시키기 위해 효모 세포 표면에 발현된 ACE2 ECD (Q18-N720)를 염색하였다. 1차 염색은 항원 결합 친화도를 확인할 수 있는 200~500nM biotinylated SARS-CoV-2 spike protein RBD-his와 ACE2 라이브러리의 발현을 확인할 수 있는 1:500 anti-V5 antibody(Invitrogen, cat#R960-25)를 FACS buffer(0.1% BSA in pH7.4 PBS buffer)로 희석하여 실온에서 30분간 rotational mixer에서 인큐베이션하여 수행하였다. 1차 염색이 끝난 후 FACS buffer로 washing한 후 1:100 비율로 Streptavidin, R-Phycoerythrin Conjugate (SAPE) (Invitrogen, cat#S866) 와 Anti-Mouse IgG (H+L)-FITC antibody (Invitrogen/Cat No.11-4011-85)를 FACS buffer에 희석하여 빛이 차단된 4℃에서 20분간 인큐베이션하였다. 2차 염색이 끝난 cell은 FACS buffer로 2번 washing 후 FACS buffer 로 resuspension 하여 분석 샘플을 준비하였다. 각각의 라이브러리는 wild type ACE2의 결합 친화도보다 높은 부분을 0.1 ~ 1% 사이에서 게이팅(gating)하여, 200nM의 항원 농도 조건 하에서 초당 15,000 ~ 25,000 개의 속도로 2~3 round sorting 하였다.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. After washing with FACS buffer after primary staining, Streptavidin, R-Phycoerythrin Conjugate (SAPE) (Invitrogen, cat#S866) and Anti-Mouse IgG (H+L)-FITC antibody (Invitrogen/Cat) at a ratio of 1:100 No.11-4011-85) was diluted in FACS buffer and incubated for 20 minutes at 4°C blocked from light. After secondary staining, the cells were washed twice with FACS buffer and then resuspensioned with FACS buffer to prepare analysis samples. Each library was gated between 0.1 and 1% of a portion higher than the binding affinity of wild type ACE2, and 2 to 3 rounds were performed at a rate of 15,000 to 25,000 per second under the condition of an antigen concentration of 200 nM.
실시예 3: 향상된 결합 친화력을 가지는 ACE2 변이체 개별 클론의 항원결합 친화도 및 서열 분석Example 3: Antigen-binding affinity and sequence analysis of individual clones of ACE2 variants with improved binding affinity
실시예 2에서 최종적으로 선별된 세포를 일부 SDCAA::Km plate에서 배양하였다. 콜로니를 picking 하여 SDCAA media에서 키운 후 SGCAA 배지로 induction 하여 위와 같은 방법으로 FACS 분석을 진행하였다. wild type ACE2 (Q18-N720)과 PE histogram을 비교하여 결합 친화도가 향상된 클론들을 선별하여 함께 염색하고 FACS로 비교분석 하였다(도 5).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).
SDCAA::Km plate에서 colony picking 하여 SDCAA::Km media 로 키운 yeast cell을 ZymoPrep Yeast Plasmid MiniPrep II Kit(zymo research, Cat#D2004-50) 로 DNA prep을 하였다. 추출된 DNA는 E.coli Stellar™ Competent Cells 에 형질전환한 후 서열 분석을 진행하였다. 각 라이브러리에서 도출된 향상된 결합 친화력을 가지는 ACE2 변이체의 아미노산 변이 정보는 다음 표 4와 같다.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 Stellar™ 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 mutatation librarypoint mutation library
(변이 잔기 번호)(variant residue number) 		클론명clone name 변이 정보mutation information
1919 19_20319_203 S19WS19W
19_20419_204 S19PS19P
2727 27_10227_102 T27WT27W
27_10627_106 T27DT27D
27_10727_107 T27AT27A
27_11427_114 T27YT27Y
27_20227_202 T27LT27L
27_20527_205 T27MT27M
27_20727_207 T27CT27C
3030 30_10130_101 D30VD30V
3434 34_10134_101 H34AH34A
34_11034_110 H34VH34V
3535 35_20235_202 E35VE35V
7979 79_10179_101 L79YL79Y
79_10579_105 L79VL79V
325325 325_102325_102 Q325PQ325P
330330 330_101330_101 N330YN330Y
330_105330_105 N330FN330F
실시예 4: 향상된 결합 친화력을 가지는 ACE2 변이체 기반의 ACE2 error prone 라이브러리 제작 및 변이체 도출Example 4: ACE2 error prone library production and variant derivation based on ACE2 variants with improved binding affinity
실시예 4-1: ACE2 error prone 라이브러리 제작Example 4-1: ACE2 error prone library production
NNK codon library(점 돌연변이 라이브러리)로부터 도출된 Affinity 향상에 영향을 주는 변이를 조합하여 ACE2 (Q18-D615) 주형에 8개의 변이 (S19P, T27L, D30V, H34A, E35V, L79V, Q325P, N330Y)를 갖는 ACE2_8mut 템플릿을 만들고 이 템플릿으로 하여 ACE2_8mut 템플릿의 Q18-L100 내에서의 error prone 라이브러리, ACE2_8mut 템플릿의 Q18-D615 내에서의 error prone 라이브러리 2종류의 라이브러리를 제작하였다. 위와 같은 방법으로 ACE2 (Q18-D615) 주형에 1개의 변이(H34A)를 갖는 ACE2_H34A 템플릿으로 2 종류의 error prone 라이브러리를 제작하였다(도 6). 각 템플릿에 특이적인 프라이머를 제작하고 PCR ramdom mutagenesis kit (TAKARA, cat no. 630703)을 사용하여 error rate 0.23%, 0.48%, 0.81% 라이브러리를 각각 제작하였다. 형질전환 및 배양은 위와 같은 방법으로 진행하였다.8 mutations (S19P, T27L, D30V, H34A, E35V, L79V, Q325P, N330Y) were added to the ACE2 (Q18-D615) template by combining the mutations that affect affinity improvement derived from the NNK codon library (point mutation library). To make an ACE2_8mut template with this template, an error prone library in Q18-L100 of the ACE2_8mut template and an error prone library in Q18-D615 of the ACE2_8mut template were produced as this template. In the same manner as above, two types of error prone libraries were prepared using the ACE2_H34A template having one mutation (H34A) in the ACE2 (Q18-D615) template (FIG. 6). 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.
실시예 4-2: ACE2 error prone 라이브러리를 사용한 sorting 및Example 4-2: sorting and using the ACE2 error prone library enrichmentenrichment
주형이 다른 각 error prone 라이브러리는 FACS sorting을 통해 항원 결합 친화도를 향상시키기 위해 yeast cell 표면에 발현된 ACE2 (Q18-D615)를 염색하였다. 염색 방법은 실시예 2와 동일하며, 항원 농도를 0.5nM 로 낮추어 4번의 round sorting을 수행하였다.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.
실시예 4-3: 개별 클론의 항원 결합 친화도 및 서열 분석Example 4-3: Antigen Binding Affinity and Sequence Analysis of Individual Clones
Error prone 라이브러리에서 최종 sorting(4 라운드)된 개별클론을 FACS에서 분석하였다. 염색 방법은 실시예 2와 동일하며, 각 라이브러리의 템플릿이 되는 H34A 또는 8mut (S19P, T27L, D30V, H34A, E35V, L79V, Q325P, N330Y)와 비교하였다(도 7A 내지 도 7D). 이어서, error prone 라이브러리의 개별클론 FACS 분석 후 SARS-CoV-2 S 단백질 RBD에 높은 결합 친화도를 갖는 것으로 선별된 클론들의 서열을 분석하였다. 선별된 ACE2 변이체의 아미노산 변이 정보는 다음 표 5와 같다.Individual clones finally sorted (4 rounds) in the error prone library were analyzed by FACS. The staining method was the same as in Example 2, and compared with H34A or 8mut (S19P, T27L, D30V, H34A, E35V, L79V, Q325P, N330Y) serving as a template for each library ( FIGS. 7A to 7D ). Then, after FACS analysis of individual clones of the error prone library, the sequences of clones selected to have high binding affinity to the SARS-CoV-2 S protein RBD were analyzed. The amino acid mutation information of the selected ACE2 mutant is shown in Table 5 below.
No.No. 라이브러리 종류library type 클론 번호clone number 변이 정보mutation information
1One 8mut_err 100 A.A 8mut_err 100 A.A 22 K26N, K31RK26N, K31R
22 44 K26N, L91PK26N, L91P
33 8mut_err Full length8mut_err Full length 22 N250K N250K
44 55 N250K,G448VN250K, G448V
55 66 G448V G448V
66 99 K74,V185AK74,V185A
77 H34A_err 100A.AH34A_err 100A.A R4-2R4-2 N49D N49D
88 H34A_err Full lengthH34A_err Full length R4-1R4-1 L79I, L91P, S280R, S602TL79I, L91P, S280R, S602T
99 R4-3R4-3 L79I, L91PL79I, L91P
실시예 5: 선별된 ACE2 변이체의 클로닝 및 제조Example 5: Cloning and Preparation of Selected ACE2 Variants
Fc 도메인과 융합된 ACE2 변이체의 생산을 위하여 ACE2-GS linker-Fc_pcDNA3.3 plasmid를 구축하였다. 동물세포 발현벡터인 pcDNA3.3에 클로닝 하기위해 wild type ACE2 서열의 C' 말단에는 EcoRI, N' 말단에는 BamHI 제한효소 부위를 삽입하고 양 말단에 pcDNA3.3 vector와 homologous한 서열을 추가하여 IDT에서 합성하였다 신호 펩타이드(signal peptide)는 human ACE2의 신호펩타이드(seq ID:Q9BYF1)를 사용하였다.An ACE2-GS linker-Fc_pcDNA3.3 plasmid was constructed for the production of ACE2 variants fused with the Fc domain. For cloning into pcDNA3.3, 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.
각 개별 클론은 변이 위치에 따라 제한효소 부위(restriction enzyme site)를 다르게 하여 합성하고 클로닝하였다. 100번째 아미노산 내의 변이는 BspEI 을 양 말단에 합성하여, BspEI cut vector와 클로닝하였으며, 100번 아미노산 이후의 변이는 Fragment 1, 2로 나누어 assemble 되도록 합성하고 EcoRI, BamHI cut vector를 사용하여 클로닝하였다. 또한 도출된 변이의 조합으로 이루어진 변이체들도 추가로 합성하였으며 효용성에 따라 Fc를 융합하거나 또는 Fc-his tag을 융합하여 제작하였다. 합성이 완료된 insert gene은 In-Fusion® HD Cloning Kit(Clontech)을 사용하여 각각의 linearized vector을 넣어 클로닝 하고 sequencing primer는 CmV Forward, pcDNA3.3 reverse primer(표 15)를 사용하여 확인하였다(도 8). 최종적으로 선택되어 클로닝된 ACE2 변이체의 변이 정보는 다음 표 6과 같다.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 nameACE2 Variants name Mutation SitesMutation Sites 서열번호SEQ ID NO:
ACE2 wild type
(M1~D615)ACE2 wild type
(M1-D615) 		-- 22
ACE2.V.06ACE2.V.06 H34AH34A 33
ACE2.V.07ACE2.V.07 H34VH34V 44
ACE2.V.08ACE2.V.08 S19WS19W 55
ACE2.V.09ACE2.V.09 S19PS19P 66
ACE2.V.10ACE2.V.10 T27DT27D 77
ACE2.V.11ACE2.V.11 T27YT27Y 88
ACE2.V.12ACE2.V.12 T27LT27L 99
ACE2.V.13ACE2.V.13 T27CT27C 1010
ACE2.V.14ACE2.V.14 D30VD30V 1111
ACE2.V.15ACE2.V.15 E35VE35V 1212
ACE2.V.16ACE2.V.16 L79YL79Y 1313
ACE2.V.17ACE2.V.17 L79VL79V 1414
ACE2.V.18ACE2.V.18 Q325PQ325P 1515
ACE2.V.19ACE2.V.19 N330YN330Y 1616
ACE2.V.20ACE2.V.20 N330FN330F 1717
ACE2.V.21ACE2.V.21 S19P, T27L, H34A, L79V, N330YS19P, T27L, H34A, L79V, N330Y 1818
ACE2.V.22ACE2.V.22 S19P, T27L,D30V, H34A, E35V, L79V, Q325P, N330YS19P, T27L, D30V, H34A, E35V, L79V, Q325P, N330Y 1919
ACE2.V.23ACE2.V.23 N330HN330H 2020
ACE2.V.24ACE2.V.24 H34A, N330YH34A, N330Y 2121
ACE2.V.25ACE2.V.25 H34V, N330HH34V, N330H 2222
ACE2.V.26ACE2.V.26 T27Y, N330YT27Y, N330Y 2323
ACE2.V.27ACE2.V.27 T27Y, H34AT27Y, H34A 2424
ACE2.V.28ACE2.V.28 T27Y, H34A, N330YT27Y, H34A, N330Y 2525
ACE2.V.29ACE2.V.29 H34V, N330YH34V, N330Y 2626
ACE2.V.30ACE2.V.30 K26NK26N 2727
ACE2.V.31ACE2.V.31 K31RK31R 2828
ACE2.V.32ACE2.V.32 L91PL91P 2929
ACE2.V.33ACE2.V.33 K26N, K31R, L91PK26N, K31R, L91P 3030
ACE2.V.34ACE2.V.34 R514QR514Q 3131
ACE2.V.35ACE2.V.35 L79IL79I 3232
ACE2.V.36ACE2.V.36 T27Y, H34A, L79YT27Y, H34A, L79Y 3333
ACE2.V.37ACE2.V.37 T27Y, H34A, L79Y, N330YT27Y, H34A, L79Y, N330Y 3434
ACE2.V.38ACE2.V.38 T27Y, H34A, L79V, N330YT27Y, H34A, L79V, N330Y 3535
ACE2.V.39ACE2.V.39 T27D, H34A, L79V, N330YT27D, H34A, L79V, N330Y 3636
ACE2.V.40ACE2.V.40 T27Y, H34A, L79Y, L91P, N330YT27Y, H34A, L79Y, L91P, N330Y 3737
ACE2.V.41ACE2.V.41 T27Y, H34A, L79V, L91P, N330YT27Y, H34A, L79V, L91P, N330Y 3838
ACE2.V.42ACE2.V.42 T27D, H34A, L79V, L91P, N330YT27D, H34A, L79V, L91P, N330Y 3939
ACE2.V.43ACE2.V.43 S19P, T27D, H34A, L79V, N330YS19P, T27D, H34A, L79V, N330Y 4040
ACE2.V.44ACE2.V.44 S19P, T27D, H34A, L79V, L91P, N330YS19P, T27D, H34A, L79V, L91P, N330Y 4141
ACE2.V.45ACE2.V.45 S19P, T27Y, H34A, L79Y, N330YS19P, T27Y, H34A, L79Y, N330Y 4242
ACE2.V.46ACE2.V.46 S19P, T27Y, H34A, L79Y, L91P, N330YS19P, T27Y, H34A, L79Y, L91P, N330Y 4343
ACE2.V.47ACE2.V.47 S19P, T27Y, H34A, L79V, N330YS19P, T27Y, H34A, L79V, N330Y 4444
ACE2.V.48ACE2.V.48 S19P, T27Y, H34A, L79V, L91P, N330YS19P, T27Y, H34A, L79V, L91P, N330Y 4545
실시예 6: ACE2 변이체의 생산 Example 6: Production of ACE2 variants
결합 친화도 향상을 위해 디자인된 ACE2 변이체가 도입된 플라스미드를 Expi293 expression system (Invitrogen)을 이용하여 발현하였으며, 이를 AktaPure (GE healthcare)와 AktaPrime purifier (GE healthcare) 및 MabselectSURE컬럼 (GE healthcare, Cat#11-0034-95)를 이용하여 정제하였다. 정제된 항체는 Desalting컬럼 (GE healthcare, Cat#17-1408-01)를 통하여 PBS로 buffer change하였으며, Multiskan GO (Thermo)를 통하여 농도를 측정하였다. 각 ACE2 변이체의 생산량은 다음 표 7과 같다.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.
ACE2 variantsACE2 variants Yield (mg)Yield (mg) RatioRatio
ACE2 wild typeACE2 wild type 0.8470.847 1One
ACE2.V.06ACE2.V.06 0.9570.957 1.1301.130
ACE2.V.07ACE2.V.07 0.8210.821 0.9690.969
ACE2.V.08ACE2.V.08 0.3930.393 0.4640.464
ACE2.V.09ACE2.V.09 0.8900.890 1.0511.051
ACE2.V.10ACE2.V.10 0.9760.976 1.1521.152
ACE2.V.11ACE2.V.11 1.1371.137 1.3421.342
ACE2.V.12ACE2.V.12 0.8300.830 0.9800.980
ACE2.V.13ACE2.V.13 0.8790.879 1.0381.038
ACE2.V.14ACE2.V.14 0.8000.800 0.9450.945
ACE2.V.15ACE2.V.15 1.1181.118 1.3201.320
ACE2.V.16ACE2.V.16 0.9000.900 1.0631.063
ACE2.V.17ACE2.V.17 0.8400.840 0.9920.992
ACE2.V.18ACE2.V.18 1.041.04 1.2281.228
ACE2.V.19ACE2.V.19 1.0361.036 1.2231.223
ACE2.V.20ACE2.V.20 0.1890.189 0.2230.223
ACE2.V.21ACE2.V.21 1.0701.070 1.2631.263
ACE2.V.22ACE2.V.22 0.1610.161 0.1900.190
ACE2.V.23ACE2.V.23 0.4200.420 0.4960.496
ACE2.V.24ACE2.V.24 0.5660.566 0.6680.668
ACE2.V.25ACE2.V.25 0.9050.905 1.0681.068
ACE2.V.26ACE2.V.26 0.0320.032 0.0380.038
ACE2.V.27ACE2.V.27 0.0570.057 0.0670.067
ACE2.V.28ACE2.V.28 0.9960.996 1.1761.176
ACE2.V.29ACE2.V.29 0.3540.354 0.4180.418
ACE2.V.30ACE2.V.30 0.1150.115 0.1360.136
ACE2.V.31ACE2.V.31 0.1410.141 0.1660.166
ACE2.V.32ACE2.V.32 0.1300.130 0.1530.153
ACE2.V.33ACE2.V.33 1.3911.391 1.6421.642
ACE2.V.34ACE2.V.34 Low expressionlow expression
ACE2.V.35ACE2.V.35
ACE2.V.36ACE2.V.36
ACE2.V.37ACE2.V.37 1.0321.032 1.2181.218
ACE2.V.38ACE2.V.38 0.7010.701 0.8280.828
ACE2.V.39ACE2.V.39 1.0561.056 1.2471.247
ACE2.V.40ACE2.V.40 0.5680.568 0.6710.671
ACE2.V.41ACE2.V.41 0.5890.589 0.6950.695
ACE2.V.42ACE2.V.42 0.6300.630 0.7440.744
ACE2.V.43ACE2.V.43 0.3920.392 0.4630.463
ACE2.V.44ACE2.V.44 0.4160.416 0.4910.491
ACE2.V.45ACE2.V.45 0.5400.540 0.6380.638
ACE2.V.46ACE2.V.46 0.5040.504 0.5950.595
ACE2.V.47ACE2.V.47 0.5130.513 0.6060.606
ACE2.V.48ACE2.V.48 0.5800.580 0.6850.685
실시예 7: ACE2 변이체의 특성분석Example 7: Characterization of ACE2 variants
실시예 7-1: SDS-PAGE 분석Example 7-1: SDS-PAGE analysis
생산된 ACE2 변이체를 (3 ug)를 LDS sample buffer (Invitrogen, Cat#B0007)를 넣어준 후 reducing condition group에는 sample reducing agent (Invitrogen, Cat#B0004)를 넣어주고 70 °C에서 10분동안 열처리하여 샘플을 준비하였다. SDS running buffer (Bio-rad, Cat#1610732)를 넣은, Mini gel tank에 Mini-PROTEIN TGX Stain-Free Gel (Bio-rad, Cat#456-8096)을 설치한 후 power supply (Bio-rad)를 통해 160V, 30분간 샘플을 running 하였다. Sample running이 완료된 겔은 분리 후 Chemidoc (Bio-rad)를 통해 분석하였다(도 9a-9b).After adding the LDS sample buffer (Invitrogen, Cat#B0007) to the produced ACE2 variant (3 ug), add a sample reducing agent (Invitrogen, Cat#B0004) to the reducing condition group, and heat treatment at 70 °C for 10 minutes. Samples were prepared. After installing Mini-PROTEIN TGX Stain-Free Gel (Bio-rad, Cat#456-8096) in the mini gel tank with SDS running buffer (Bio-rad, Cat#1610732), turn on the power supply (Bio-rad). Through 160V, the sample was run for 30 minutes. After sample running, the gel was separated and analyzed by Chemidoc (Bio-rad) (FIGS. 9a-9b).
실시예 7-2: ACE2 변이체와 SARS-CoV-2 spike protein RBD 의 결합 친화도 분석Example 7-2: Binding affinity analysis of ACE2 mutant and SARS-CoV-2 spike protein RBD
디자인된 ACE2 변이체를 20 ug/mL로 희석한 뒤, Human capture kit (GE healthcare, Cat#BR-1008-39)를 이용하여 anti-human Fc antibody를 고정한 CM5 chip (GE Healthcare, Cat#BR-1005-30)에 10 uL/min 유속으로, 60초 흘려주어 ligand를 고정하였다. 그 후 analyte인 SARS-CoV-2_His (Sino)을 100, 50, 25, 12.5, 6.25, 3.125 nM로 단계 희석(serial dilution)하였고 association time을 150초, dissociation time을 240초로 injection하였다. Biacore T200 (GE healthcare) 장비를 통하여 측정된 sensorgram을 1:1 결합 모델로 fitting하여 결합 친화도를 분석하였다(표 8 및 표 9).After diluting the designed ACE2 variant to 20 ug/mL, a CM5 chip (GE Healthcare, Cat#BR-1005) immobilized with an anti-human Fc antibody using a human capture kit (GE healthcare, Cat#BR-1008-39) -30) at a flow rate of 10 uL/min, for 60 seconds, the ligand was immobilized. After that, 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).
AntibodyAntibody ka (1/Ms)ka (1/Ms) kd (1/s) kd (1/s) KD (M)KD (M)
ACE2 wtACE2 wt 3.873E+53.873E+5 0.010720.01072 2.768E-82.768E-8
ACE2.V.06ACE2.V.06 2.837E+52.837E+5 0.001430.00143 5.048E-95.048E-9
ACE2.V.07ACE2.V.07 2.574E+52.574E+5 0.0035420.003542 1.376E-81.376E-8
ACE2.V.08ACE2.V.08 6.953E+56.953E+5 0.0083760.008376 1.205E-81.205E-8
ACE2.V.09ACE2.V.09 4.317E+54.317E+5 0.0046620.004662 1.080E-81.080E-8
ACE2.V.10ACE2.V.10 2.307E+52.307E+5 0.0029220.002922 1.266E-81.266E-8
ACE2.V.11ACE2.V.11 6.452E+56.452E+5 0.0050270.005027 7.791E-97.791E-9
ACE2.V.12ACE2.V.12 5.801E+55.801E+5 0.0037290.003729 6.429E-96.429E-9
ACE2.V.13ACE2.V.13 2.301E+52.301E+5 0.0043280.004328 1.881E-81.881E-8
ACE2.V.14ACE2.V.14 3.129E+53.129E+5 0.0066400.006640 2.122E-82.122E-8
ACE2.V.15ACE2.V.15 3.860E+53.860E+5 0.0067130.006713 1.739E-81.739E-8
ACE2.V.16ACE2.V.16 6.088E+56.088E+5 0.0060430.006043 9.927E-99.927E-9
ACE2.V.17ACE2.V.17 4.707E+54.707E+5 0.0032340.003234 6.871E-96.871E-9
ACE2.V.18ACE2.V.18 4.235E+54.235E+5 0.0051260.005126 1.210E-81.210E-8
ACE2.V.19ACE2.V.19 4.190E+54.190E+5 0.0029250.002925 6.981E-96.981E-9
ACE2.V.20ACE2.V.20 3.640E+53.640E+5 0.0043340.004334 1.191E-81.191E-8
ACE2.V.21ACE2.V.21 2.849E+52.849E+5 0.00019120.0001912 6.712E-106.712E-10
ACE2.V.22ACE2.V.22 2.946E+52.946E+5 0.00022070.0002207 7.494E-107.494E-10
ACE2.V.23ACE2.V.23 4.033E+54.033E+5 0.0051360.005136 1.274E-81.274E-8
ACE2.V.24ACE2.V.24 1.613E+51.613E+5 0.00056220.0005622 3.485E-93.485E-9
-ACE2.V.25-ACE2.V.25 3.609E+53.609E+5 0.0017000.001700 4.712E-94.712E-9
ACE2.V.26ACE2.V.26 2.548E+52.548E+5 0.0017550.001755 6.889E-96.889E-9
ACE2.V.27ACE2.V.27 9.470E+49.470E+4 0.0018920.001892 1.998E-81.998E-8
ACE2.V.28ACE2.V.28 4.906E+54.906E+5 0.00022160.0002216 4.517E-104.517E-10
ACE2.V.29ACE2.V.29 5.221E+55.221E+5 0.0010050.001005 1.925E-91.925E-9
ACE2.V.39ACE2.V.39 2.142E+52.142E+5 0.00011460.0001146 5.347E-105.347E-10
ACE2.V.40ACE2.V.40 1.586E+61.586E+6 0.00017620.0001762 1.111E-101.111E-10
ACE2.V.41ACE2.V.41 1.091E+61.091E+6 0.00013620.0001362 1.249E-101.249E-10
ACE2.V.42ACE2.V.42 3.242E+53.242E+5 0.00012540.0001254 3.869E-103.869E-10
AntibodyAntibody ka (1/Ms)ka (1/Ms) kd (1/s) kd (1/s) KD (M)KD (M)
ACE2 wtACE2 wt 2.777E+52.777E+5 0.0094950.009495 3.419E-83.419E-8
ACE2.V.43ACE2.V.43 2.394E+52.394E+5 0.00016210.0001621 6.772E-106.772E-10
ACE2.V.44ACE2.V.44 2.775E+52.775E+5 0.0000650.000065 2.342E-102.342E-10
ACE2.V.45ACE2.V.45 8.423E+58.423E+5 0.00014880.0001488 1.767E-101.767E-10
ACE2.V.46ACE2.V.46 1.589E+61.589E+6 0.00011840.0001184 7.451E-117.451E-11
ACE2.V.47ACE2.V.47 5.675E+55.675E+5 0.00011590.0001159 2.042E-102.042E-10
ACE2.V.48ACE2.V.48 9.205E+59.205E+5 0.00010080.0001008 1.096E-101.096E-10
ACE2.V.46에 FcγR 결합 친화도를 낮춘 Fc 변이체 (L234A, L235A, K322A)를 융합한 형태로 제작하여 마찬가지로 SARS-CoV-2 RBD와의 결합을 확인하였다(표 10).ACE2.V.46 was prepared in the form of a fusion of Fc mutants (L234A, L235A, K322A) with lower FcγR binding affinity, and similarly, binding to SARS-CoV-2 RBD was confirmed (Table 10).
AntibodyAntibody ka (1/Ms)ka (1/Ms) kd (1/s) kd (1/s) KD (M)KD (M)
ACE2.V.46-Fc (AAA)ACE2.V.46-Fc (AAA) 4.873E+64.873E+6 1.105E-41.105E-4 2.268E-112.268E-11
실시예 7-3: ACE2 변이체와 SARS-CoV-2 spike protein RBD 변이체(영국발 변종 및 남아공발 변종)의 결합 친화도 분석Example 7-3: Binding affinity analysis of ACE2 mutant and SARS-CoV-2 spike protein RBD mutant (variant from UK and strain from South Africa)
최근 발견된 SARS-CoV-2 spike protein RBD 변이체 2종 (영국발, 남아공발)에 대한 결합 친화도를 분석하기 위해 analyte로 각각 RBD 변이체를 이용하여 위와 같은 조건에서 분석하였다(표 11 및 표 12).To analyze the binding affinity of two recently discovered SARS-CoV-2 spike protein RBD mutants (from UK and South Africa), each RBD mutant was used as an analyte and analyzed under the same conditions as above (Table 11 and Table 12). ).
영국발 변이체 (SARS-CoV-2 RBD_N501Y)UK variant (SARS-CoV-2 RBD_N501Y)
AntibodyAntibody ka (1/Ms)ka (1/Ms) kd (1/s) kd (1/s) KD (M)KD (M)
ACE2 wtACE2 wt 6.779E+56.779E+5 0.00085180.0008518 1.257E-91.257E-9
ACE2.V.46ACE2.V.46 4.378E+64.378E+6 0.000026740.00002674 6.108E-126.108E-12
남아공발 변이체 (SARS-CoV-2 RBD_N501Y, K417N, E484K))variants from South Africa (SARS-CoV-2 RBD_N501Y, K417N, E484K))
AntibodyAntibody ka (1/Ms)ka (1/Ms) kd (1/s)kd (1/s) KD (M)KD (M)
ACE2 wtACE2 wt 5.577E+55.577E+5 0.0020600.002060 3.694E-93.694E-9
ACE2.V.46ACE2.V.46 3.592E+63.592E+6 0.000092840.00009284 2.585E-112.585E-11
실시예 7-4: 크기 배제 크로마토그래피(Size exclusion chromatography) 분석Example 7-4: Size exclusion chromatography analysis
생산된 ACE2 변이체를 Superdex200 Increase 10/300 GL (GE healthcare, Cat#.28-9909-44)을 연결한 AKTA pure를 이용하여 분석하였다. 먼저 분석 전 PBS를 0.3 mL/min 유속으로 흘려주어 column equilibration한다. 그후 0.5 mL standard와 샘플을 1 mL 실린지(syringe)를 이용하여 FPLC장비에 주입하고, 샘플이 컬럼을 통과하도록 하였다. UNICORN (GE healthcare) 프로그램을 이용하여 분석하였다(도 10a 내지 10h).The produced ACE2 variant was analyzed using AKTA pure connected to Superdex200 Increase 10/300 GL (GE healthcare, Cat#.28-9909-44). First, before analysis, column equilibration is performed by flowing PBS at a flow rate of 0.3 mL/min. Then, 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).
실시예 7-5: ACE2 변이체의 SARS-CoV-2 중화능 분석Example 7-5: SARS-CoV-2 neutralizing ability analysis of ACE2 variants
ACE2 wild type과 변이체들의 SARS-CoV-2 RBD에 대한 중화능력을 비교하기 위해 In Vitro Neutralizing Antibody Test (IVnAT) (수젠텍, cat no.CONE001E)를 이용하였다. ACE2 변이체를 단계 희석하여 RBD-HRP와 1:1 반응시켜 37℃에서 15분동안 사전 인큐베이션한 후, hACE2 코팅된 플레이트에 처리하여 농도에 따른 중화능을 확인하였다(도 11 및 도 12).In Vitro Neutralizing Antibody Test (IVnAT) (Sugentech, cat no.CONE001E) was used to compare the neutralizing ability of ACE2 wild type and mutants against 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 ).
도 11 및 도12에 도시된 것과 같이, 본 발명의 ACE2 변이체가 야생형 ACE2보다 낮은 농도에서도 현저히 뛰어난 코로나바이러스 RBD의 ACE2 결합 억제 및 높은 중화능을 나타내는 것을 확인하였다.11 and 12, it was confirmed that the ACE2 mutant of the present invention exhibited significantly superior ACE2 binding inhibition and high neutralizing ability of coronavirus RBD even at a lower concentration than wild-type ACE2.
실시예 7-6: ACE2 변이체의 SARS-CoV-2 변종(영국발, 남아공발) 중화능 분석Example 7-6: Analysis of neutralizing ability of SARS-CoV-2 mutant (from UK, South Africa) of ACE2 mutant
SARS-CoV-2 RBD 변이체 2종 (영국발(N501Y), 남아공발(N501Y, K417N, E484K))에 대한 중화능을 확인하기 위해 SARS-CoV-2 inhibitor screening kit (Acro, cat no.EP-105)를 이용하였다. RBD 변이체를 코팅한 microplate에 biotinylated hACE2와 serial diluted ACE2 변이체를 1:1로 혼합한 용액을 반응시켜 37℃, 1시간 인큐베이션하였다. 그 후 streptavidin HRP를 37℃, 1시간 처리하고 TMB로 발색하여 ACE2 농도에 따른 SARS-CoV-2 RBD 중화능을 확인하였다(도 13 및 도 14).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 ).
도 13 및 도 14에 도시된 것과 같이, 본 발명의 ACE2 변이체는 SARS-CoV-2 RBD 변이체 2종에 대해서도 야생형 ACE2에 비해 현저히 뛰어난 중화능을 나타내었다.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.
실시예 7-7: ACE2 변이체의 촉매 활성 분석Example 7-7: Analysis of catalytic activity of ACE2 variants
안지오텐신 변환 효소 II(ACE2) 변이체의 촉매 활성을 확인하기 위하여 Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric) (Biovision, cat no.K897)를 이용하였다. 5nM 또는 10nM의 ACE2에 ACE2 substrate 를 넣어 효소 반응을 진행시키고 생산물을 Fluoroskan Ascent FL 로 RFU(Ex/Em=355nm/460nm) 값을 20분동안 10초 간격으로 측정하였다(도 15 및 도 16).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 ACE2 substrate was put into 5nM or 10nM of ACE2 to proceed with the enzymatic reaction, and the RFU (Ex/Em=355nm/460nm) value of the product was measured with Fluoroskan Ascent FL at 10 second intervals for 20 minutes ( FIGS. 15 and 16 ).
최종적으로, Human ACE2 engineering을 수행하여 결합 친화도(binding affinity)이 가장 향상된 ACE2.V.46 변이체를 선별하였고 결합 친화도는 약 460배, 중화능은 약 130배 증가하면서 ACE2 효소활성은 유지됨을 확인하였다. 또한, ACE2.V.46은 영국 및 남아공 변이체에 대해서도 각각 약 206배, 143배 결합 친화도가 증가하였으며, 변이체에 대해서도 향상된 중화능을 보임을 확인하였다.Finally, human ACE2 engineering was performed to select the ACE2.V.46 mutant with the most improved binding affinity, and it was found that the ACE2 enzymatic activity was maintained while the binding affinity increased by about 460 times and the neutralization ability by about 130 times. Confirmed. In addition, it was confirmed that ACE2.V.46 increased binding affinity by about 206-fold and 143-fold to the British and South African mutants, respectively, and showed improved neutralizing ability for the mutant.
또한 본 발명의 실시예에서 확인하고 있는 것과 같이, ACE2.V.46뿐만 아니라, 라이브러리로부터 sorting된 개별 클론(표 4 및 표 5)과 ACE2.V.06 내지 ACE2.V.48을 포함하는 본 발명의 ACE2 변이체는 야생형 ACE2에 비해 코로나바이러스 스파이크 단백질의 RBD에 현저히 높은 결합친화도를 가지며, 코로나바이러스에 대한 뛰어난 중화능을 나타내어 바이러스의 세포 내 침입 및 증폭을 효과적으로 억제할 수 있다.In addition, as confirmed in the Examples of the present invention, 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.
실시예 8: ACE2 변이체의 약동학적 특성 분석Example 8: Pharmacokinetic Characterization of ACE2 Variants
Balb/c mouse에 생산된 ACE2-Fc 를 I.V injection으로 10mpk 를 주입하여 in vivo 실험을 진행하였다. 총 14번의 time point로 나누어서 5min, 10min, 15min, 20min, 45min, 1hr, 2hr, 4hr, 8hr, 24hr, 48hr, 96hr, 168hr, 240hr 로 100ul bleeding 하여 샘플 채취를 한 후 혈청을 분리하여 잔존하는 ACE2의 양을 ELISA로 확인하였다. 그리고 24hr 에서의 샘플에서 ACE2 변이체의 촉매 활성도 측정 하였다.In vivo experiments were carried out by injecting 10mpk of ACE2-Fc produced in Balb/c mice by I.V injection. Divide the total of 14 time points and collect the sample by bleeding 100ul at 5min, 10min, 15min, 20min, 45min, 1hr, 2hr, 4hr, 8hr, 24hr, 48hr, 96hr, 168hr, 240hr to separate the serum and ACE2 remaining was confirmed by ELISA. And the catalytic activity of the ACE2 variant was also measured in the sample at 24 hr.
실시예 8-1: ACE2.V.46(EU129) 의 in vivo 안정성 시험Example 8-1: In vivo stability test of ACE2.V.46 (EU129)
96wells immune plate에 anti-Human IgG (Fc specific) antibody(Sigma, Cat# I2136)를 2ug/ml 농도로 100ul/well에 4℃에서 overnight 코팅하여 준비하였다. 5% skim milk로 1hr, RT 에서 blocking 한 후 ACE2.V.46(EU129) 의 혈청 샘플을 각각 standard curve에 rage에 맞는 농도로 1X PBS에 희석하여 준비하고 ACE2 혈청 샘플을 100ul/well, 1hr, RT로 처리하였다. 그 후 20nM biotinylated RBD-his로 ACE2 binding을 1hr, RT에서 결합시킨 후 1:5000(v/v) HRP-Conjugated Streptavidin 을 동일하게 반응시켰다. 반응은 TMB substrate를 사용하였고 5-10min develop 시킨 뒤 stop solution 을 넣고 450nm 에서 분석하였다(도 17).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℃ 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. Thereafter, 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. For the reaction, TMB substrate was used, and after 5-10 min of development, a stop solution was added and analyzed at 450 nm (FIG. 17).
도 17과 같이, 본 발명의 ACE2 변이체는 in vivo에서도 야생형 ACE2와 동일한 수준 또는 장시간 경과시 유의미하게 향상된 수준의 안정성을 나타내는 것을 확인하였다.17, it was confirmed that 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.
실시예 8-2: ACE2 변이체의 촉매 활성 평가Example 8-2: Evaluation of catalytic activity of ACE2 variants
In vivo 실험의 혈청 샘플에 있는 안지오텐신 변환 효소 II(ACE2) 변이체의 촉매활성을 확인하기 위하여 Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric) (Biovision, cat no.K897)를 이용하였다. ACE2 pk test를 통하여 확인된 ACE2 잔존량을 토대로 24hr의 혈청을 ACE2 5nM로 1X PBS에 희석하여 샘플을 준비하였다. ACE2에 ACE2 substrate(MCA-APK) 를 넣어 효소 반응을 진행시키고 생산물을 Fluoroskan Ascent FL 로 RFU(Ex/Em=355nm/460nm) 값을 20분동안 10초 간격으로 측정하였다(도 18). 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. ACE2 substrate (MCA-APK) was put into ACE2 to proceed with the enzymatic reaction, and the RFU (Ex/Em=355nm/460nm) value of the product was measured with Fluoroskan Ascent FL at 10 second intervals for 20 minutes (FIG. 18).
도 18에 도시된 것과 같이, 본 발명의 ACE2 변이체의 주입 후 마우스 체내에서 장시간이 도과한 뒤에도, 유의미하게 효소활성이 유지됨을 확인하였다.As shown in FIG. 18 , it was confirmed that the enzyme activity was significantly maintained even after a long period of time in the mouse body after injection of the ACE2 variant of the present invention.
실시예 9: 세포수준에서 실제 코로나바이러스의 감염 및 증폭 억제효과Example 9: Inhibition of infection and amplification of actual coronavirus at the cellular level
본 발명자들은 실제 세포 수준에서의 본 발명의 ACE2 변이체들의 SARS-CoV-2 바이러스에 대한 항바이러스 효과를 입증하기 위하여, 본 발명의 ACE2 변이체들 중 ACE2.V.06-Fc(이하, ACE2.V.06) 및 ACE2.V.41-Fc(이하, ACE2.V.46)과 ACE2 WT-Fc(이하, ACE2-WT) 단백질을 사용하여 SARS-CoV-2 중화(neutralization) 실험을 실시하였다.In order to demonstrate the antiviral effect of the ACE2 variants of the present invention against SARS-CoV-2 virus at the actual cellular level, the present inventors have investigated ACE2.V.06-Fc (hereinafter, ACE2.V .06) and ACE2.V.41-Fc (hereinafter, ACE2.V.46) and ACE2 WT-Fc (hereinafter, ACE2-WT) proteins were used to conduct a SARS-CoV-2 neutralization experiment.
세포주에 SARS-CoV-2와 함께 외부 ACE2-WT 또는 본 발명의 ACE2 변이체들을 처리했을 때, 외부 ACE2-WT 또는 본 발명의 ACE2-변이체의 SARS-CoV-2와 세포의 ACE2 간의 결합을 억제하는 효능을 확인하였다.When a cell line was treated with exogenous ACE2-WT or ACE2 variants of the present invention along with SARS-CoV-2, the binding between SARS-CoV-2 of exogenous ACE2-WT or ACE2-mutant of the present invention and cell ACE2 was inhibited. Efficacy was confirmed.
SARS-CoV-2 실제 바이러스에 ACE2 Wt-Fc 또는 변이 단백질인 ACE2.V.06-Fc 및 ACE2.V.41-Fc을 다양한 MOI로 혼합하여 바이러스의 중화를 유도한 후, 원숭이 신장세포인 Vero E6 세포주에 감염을 유도하였다. 상온에서 1시간 배양 후에 바이러스를 제거하고 OPTI-MEM 배지로 세포를 세척해 세포에 결합하지 않은 바이러스를 제거한 뒤, 새로운 배지를 첨가하여 24시간 배양하고, 세포를 포집 후 바이러스 단백질과 RNA 양을 측정하였다.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
간략하게는 다음과 같다: SARS-CoV-2(국가병원체자원은행 제공, NCCP43331)를 배양하고, 배양된 SARS-CoV-2(MOI=1, 0.1, 0.01)에 다양한 농도의 ACE2-WT, ACE2-V.06, 또는 ACE2-V.41을 혼합하여 상온에서 1시간 반응시켰다. VeroE6 세포에서 배양액을 제거한 후 혼합액을 처리하고 1시간 동안 감염을 유도하였다. 바이러스 혼합액을 제거하고 OPTI-MEM을 사용하여 세포를 세척 및 잔여 바이러스를 제거하고, 10% FBS가 포함된 DMEM을 넣어 24시간 또는 48시간 배양하였다.Briefly, SARS-CoV-2 (provided by the National Pathogen Resources Bank, NCCP43331) was cultured, and various concentrations of ACE2-WT, ACE2 were added to the cultured SARS-CoV-2 (MOI=1, 0.1, 0.01). -V.06 or ACE2-V.41 was mixed and reacted at room temperature for 1 hour. After removing the culture medium from VeroE6 cells, the mixture was treated and infection was induced for 1 hour. After removing the virus mixture, cells were washed using OPTI-MEM to remove residual virus, and DMEM containing 10% FBS was added and cultured for 24 hours or 48 hours.
세포 변성 효과 분석(Cytopathic effect assay)을 통해 SARS-CoV-2에 감염된 VeroE6 세포주에서 나타나는 세포 변성 효과와 ACE2 단백질 처리에 의한 CPE 감소 현상을 광학현미경 하에서 관찰하였다.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.
또한, SARS-CoV-2에 감염된 VeroE6 세포주에 lysis buffer(Cell culture lysis reagent, Promega)를 첨가하여 세포를 용해하고, SARS-CoV-2 nucleoprotein (Sino Biological, China)을 감지하는 항체를 사용하는 웨스턴 블롯팅을 통해 발현된 바이러스 단백질 양을 측정하였다.In addition, lysis buffer (Cell culture lysis reagent, Promega) is added to the VeroE6 cell line infected with SARS-CoV-2 to lyse the cells, and Western using an antibody that detects SARS-CoV-2 nucleoprotein (Sino Biological, China) The amount of expressed viral protein was determined through blotting.
또한, SARS-CoV-2 RNA 양 측정을 통해 ACE2 단백질이 SARS-CoV-2 바이러스와 결합하여 제거되는 중화(neutralization) 효능 측정 및 IC50 측정하였다. VeroE6 세포를 NucleoZol (MN)을 사용하여 용해, total RNA를 분리, cDNA synthesis kit (Toyobo, Japan)를 사용하여 cDNA를 합성하고 프라이머(서열번호 107; sense, 5'-GTG AAA TGG TCA TGT GTG GCG G-3'및 서열번호 108; antisense, 5'-CAA ATG TTA AAA ACA CTA TTA GCA TA-3')를 이용하여 qRT-PCR (SYBR Green Master Mix, Bio-Rad Laboratories)을 실시하였다.In addition, by measuring the amount of SARS-CoV-2 RNA, neutralization efficacy and IC 50 were measured in which the ACE2 protein was removed by binding to the SARS-CoV-2 virus. 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').
8-1. SARS-CoV-2에 대한 ACE2-V.06의 항바이러스 효과8-1. Antiviral effect of ACE2-V.06 on SARS-CoV-2
본 발명자들은 SARS-CoV-2에 대한 ACE2-V.06의 항바이러스 효과를 확인하였다.The present inventors confirmed the antiviral effect of ACE2-V.06 on SARS-CoV-2.
그 결과, 도 19에 나타낸 바와 같이, SARS-CoV-2(MOI=1)에 다양한 농도의 ACE2-V.06을 처리하여 CPE를 관찰한 경우, 5ug/ml이상의 ACE2-V.06 처리군에서 SARS-CoV-2에 의해서 생성되는 CPE가 나타나지 않았다. As a result, as shown in FIG. 19, when SARS-CoV-2 (MOI = 1) was treated with ACE2-V.06 at various concentrations and CPE was observed, in the ACE2-V.06 treatment group of 5 ug/ml or more There was no CPE produced by SARS-CoV-2.
또한, 도 20에 나타낸 바와 같이, SARS-CoV-2(MOI=0.1)에 다양한 농도의 ACE2-V.06을 처리하여 CPE를 관찰한 경우, 1ug/ml ACE2-V.06 처리군에서 CPE가 감소되는 현상이 나타나고, 5ug/ml이상의 ACE2-V.06 처리군에서 SARS-CoV-2에 의해서 생성되는 CPE가 현저히 억제 또는 관찰되지 않았다. In addition, as shown in Figure 20, when SARS-CoV-2 (MOI = 0.1) was treated with ACE2-V.06 at various concentrations to observe CPE, CPE was increased in the 1ug/ml ACE2-V.06 treatment group. A decrease was observed, and CPE produced by SARS-CoV-2 was not significantly inhibited or observed in the ACE2-V.06 treatment group of 5 ug/ml or more.
또한, 도 21에 나타낸 바와 같이, SARS-CoV-2 (MOI=0.1 또는 1)에 다양한 농도의 ACE2-V.06을 처리하고 웨스턴 블롯으로 바이러스 핵단백질(nucleoprotein)의 양을 측정한 경우, 5ug/ml ACE2-V.06 처리군에서 SARS-CoV-2 nucleoprotein 발현양이 현저히 감소되고, 그 이상의 농도에서는 바이러스 단백질이 검출되지 않았다.In addition, as shown in FIG. 21, when SARS-CoV-2 (MOI=0.1 or 1) was treated with various concentrations of ACE2-V.06 and the amount of viral nucleoprotein was measured by Western blot, 5ug In the /ml ACE2-V.06 treatment group, the expression level of SARS-CoV-2 nucleoprotein was significantly reduced, and no viral protein was detected at a concentration higher than that.
또한, 도 22에 나타낸 바와 같이, SARS-CoV-2 (MOI=0.01)에 다양한 농도의 ACE2-WT 또는 ACE2-V.06을 처리하여 CPE를 관찰한 경우, ACE2-WT과 ACE2-V.06 모두 1ug/ml에서 CPE 현상이 감소되었으며, WT과 V.06 처리에 의한 CPE 차이는 크게 나타나지 않았다.In addition, as shown in Figure 22, when SARS-CoV-2 (MOI = 0.01) was treated with various concentrations of ACE2-WT or ACE2-V.06 to observe CPE, ACE2-WT and ACE2-V.06 In all, the CPE phenomenon was reduced at 1ug/ml, and there was no significant difference in CPE between WT and V.06 treatment.
또한, 도 23에 나타낸 바와 같이, SARS-CoV-2 (MOI=0.01)에 다양한 농도의 ACE2-WT 또는 ACE2-V.06을 처리하고 웨스턴 블롯을 실시한 경우(상단 패널); 및 동일한 실험군에서 세포 배양액을 포집하여 TCID50 방법을 사용하여 세포 밖으로 배출되는 바이러스 양을 측정한 경우(하단 패널), ACE2-WT 처리에 의한 농도의존적인 nucleoprotein의 감소 현상이 관찰되었으나 10ug/ml에서 nucleoprotein이 존재하는 것으로 보아 완벽한 항바이러스 효능이 나타나지 않았다. 반면, ACE2-V.06의 10 ug/ml 처리군에서 바이러스 단백질이 관찰되지 않았으므로 WT에 비해 항바이러스 효능이 높은 것을 알 수 있다. 동일한 샘플에서 세포 밖으로 배출되는 바이러스 양을 측정한 결과에서도 웨스턴 블롯의 결과와 동일하게 ACE2-V.06의 10 ug/ml 처리군에서 바이러스 배출이 현저히 낮게 나타났다.In addition, as shown in FIG. 23, SARS-CoV-2 (MOI=0.01) was treated with various concentrations of ACE2-WT or ACE2-V.06 and Western blot was performed (upper panel); And when the amount of virus excreted out of the cell was measured using the TCID50 method by collecting the cell culture medium from the same experimental group (lower panel), a concentration-dependent reduction of nucleoprotein by ACE2-WT treatment was observed, but at 10ug/ml, nucleoprotein It was found that there was no complete antiviral efficacy. On the other hand, since no viral protein was observed in the 10 ug/ml treatment group of ACE2-V.06, it can be seen that the antiviral efficacy was higher than that of WT. As a result of measuring the amount of virus excreted out of the cell from the same sample, the virus excretion was significantly lower in the 10 ug/ml treatment group of ACE2-V.06, similar to the result of western blot.
또한, 도 24에 나타낸 바와 같이, SARS-CoV-2 (MOI=0.1, 0.01)에 다양한 농도의 ACE2-WT 또는 ACE2-V.06을 처리하여 CPE를 관찰한 경우, 24시간 배양에서는 SARS-CoV-2 감염에 의한 CPE가 관찰되지 않았고, ACE2-WT과 ACE2-V.06를 처리한 모든 세포에서도 CPE가 관찰되지 않았다.In addition, as shown in Figure 24, when SARS-CoV-2 (MOI = 0.1, 0.01) was treated with various concentrations of ACE2-WT or ACE2-V.06 to observe CPE, in culture for 24 hours, SARS-CoV CPE by -2 infection was not observed, and CPE was not observed in all cells treated with ACE2-WT and ACE2-V.06.
또한, 도 25에 나타낸 바와 같이, SARS-CoV-2 (MOI=0.1, 0.01)에 다양한 농도의 ACE2-WT 또는 ACE2-V.06을 처리하고 nucleoprotein 발현양을 Western blot의 방법으로 측정한 경우, 24시간 배양에서는 바이러스 MOI=0.1에서 nucleoprotein의 발현을 확인하였고 ACE2-WT의 1ug/ml 농도에서도 이 단백질의 양이 현저히 감소되었으며 5ug/ml이상에서 소량의 바이러스 단백질이 관찰되었다. ACE2-V.06의 처리된 모든 농도에서 nucleoprotein이 감지되지 않았다. MOI=0.01 SARS-CoV-2 감염에서는 24시간에 바이러스 단백질이 감지되지 않았다. In addition, as shown in FIG. 25 , when SARS-CoV-2 (MOI=0.1, 0.01) was treated with various concentrations of ACE2-WT or ACE2-V.06 and the nucleoprotein expression level was measured by the Western blot method, In 24-hour culture, expression of nucleoprotein was confirmed at virus MOI = 0.1, and the amount of this protein was significantly reduced even at 1ug/ml concentration of ACE2-WT, and a small amount of viral protein was observed at 5ug/ml or higher. No nucleoprotein was detected in all treated concentrations of ACE2-V.06. MOI=0.01 In SARS-CoV-2 infection, no viral protein was detected at 24 h.
또한, 도 26에 나타낸 바와 같이, SARS-CoV-2 (MOI=0.1)에 다양한 농도의 ACE2-WT 또는 ACE2-V.06을 처리하고 분리된 총 RNA에서 정량-PCR의 방법으로 바이러스 RNA 양을 측정한 경우, WT에 비해 V.06 돌연변이 단백질이 중화 효능이 크게 나타났다. In addition, as shown in FIG. 26, SARS-CoV-2 (MOI=0.1) was treated with various concentrations of ACE2-WT or ACE2-V.06 and the amount of viral RNA was measured from the isolated total RNA by quantitative-PCR. When measured, the neutralizing effect of the V.06 mutant protein was greater than that of the WT.
8-2. SARS-CoV-2에 대한 ACE2-V.41의 항바이러스 효과8-2. Antiviral effect of ACE2-V.41 on SARS-CoV-2
본 발명자들은 SARS-CoV-2에 대한 ACE2-V.41의 항바이러스 효과를 확인하였다.The present inventors confirmed the antiviral effect of ACE2-V.41 on SARS-CoV-2.
그 결과, 도 27에 나타낸 바와 같이, SARS-CoV-2 (MOI=0.1)에 다양한 농도의 ACE2-WT 또는 ACE2-V.41을 처리하고 CPE를 관찰한 경우, 24시간 배양에서는 SARS-CoV-2 감염에 의한 CPE가 관찰되지 않았다. ACE2-WT과 ACE2-V.41를 처리한 모든 세포에서도 CPE가 관찰되지 않았다. 48시간 배양한 실험군에서 SARS-CoV-2 감염에 의해 발생한 CPE를 ACE2-WT은 10ug/ml의 농도에서는 CPE가 감소하는 현상이 나타났다. ACE2-V.41 처리군에서는 1ug/ml 이상이 농도에서 CPE가 현저히 감소 또는 없어지는 현상이 관찰되었다. As a result, as shown in FIG. 27, when SARS-CoV-2 (MOI=0.1) was treated with various concentrations of ACE2-WT or ACE2-V.41 and CPE was observed, in culture for 24 hours, SARS-CoV- 2 No CPE due to infection was observed. CPE was not observed in all cells treated with ACE2-WT and ACE2-V.41. In the experimental group cultured for 48 hours, CPE caused by SARS-CoV-2 infection was decreased at a concentration of 10 ug/ml in ACE2-WT. In the ACE2-V.41 treatment group, a phenomenon in which CPE was significantly reduced or disappeared was observed at a concentration of 1 ug/ml or more.
또한, 도 28에 나타낸 바와 같이, SARS-CoV-2 (MOI=0.1)에 다양한 농도의 ACE2-WT 또는 ACE2-V.41을 처리하고 Western blot의 방법으로 nucleoprotein의 양을 측정한 경우, 24시간 배양에서는 ACE2-WT의 처리에 의해서 농도의존적인 nucleoprotein의 감소가 나타났으며 10ug/ml 처리군에서는 감지되지 않았다. 반면, ACE2-V.41의 1ug/ml 이상의 농도를 처리한 실험군에서 nucleoprotein이 감지되지 않아 WT에 비해 항바이러스 효능이 높은 것을 알 수 있다. In addition, as shown in FIG. 28, when SARS-CoV-2 (MOI=0.1) was treated with various concentrations of ACE2-WT or ACE2-V.41 and the amount of nucleoprotein was measured by Western blot, 24 hours In culture, a concentration-dependent decrease in nucleoprotein was observed by treatment with ACE2-WT, and was not detected in the 10 ug/ml treatment group. On the other hand, it can be seen that nucleoprotein was not detected in the experimental group treated with a concentration of 1ug/ml or more of ACE2-V.41, indicating that the antiviral efficacy was higher than that of WT.
48시간 배양한 실험군에서 SARS-CoV-2 감염에 의해 증가된 nucleoprotein이 ACE2-WT을 처리한 모든 실험군에서 감소 현상이 나타나지 않았다. 반면, ACE2-V.41을 1ug/ml 이상을 처리한 실험군에서 nucleoprotein이 감지 되지 않아 WT에 비해 항바이러스 효능을 높은 것을 알 수 있다. In the experimental group cultured for 48 hours, the nucleoprotein increased by SARS-CoV-2 infection did not decrease in all the experimental groups treated with ACE2-WT. On the other hand, 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.
또한, 도 29에 나타낸 바와 같이, SARS-CoV-2 (MOI=0.1)에 다양한 농도의 ACE2-WT 또는 ACE2-V.41을 처리하고 총 RNA를 분리하여 RdRp를 타겟으로 하는 프라이머를 사용한 정량 PCR을 실시한 경우, ACE2-V.41 처리군에서 ACE2-WT 처리군에 비하여 저농도에서 SARS-CoV-2 RNA 양을 현저히 감소시키는 것이 관찰되었다.In addition, as shown in FIG. 29, SARS-CoV-2 (MOI=0.1) was treated with various concentrations of ACE2-WT or ACE2-V.41, total RNA was isolated, and quantitative PCR using primers targeting RdRp , it was observed that the ACE2-V.41 treated group significantly reduced the amount of SARS-CoV-2 RNA at a low concentration compared to the ACE2-WT treated group.
또한, ACE2-WT과 ACE2-V.41의 SARS-CoV-2에 대한 항바이러스 효능을 비교한 반복 실험을 실시하였다(도 30 내지 도 33).In addition, repeated experiments comparing the antiviral efficacy of ACE2-WT and ACE2-V.41 against SARS-CoV-2 were performed ( FIGS. 30 to 33 ).
그 결과, 도 30에 나타낸 바와 같이, SARS-CoV-2 (MOI=0.1)에 다양한 농도의 ACE2-WT 또는 ACE2-V.41을 처리하여 48시간 배양한 후 CPE를 관찰하였다. 그 결과, 48시간 배양한 실험군에서 SARS-CoV-2 감염에 의해 발생한 CPE가 ACE2-WT 10ug/ml의 농도에서 CPE가 감소하는 현상이 관찰되었다. ACE2-V.41이 처리된 실험군에서는 0.5ug/ml 이상의 농도에서 CPE가 현저히 감소 또는 없어지는 현상이 관찰되었다.As a result, as shown in FIG. 30, SARS-CoV-2 (MOI=0.1) was treated with ACE2-WT or ACE2-V.41 at various concentrations, and CPE was observed after culturing for 48 hours. As a result, it was observed that 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. In the experimental group treated with ACE2-V.41, a phenomenon in which CPE was significantly reduced or disappeared was observed at a concentration of 0.5ug/ml or more.
또한, 도 31에 나타낸 바와 같이, SARS-CoV-2 (MOI=0.1)에 다양한 농도의 ACE2-WT 또는 ACE2-V.41을 처리하고 24시간 또는 48시간 배양하고 Western blot의 방법으로 nucleoprotein의 양을 측정한 경우, 24시간 배양에서는 ACE2-WT의 처리에 의해서 농도의존적인 nucleoprotein의 감소가 나타났으며, 5ug/ml을 처리한 실험군에서는 감지되지 않았다. 반면, ACE2-V.41의 0.5ug/ml 이상의 농도를 처리한 실험군에서 nucleoprotein이 감지되지 않아 WT에 비해 높은 항바이러스 효능이 관찰되었다. 48시간 배양한 실험군에서 SARS-CoV-2 감염에 의해 증가된 nucleoprotein이 ACE2-WT을 처리한 모든 실험군에서 감소 현상이 나타나지 않았다. 그러나, ACE2-V.41 0.5ug/ml 이상을 처리한 실험군에서는 nucleoprotein이 감지 되지 않아 WT에 비해 높은 항바이러스 효능을 나타났다. In addition, as shown in Figure 31, SARS-CoV-2 (MOI = 0.1) was treated with ACE2-WT or ACE2-V.41 at various concentrations, cultured for 24 hours or 48 hours, and the amount of nucleoprotein by Western blot method. , 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. On the other hand, 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. In the experimental group cultured for 48 hours, the nucleoprotein increased by SARS-CoV-2 infection did not decrease in all the experimental groups treated with ACE2-WT. However, in the experimental group treated with ACE2-V.41 0.5ug/ml or more, nucleoprotein was not detected, so it showed higher antiviral efficacy than WT.
또한, 도 32에 나타낸 바와 같이, SARS-CoV-2 (MOI=0.1)에 다양한 농도의 ACE2-WT 또는 ACE2-V.41을 처리하고 24시간 배양하고 세포를 포집하여 total RNA를 분리, RdRp를 타겟으로 하는 primer를 사용하여 정량 PCR을 실시했을 때, ACE2-V.41를 처리한 실험군에서 ACE2-WT을 처리한 실험군에 비하여 저농도에서 SARS-CoV-2 RNA 양을 현저히 감소시킴이 관찰되었다. In addition, as shown in FIG. 32, SARS-CoV-2 (MOI=0.1) was treated with ACE2-WT or ACE2-V.41 at various concentrations, cultured for 24 hours, cells were collected, total RNA was isolated, and RdRp was When quantitative PCR was performed using a target primer, it was observed that the amount of SARS-CoV-2 RNA was significantly reduced in the experimental group treated with ACE2-V.41 compared to the experimental group treated with ACE2-WT.
또한, 도 33에 나타낸 바와 같이, 상기 데이터들을 기반으로 통계 프로그램을 사용하여 하기와 같이 SARS-CoV-2 중화에 필요한 ACE2-WT 또는 V.41의 IC50값을 산출하였다:In addition, as shown in FIG. 33 , using a statistical program based on the above data, IC 50 values of ACE2-WT or V.41 required for SARS-CoV-2 neutralization were calculated as follows:
1차 실험: ACE2-WT ( IC50 = 1.56 ug/ml)Primary experiment: ACE2-WT (IC 50 = 1.56 ug/ml)
ACE2-V.41 ( IC50 = 0.17 ug/ml)ACE2-V.41 (IC 50 = 0.17 ug/ml)
2차 실험: ACE2-WT ( IC50 = 0.23 ug/ml)Second experiment: ACE2-WT (IC 50 = 0.23 ug/ml)
ACE2-V.41 ( IC50 = 0.079 ug/ml).ACE2-V.41 (IC 50 = 0.079 ug/ml).
정리하면, ACE2-WT, ACE2-V.06 및 ACE2-V.41 단백질을 SARS-CoV-2 바이러스와 혼합하여 바이러스와 단백질의 결합을 유도하고 이후 VeroE6 세포주에 처리한 결과, 상기 단백질 모두에서 농도 의존적으로 중화 효능이 나타났고, 모든 단백질에서 농도를 증가시켰을 때 CPE의 감소가 나타났으며, SARS-CoV-2 nucleoprotein과 RNA양의 감소가 유도되었다. 또한, ACE2-WT과 ACE2-V.06 단백질의 SARS-CoV-2에 대한 항바이러스 효능을 관찰하였을 때, ACE2-V.06이 WT에 비하여 저농도에서 중화 효능이 큰 것으로 나타났다. 또한, ACE2-WT과 ACE2-V.41 단백질의 SARS-CoV-2에 대한 항바이러스 효능을 관찰하였을 때, ACE2-V.41이 WT에 비하여 저농도에서 중화 효능이 큰 것으로 나타났다. SARS-CoV-2에 감염된 VeroE6 세포에서 바이러스 단백질 양을 48시간에 측정하였을 때, ACE2-WT의 경우 10ug/ml을 처리한 군에서도 바이러스 단백질이 감지되었으나, ACE2-V.41이 0.5ug/ml 이상 처리된 실험군에서는 감지되지 않은 것으로 보았을 때, 인 비트로 세포 모델에서 ACE2-V.41은 SARS-CoV-2에 대해 높은 효율로 중화 효과를 나타낸다. 또한, ACE2-WT의 IC50는 두 번의 실험에서 1.56 ug/ml과 0.23 ug/ml이 측정되었으며, ACE-V.41의 IC50는 0.17ug/ml과 0.079ug/ml로 각각 측정되어 ACE2-WT에 비하여 ACE2-V.41이 각각의 실험 결과에서 낮은 농도에서도 중화 효능이 나타남을 알 수 있다.In summary, 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. In addition, when the antiviral efficacy of ACE2-WT and ACE2-V.06 protein against SARS-CoV-2 was observed, it was found that ACE2-V.06 had a greater neutralizing effect at a low concentration than WT. In addition, when the antiviral efficacy of ACE2-WT and ACE2-V.41 protein against SARS-CoV-2 was observed, it was found that ACE2-V.41 had a greater neutralizing effect at a low concentration than WT. When 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. In addition, the 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. Compared to WT, it can be seen that ACE2-V.41 exhibits neutralizing efficacy even at low concentrations in each experimental result.
즉, 상기 데이터들은 ACE2-WT를 처리한 경우에 비해, 본 발명의 ACE2 변이체인 ACE2.V.06 또는 ACE2.V.41를 처리한 경우 현저히 낮은 농도에서 높은 바이러스 중화율로, 뛰어난 코로나바이러스(SARS-CoV-2) 감염 및 증폭 억제 효과를 나타낼 수 있음을 입증한다.That is, the data show that, compared to the case of treatment with ACE2-WT, when treated with ACE2.V.06 or ACE2.V.41, which are the ACE2 variants of the present invention, high virus neutralization rate at a significantly lower concentration, excellent coronavirus ( SARS-CoV-2) demonstrates that it can exhibit the effect of inhibiting infection and amplification.
따라서, 본 발명의 ACE2 변이체는 SARS-CoV-2에 대한 효과적인 치료제로 활용될 수 있을 것으로 기대된다.Therefore, it is expected that the ACE2 variant of the present invention can be utilized as an effective therapeutic agent for SARS-CoV-2.
서열 정보sequence information
이름name 서열order 서열번호SEQ ID NO:
ACE2 wild type(full sequence)ACE2 wild type (full sequence) UniProtKB seq ID : Q9BYF1UniProtKB seq ID: Q9BYF1 1One
ACE2 wild type (M1~D615)ACE2 wild type (M1~D615) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 22
ACE2.V.06 (H34A)ACE2.V.06 (H34A) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 33
ACE2.V.07 (H34V)ACE2.V.07 (H34V) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNVEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNVEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 44
ACE2.V.08 (S19W)ACE2.V.08 (S19W) MSSSSWLLLSLVAVTAAQWTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQWTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 55
ACE2.V.09 (S19P)ACE2.V.09 (S19P) MSSSSWLLLSLVAVTAAQPTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQPTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 66
ACE2.V.10 (T27D)ACE2.V.10 (T27D) MSSSSWLLLSLVAVTAAQSTIEEQAKDFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKDFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 77
ACE2.V.11 (T27Y)ACE2.V.11 (T27Y) MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 88
ACE2.V.12 (T27L)ACE2.V.12 (T27L) MSSSSWLLLSLVAVTAAQSTIEEQAKLFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKLFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 99
ACE2.V.13 (T27C)ACE2.V.13 (T27C) MSSSSWLLLSLVAVTAAQSTIEEQAKCFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKCFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 1010
ACE2.V.14 (D30V)ACE2.V.14 (D30V) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLVKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLVKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 1111
ACE2.V.15 (E35V)ACE2.V.15 (E35V) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHVAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHVAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 1212
ACE2.V.16 (L79Y)ACE2.V.16 (L79Y) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTYAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTYAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 1313
ACE2.V.17 (L79V)ACE2.V.17 (L79V) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 1414
ACE2.V.18 (Q325P)ACE2.V.18 (Q325P) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTPGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTPGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 1515
ACE2.V.19 (N330Y)ACE2.V.19 (N330Y) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 1616
ACE2.V.20 (N330F)ACE2.V.20 (N330F) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEFSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEFSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 1717
ACE2.V.21 (S19P, T27L, H34A, L79V, N330Y)ACE2.V.21 (S19P, T27L, H34A, L79V, N330Y) MSSSSWLLLSLVAVTAAQPTIEEQAKLFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQPTIEEQAKLFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 1818
ACE2.V.22 (S19P, T27L, D30V, H34A, E35V, L79V, Q325P, N330Y)ACE2.V.22 (S19P, T27L, D30V, H34A, E35V, L79V, Q325P, N330Y) MSSSSWLLLSLVAVTAAQPTIEEQAKLFLVKFNAVAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTPGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQPTIEEQAKLFLVKFNAVAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTPGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 1919
ACE2.V.23 (N330H)ACE2.V.23 (N330H) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEHSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEHSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 2020
ACE2.V.24 (H34A, N330Y)ACE2.V.24 (H34A, N330Y) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 2121
ACE2.V.25 (H34V, N330H)ACE2.V.25 (H34V, N330H) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNVEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEHSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNVEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEHSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 2222
ACE2.V.26 (T27Y, N330Y)ACE2.V.26 (T27Y, N330Y) MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 2323
ACE2.V.27 (T27Y, H34A)ACE2.V.27 (T27Y, H34A) MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 2424
ACE2.V.28 (T27Y, H34A, N330Y)ACE2.V.28 (T27Y, H34A, N330Y) MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 2525
ACE2.V.29 (H34V, N330Y)ACE2.V.29 (H34V, N330Y) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNVEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNVEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 2626
ACE2.V.30 (K26N)ACE2.V.30 (K26N) MSSSSWLLLSLVAVTAAQSTIEEQANTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQANTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 2727
ACE2.V.31 (K31R)ACE2.V.31 (K31R) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDRFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDRFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 2828
ACE2.V.32 (L91P)ACE2.V.32 (L91P) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 2929
ACE2.V.33 (K26N, K31R, L91P)ACE2.V.33 (K26N, K31R, L91P) MSSSSWLLLSLVAVTAAQSTIEEQANTFLDRFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQANTFLDRFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 3030
ACE2.V.34 (R514Q)ACE2.V.34 (R514Q) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIQYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIQYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 3131
ACE2.V.35 (L79I)ACE2.V.35 (L79I) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTIAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTIAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 3232
ACE2.V.36 (T27Y, H34A, L79Y)ACE2.V.36 (T27Y, H34A, L79Y) MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTYAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTYAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 3333
ACE2.V.37 (T27Y, H34A, L79Y, N330Y)ACE2.V.37 (T27Y, H34A, L79Y, N330Y) MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTYAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTYAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 3434
ACE2.V.38 (T27Y, H34A, L79V, N330Y)ACE2.V.38 (T27Y, H34A, L79V, N330Y) MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 3535
ACE2.V.39 (T27D, H34A, L79V, N330Y)ACE2.V.39 (T27D, H34A, L79V, N330Y) MSSSSWLLLSLVAVTAAQSTIEEQAKDFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKDFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 3636
ACE2.V.40 (T27Y, H34A, L79Y, L91P, N330Y)ACE2.V.40 (T27Y, H34A, L79Y, L91P, N330Y) MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTYAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTYAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 3737
ACE2.V.41 (T27Y, H34A, L79V, L91P, N330Y)ACE2.V.41 (T27Y, H34A, L79V, L91P, N330Y) MSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 3838
ACE2.V.42 (T27D, H34A, L79V, L91P, N330Y)ACE2.V.42 (T27D, H34A, L79V, L91P, N330Y) MSSSSWLLLSLVAVTAAQSTIEEQAKDFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQSTIEEQAKDFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 3939
ACE2.V.43 (S19P, T27D, H34A, L79V, N330Y)ACE2.V.43 (S19P, T27D, H34A, L79V, N330Y) MSSSSWLLLSLVAVTAAQPTIEEQAKDFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQPTIEEQAKDFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 4040
ACE2.V.44 (S19P, T27D, H34A, L79V, L91P, N330Y)ACE2.V.44 (S19P, T27D, H34A, L79V, L91P, N330Y) MSSSSWLLLSLVAVTAAQPTIEEQAKDFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQPTIEEQAKDFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 4141
ACE2.V.45 (S19P, T27Y, H34A, L79Y, N330Y)ACE2.V.45 (S19P, T27Y, H34A, L79Y, N330Y) MSSSSWLLLSLVAVTAAQPTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTYAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQPTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTYAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 4242
ACE2.V.46 (S19P, T27Y, H34A, L79Y, L91P, N330Y)ACE2.V.46 (S19P, T27Y, H34A, L79Y, L91P, N330Y) MSSSSWLLLSLVAVTAAQPTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTYAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQPTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTYAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 4343
ACE2.V.47 (S19P, T27Y, H34A, L79V, N330Y)ACE2.V.47 (S19P, T27Y, H34A, L79V, N330Y) MSSSSWLLLSLVAVTAAQPTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQPTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 4444
ACE2.V.48 (S19P, T27Y, H34A, L79V, L91P, N330Y)ACE2.V.48 (S19P, T27Y, H34A, L79V, L91P, N330Y) MSSSSWLLLSLVAVTAAQPTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADMSSSSWLLLSLVAVTAAQPTIEEQAKYFLDKFNAEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTVAQMYPLQEIQNPTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWEYSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD 4545
human IgG1 Fchuman IgG1 Fc DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKKNQVSLTCLDWSDNGQPQSDIKVYTLPPSRDELTKKNQVSLTCLDWSDNGQFQSDIKFSKSPVWSDNGWFFN 4646
human IgG1 Fc variants(L234A, L235A, K322A)human IgG1 Fc variants (L234A, L235A, K322A) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKKKNQVSLTCLDWSDNGQPQSDIKVYTLPPSRDELTKKNQVSLTCLDWSDNGQFQSDIKFSDKVVSDNGGFFYPHDKFSDKVESRDGPFNWYTKSKSVVESRDGGFN 4747
G4S linkerG4S linker GSGS 4848
point mutation 라이브러리 제조에 사용된 프라이머Primers used to prepare the point mutation library
Primer명Primer name 서열(5'-3')sequence (5'-3') 서열 번호SEQ ID NO:
pYD5 1FpYD5 1F TACATTTTCAATTAAGATGCAGTTTACATTTTCAATTAAGATGCAGTT 4949
pYD5 2RpYD5 2R ATCCACCACCACCCGTAGAATCGAATCCACCACCACCCGTAGAATCGA 5050
>ACE2#19_2F>ACE2#19_2F GTTATTGCTAGCGTTTTAGCACAG NNK ACAATAGAGGAGCAAGCTAAAACCGTTATTGCTAGCGTTTTAGCACAG NNK ACAATAGAGGAGCAAGCTAAAACC 5151
>ACE2#19_1R>ACE2#19_1R CTGTGCTAAAACGCTAGCAATAACCTGTGCTAAAACGCTAGCAATAAC 5252
>ACE2#24_2F>ACE2#24_2F TTAGCACAGTCCACAATAGAGGAG NNK GCTAAAACCTTTTTGGACAAGTTTTTAGCACAGTCCACAATAGAGGAG NNK GCTAAAACCTTTTTGGACAAGTTTT 5353
>ACE2#24_1R>ACE2#24_1R CTCCTCTATTGTGGACTGTGCTAACTCCTCTATTGTGGACTGTGCTAA 5454
>ACE2#27_2F>ACE2#27_2F TCCACAATAGAGGAGCAAGCTAAA NNK TTTTTGGACAAGTTTAATCATGAATCCACAATAGAGGAGCAAGCTAAA NNK TTTTTGGACAAGTTTAATCATGAA 5555
>ACE2#27_1R>ACE2#27_1R TTTAGCTTGCTCCTCTATTGTGGATTTAGCTTGCTCCTCTATTGTGGA 5656
>ACE2#28_2F>ACE2#28_2F ACAATAGAGGAGCAAGCTAAAACC NNK TTGGACAAGTTTAATCATGAAGCCACAATAGAGGAGCAAGCTAAAACC NNK TTGGACAAGTTTAATCATGAAGCC 5757
>ACE2#28_1R>ACE2#28_1R GGTTTTAGCTTGCTCCTCTATTGTGGTTTTAGCTTGCTCCTCTATTGT 5858
>ACE2#30_2F>ACE2#30_2F GAGGAGCAAGCTAAAACCTTTTTG NNK AAGTTTAATCATGAAGCCGAAGATGAGGAGCAAGCTAAAACCTTTTTG NNK AAGTTTAATCATGAAGCCGAAGAT 5959
>ACE2#30_1R>ACE2#30_1R CAAAAAGGTTTTAGCTTGCTCCTCCAAAAAGGTTTTAGCTTGCTCCTC 6060
>ACE2#31_2F>ACE2#31_2F GAGCAAGCTAAAACCTTTTTGGAC NNK TTTAATCATGAAGCCGAAGATTTGGAGCAAGCTAAAACCTTTTTGGAC NNK TTTAATCATGAAGCCGAAGATTTG 6161
>ACE2#31_1R>ACE2#31_1R GTCCAAAAAGGTTTTAGCTTGCTCGTCCAAAAAGGTTTTAGCTTGCTC 6262
>ACE2#34_2F>ACE2#34_2F AAAACCTTTTTGGACAAGTTTAAT NNK GAAGCCGAAGATTTGTTCTATCAAAAAACCTTTTTGGACAAGTTTAAT NNK GAAGCCGAAGATTTGTTCTATCAA 6363
>ACE2#34_1R>ACE2#34_1R ATTAAACTTGTCCAAAAAGGTTTTATTAAACTTGTCCAAAAAGGTTTT 6464
>ACE2#35_2F>ACE2#35_2F ACCTTTTTGGACAAGTTTAATCAT NNK GCCGAAGATTTGTTCTATCAATCAACCTTTTTGGACAAGTTTAATCAT NNK GCCGAAGATTTGTTCTATCAATCA 6565
>ACE2#35_1R>ACE2#35_1R ATGATTAAACTTGTCCAAAAAGGTATGATTAAACTTGTCCAAAAAGGT 6666
>ACE2#37_2F>ACE2#37_2F TTGGACAAGTTTAATCATGAAGCC NNK GATTTGTTCTATCAATCATCTCTGTTGGACAAGTTTAATCATGAAGCC NNK GATTTGTTCTATCAATCATCTCTG 6767
>ACE2#37_1R>ACE2#37_1R GGCTTCATGATTAAACTTGTCCAAGGCTTCATGATTAAACTTGTCCAA 6868
>ACE2#38_2F>ACE2#38_2F GACAAGTTTAATCATGAAGCCGAA NNK TTGTTCTATCAATCATCTCTGGCGGACAAGTTTAATCATGAAGCCGAA NNK TTGTTCTATCAATCATCTCTGGCG 6969
>ACE2#38_1R>ACE2#38_1R TTCGGCTTCATGATTAAACTTGTCTTCGGCTTCATGATTAAACTTGTC 7070
>ACE2#41_2F>ACE2#41_2F AATCATGAAGCCGAAGATTTGTTC NNK CAATCATCTCTGGCGTCCTGGAACAATCATGAAGCCGAAGATTTGTTC NNK CAATCATCTCTGGCGTCCTGGAAC 7171
>ACE2#41_1R>ACE2#41_1R GAACAAATCTTCGGCTTCATGATTGAACAAATCTTCGGCTTCATGATT 7272
>ACE2#42_2F>ACE2#42_2F CATGAAGCCGAAGATTTGTTCTAT NNK TCATCTCTGGCGTCCTGGAACTACCATGAAGCCGAAGATTTGTTCTAT NNK TCATCTCTGGCGTCCTGGAACTAC 7373
>ACE2#42_1R>ACE2#42_1R ATAGAACAAATCTTCGGCTTCATGATAGAACAAATCTTCGGCTTCATG 7474
>ACE2#45_2F>ACE2#45_2F GAAGATTTGTTCTATCAATCATCT NNK GCGTCCTGGAACTACAACACAAACGAAGATTTGTTCTATCAATCATCT NNK GCGTCCTGGAACTACAACACAAAC 7575
>ACE2#45_1R>ACE2#45_1R AGATGATTGATAGAACAAATCTTCAGATGATTGATAGAACAAATCTTC 7676
>ACE2#79_2F>ACE2#79_2F GCGTTCCTAAAGGAGCAATCAACG NNK GCACAGATGTATCCTCTACAAGAAGCGTTCCTAAAGGAGCAATCAACG NNK GCACAGATGTATCCTCTACAAGAA 7777
>ACE2#79_1R>ACE2#79_1R CGTTGATTGCTCCTTTAGGAACGCCGTTGATTGCTCCTTTAGGAACGC 7878
>ACE2#82_2F>ACE2#82_2F AAGGAGCAATCAACGTTAGCACAG NNK TATCCTCTACAAGAAATTCAAAACAAGGAGCAATCAACGTTAGCACAG NNK TATCCTCTACAAGAAATTCAAAAC 7979
>ACE2#82_1R>ACE2#82_1R CTGTGCTAACGTTGATTGCTCCTTCTGTGCTAACGTTGATTGCTCCTT 8080
>ACE2#83_2F>ACE2#83_2F GAGCAATCAACGTTAGCACAGATG NNK CCTCTACAAGAAATTCAAAACCTTGAGCAATCAACGTTAGCACAGATG NNK CCTCTACAAGAAATTCAAAACCTT 8181
>ACE2#83_1R>ACE2#83_1R CATCTGTGCTAACGTTGATTGCTCCATCTGTGCTAACGTTGATTGCTC 8282
>ACE2#325_2F>ACE2#325_2F TCCGTCGGCTTGCCGAATATGACC NNK GGATTCTGGGAAAATAGCATGTTATCCGTCGGCTTGCCGAATATGACC NNK GGATTCTGGGAAAATAGCATGTTA 8383
>ACE2#325_1R>ACE2#325_1R GGTCATATTCGGCAAGCCGACGGAGGTCATATTCGGCAAGCCGACGGA 8484
>ACE2#329_2F>ACE2#329_2F CCGAATATGACCCAAGGATTCTGG NNK AATAGCATGTTAACGGATCCGGGTCCGAATATGACCCAAGGATTCTGG NNK AATAGCATGTTAACGGATCCGGGT 8585
>ACE2#329_1R>ACE2#329_1R CCAGAATCCTTGGGTCATATTCGGCCAGAATCCTTGGGTCATATTCGG 8686
>ACE2#330_2F>ACE2#330_2F AATATGACCCAAGGATTCTGGGAA NNK AGCATGTTAACGGATCCGGGTAATAATATGACCCAAGGATTCTGGGAA NNK AGCATGTTAACGGATCCGGGTAAT 8787
>ACE2#330_1R>ACE2#330_1R TTCCCAGAATCCTTGGGTCATATTTTCCCAGAATCCTTGGGTCATATT 8888
>ACE2#353_2F>ACE2#353_2F CACCCAACTGCGTGGGACTTAGGC NNK GGCGATTTTAGGATTCTTATGTGCCACCCAACTGCGTGGGACTTAGGC NNK GGCGATTTTAGGATTCTTATGTGC 8989
>ACE2#353_1R>ACE2#353_1R GCCTAAGTCCCACGCAGTTGGGTGGCCTAAGTCCCACGCAGTTGGGTG 9090
>ACE2#354_2F>ACE2#354_2F CCAACTGCGTGGGACTTAGGCAAA NNK GATTTTAGGATTCTTATGTGCACCCCAACTGCGTGGGACTTAGGCAAA NNK GATTTTAGGATTCTTATGTGCACC 9191
>ACE2#354_1R>ACE2#354_1R TTTGCCTAAGTCCCACGCAGTTGGTTTGCCTAAGTCCCACGCAGTTGG 9292
>ACE2#355_2F>ACE2#355_2F ACTGCGTGGGACTTAGGCAAAGGC NNK TTTAGGATTCTTATGTGCACCAAAACTGCGTGGGACTTAGGCAAAGGC NNK TTTAGGATTCTTATGTGCACCAAA 9393
>ACE2#355_1R>ACE2#355_1R GCCTTTGCCTAAGTCCCACGCAGTGCCTTTTGCCTAAGTCCCACGCAGT 9494
>ACE2#357_2F>ACE2#357_2F TGGGACTTAGGCAAAGGCGATTTT NNK ATTCTTATGTGCACCAAAGTAACATGGGACTTAGGCAAAGGCGATTTT NNK ATTCTTATGTGCACCAAAGTAACA 9595
>ACE2#357_1R>ACE2#357_1R AAAATCGCCTTTGCCTAAGTCCCAAAAATCGCCTTTGCCTAAGTCCCA 9696
>ACE2#25_2F>ACE2#25_2F GCACAGTCCACAATAGAGGAGCAA NNK AAAACCTTTTTGGACAAGTTTAATGCACAGTCCACAATAGAGGAGCAA NNK AAAACCTTTTTGGACAAGTTTAAT 9797
>ACE2#25_1R>ACE2#25_1R TTGCTCCTCTATTGTGGACTGTGCTTGCTCCTCTATTGTGGACTGTGC 9898
>ACE2#42_1F>ACE2#42_1F CATGAAGCCGAAGATTTGTTCTAT NNK TCATCTCTGGCGTCCTGGAACTACCATGAAGCCGAAGATTTGTTCTAT NNK TCATCTCTGGCGTCCTGGAACTAC 9999
>ACE2#42_1R>ACE2#42_1R ATAGAACAAATCTTCGGCTTCATGATAGAACAAATCTTCGGCTTCATG 100100
>ACE2#92_2F>ACE2#92_2F CCTCTACAAGAAATTCAAAACCTT NNK GTAAAATTGCAGTTACAAGCGTTACCTCTACAAGAAATTCAAAACCTT NNK GTAAAATTGCAGTTACAAGCGTTA 101101
>ACE2#92_1R>ACE2#92_1R AAGGTTTTGAATTTCTTGTAGAGGAAGGTTTTGAATTTCTTGTAGAGG 102102
>ACE2#386_2F>ACE2#386_2F CACATCCAATATGACATGGCCTAC NNK GCGCAGCCCTTTCTACTGAGGAACCACATCCAATATGACATGGCCTAC NNK GCGCAGCCCTTTCTACTGAGGAAC 103103
>ACE2#386_1R>ACE2#386_1R GTAGGCCATGTCATATTGGATGTGGTAGGCCATGTCATATTGGATGTG 104104
* NNK 는 NNK codon을 의미함* NNK means NNK codon
융합단백질 제조에 사용된 프라이머Primers used to prepare fusion proteins
Primer명Primer name 서열정보sequence information 서열 번호SEQ ID NO:
CmV ForwardCmV Forward CGC AAA TGG GCG GTA GGC GTGCGC AAA TGG GCG GTA GGC GTG 105105
pcDNA3.3 reversepcDNA3.3 reverse CCG TGC GTT TTA TTC TGT CTT TTT ATT GCCCCG TGC GTT TTA TTC TGT CTT TTT ATT GCC 106106
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby It will be obvious. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (23)

  1. 높은 코로나바이러스 결합 친화도 및 코로나바이러스 중화능을 가지는 안지오텐신 변환 효소 II(ACE2) 변이체.Angiotensin converting enzyme II (ACE2) variant with high coronavirus binding affinity and coronavirus neutralizing ability.
  2. 제1항에 있어서, 서열번호 2에서 S19, K26, T27, D30, K31, H34, E35, N49, K74, L79, L91, V185, N250, S280, Q325, N330, G448, R514 및 S602로 구성된 군에서 선택되는 어느 하나 이상의 아미노산에 변이를 가지는 안지오텐신 변환 효소 II(ACE2) 변이체.The group of claim 1, wherein in SEQ ID NO: 2, S19, K26, T27, D30, K31, H34, E35, N49, K74, L79, L91, V185, N250, S280, Q325, N330, G448, R514 and S602. Angiotensin converting enzyme II (ACE2) variant having a mutation in any one or more amino acids selected from.
  3. 제1항에 있어서, 서열번호 2에서 S19, T27, D30, H34, E35, L79, Q325 및 N330 아미노산에 변이를 가지는 안지오텐신 변환 효소 II(ACE2) 변이체.The angiotensin converting enzyme II (ACE2) variant according to claim 1, wherein the amino acids S19, T27, D30, H34, E35, L79, Q325 and N330 in SEQ ID NO: 2 are mutations.
  4. 제1항에 있어서, 서열번호 2에서 S19, T27, D30, H34, E35, L79, Q325 및 N330 아미노산에 변이; 및The method of claim 1, wherein in SEQ ID NO: 2, S19, T27, D30, H34, E35, L79, Q325 and N330 amino acids; and
    K26, K31, K74, L91, V185, N250 및 G448로 구성된 군에서 선택되는 어느 하나 이상의 아미노산에 변이를 가지는 안지오텐신 변환 효소 II(ACE2) 변이체.Angiotensin converting enzyme II (ACE2) variant having a mutation in any one or more amino acids selected from the group consisting of K26, K31, K74, L91, V185, N250 and G448.
  5. 제1항에 있어서, 서열번호 2에서 H34 아미노산에 변이; 및The method according to claim 1, wherein in SEQ ID NO: 2, a mutation in amino acid H34; and
    N49, L79, L91, S280 및 S602로 구성된 군에서 선택되는 어느 하나 이상의 아미노산에 변이를 가지는 안지오텐신 변환 효소 II(ACE2) 변이체.Angiotensin converting enzyme II (ACE2) variant having a mutation in any one or more amino acids selected from the group consisting of N49, L79, L91, S280 and S602.
  6. 제1항에 있어서,The method of claim 1,
    서열번호 2에서 S19W 또는 S19P; S19W or S19P in SEQ ID NO:2;
    K26N; K26N;
    T27W, T27D, T27A, T27Y, T27L, T27M 또는 T27C; T27W, T27D, T27A, T27Y, T27L, T27M or T27C;
    D30V; D30V;
    K31R; K31R;
    H34A 또는 H34V; H34A or H34V;
    E35V; E35V;
    N49D; N49D;
    K74R; K74R;
    L79I, L79Y 또는 L79V; L79I, L79Y or L79V;
    L91P; L91P;
    V185A; V185A;
    N250K; N250K;
    S280R; S280R;
    Q325P; Q325P;
    N330Y, N330H 또는 N330F; N330Y, N330H or N330F;
    G448V; G448V;
    R514Q; 및 R514Q; and
    S602T로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 안지오텐신 변환 효소 II(ACE2) 변이체.Angiotensin converting enzyme II (ACE2) variant having one or more mutations selected from the group consisting of S602T.
  7. 제1항에 있어서, 서열번호 2에서 S19W 또는 S19P, T27L, D30V, H34A, E35V, L79V, Q325P 및 N330Y로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 안지오텐신 변환 효소 II(ACE2) 변이체.According to claim 1, S19W or S19P, T27L, D30V, H34A, E35V, L79V, Q325P and angiotensin converting enzyme II (ACE2) variant having at least one mutation selected from the group consisting of N330Y in SEQ ID NO: 2.
  8. 제1항에 있어서, According to claim 1,
    서열번호 2에서 H34A의 변이; 및mutation of H34A in SEQ ID NO: 2; and
    N49D, L79I, L91P, S280R 및 S602T로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 안지오텐신 변환 효소 II(ACE2) 변이체.Angiotensin converting enzyme II (ACE2) variant having any one or more mutations selected from the group consisting of N49D, L79I, L91P, S280R and S602T.
  9. 제1항에 있어서,According to claim 1,
    서열번호 1에서 상기 안지오텐신 변환 효소 II 변이체는 서열번호 1에서 S19W 또는 S19P, T27L, D30V, H34A, E35V, L79V, Q325P 및 N330Y의 변이; 및In SEQ ID NO: 1, the angiotensin converting enzyme II variant is S19W or S19P, T27L, D30V, H34A, E35V, L79V, Q325P and N330Y in SEQ ID NO: 1; and
    K26N, K31R, K74R, L91P, V185A, N250K 및 G448V로 구성된 군에서 선택되는 어느 하나 이상의 변이를 가지는 안지오텐신 변환 효소 II(ACE2) 변이체.Angiotensin converting enzyme II (ACE2) variant having any one or more mutations selected from the group consisting of K26N, K31R, K74R, L91P, V185A, N250K and G448V.
  10. 제1항에 있어서, S19P, T27Y, H34A, L79Y, L91P 및 N330Y로 구성된 군에서 선택되는 셋 이상의 변이를 가지는 안지오텐신 변환 효소 II(ACE2) 변이체.The angiotensin converting enzyme II (ACE2) variant according to claim 1, having three or more mutations selected from the group consisting of S19P, T27Y, H34A, L79Y, L91P and N330Y.
  11. 제1항에 있어서, S19P, T27Y, H34A, L79V, L91P 및 N330Y로 구성된 군에서 선택되는 셋 이상의 변이를 가지는 안지오텐신 변환 효소 II(ACE2) 변이체.The angiotensin converting enzyme II (ACE2) variant according to claim 1, having three or more mutations selected from the group consisting of S19P, T27Y, H34A, L79V, L91P and N330Y.
  12. 제1항에 있어서, 서열번호 3 내지 서열번호 45로 구성된 군에서 선택되는 서열을 포함하는 안지오텐신 변환 효소 II(ACE2) 변이체.According to claim 1, wherein the angiotensin converting enzyme II (ACE2) variant comprising a sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 45.
  13. 제1항에 있어서, 서열번호 43으로 표시되는 서열을 포함하는 안지오텐신 변환 효소 II(ACE2) 변이체.The angiotensin converting enzyme II (ACE2) variant according to claim 1, comprising the sequence represented by SEQ ID NO: 43.
  14. 제1항 내지 제13항 중 어느 한 항의 안지오텐신 변환 효소 II(ACE2) 변이체를 포함하는 융합단백질.A fusion protein comprising an angiotensin converting enzyme II (ACE2) variant according to any one of claims 1 to 13.
  15. 제14항에 있어서, Fc 도메인을 추가로 포함하는 것을 특징으로 하는 융합단백질.The fusion protein according to claim 14, further comprising an Fc domain.
  16. 제15항에 있어서, 상기 Fc 도메인은 Fcγ 수용체(FcγR) 결합 친화도가 감소된 것을 특징으로 하는 융합단백질.The fusion protein according to claim 15, wherein the Fc domain has reduced Fcγ receptor (FcyR) binding affinity.
  17. 제15항에 있어서, 상기 Fc 도메인은 서열번호 46 또는 서열번호 47로 표시되는 서열을 포함하는 것을 특징으로 하는 융합단백질.The fusion protein according to claim 15, wherein the Fc domain comprises a sequence represented by SEQ ID NO: 46 or SEQ ID NO: 47.
  18. 제1항 내지 제13항 중 어느 한 항의 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 코딩하는 핵산.Claims 1 to 13 of any one of the angiotensin converting enzyme II variant or a nucleic acid encoding a fusion protein comprising the same.
  19. 제18항의 핵산을 포함하는 재조합 벡터.A recombinant vector comprising the nucleic acid of claim 18 .
  20. 제18항의 핵산 또는 이를 포함하는 재조합 벡터가 숙주세포에 도입된 재조합세포.A recombinant cell into which the nucleic acid of claim 18 or a recombinant vector containing the same is introduced into a host cell.
  21. 제20항의 재조합세포를 배양하여 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 발현하는 단계; 및Culturing the recombinant cell of claim 20 to express an angiotensin converting enzyme II mutant or a fusion protein comprising the same; and
    상기 발현된 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 수득하는 단계를 포함하는 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질의 제조방법Method for producing an angiotensin converting enzyme II variant or a fusion protein comprising the same, comprising obtaining the expressed angiotensin converting enzyme II variant or a fusion protein comprising the same
  22. 제1항 내지 제13항 중 어느 한 항의 안지오텐신 변환 효소 II 변이체 또는 이를 포함하는 융합단백질을 포함하는 코로나바이러스 감염증의 예방 또는 치료용 약학 조성물.Claims 1 to 13 of any one of the angiotensin converting enzyme II variant or a pharmaceutical composition for the prevention or treatment of coronavirus infection comprising a fusion protein comprising the same.
  23. 제22항에 있어서, 상기 코로나바이러스 감염증은 SARS-CoV-2 바이러스 또는 이의 변종 바이러스 감염증(COVID-19)인 것을 특징으로 하는 약학 조성물.The pharmaceutical composition according to claim 22, wherein the coronavirus infection is SARS-CoV-2 virus or a variant virus infection (COVID-19) thereof.
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