Mutant ACE2 protein

Abstract

To provide a mutant ACE2 protein having higher bindability to an S protein of a coronavirus. A mutant ACE2 protein including an amino acid sequence A represented by SEQ ID NO: 1 or an amino acid sequence C obtained by mutating an amino acid sequence B having 70% or greater identity with the amino acid sequence A, the amino acid sequence C including a substitution ax of at least two amino acids selected from the group consisting of T20, A25, K26, K31, H34, E35, Q60, N64, S70, T78, L79, N90, T92, and Q101 in the amino acid sequence A or a substitution ay corresponding to the substitution ax in the amino acid sequence B.

Description

[Technical Field]

[0001] The present invention relates to mutant ACE2 proteins and the like. [Background technology]

[0002] Coronaviruses are a species of virus belonging to the Coronavirinae subfamily of the Coronaviridae family. They are positive-strand RNA viruses that infect humans and animals, causing respiratory, gastrointestinal, vascular, or neurological diseases. Coronaviruses can spread from infected host animals and cause large-scale human epidemics. Recent examples of coronaviruses include SARS coronavirus 1 (SARS-CoV-1), which caused epidemics in 2002 and 2003, and Middle East respiratory syndrome coronavirus (MERS-CoV). Recently, a global pandemic of SARS coronavirus 2 (SARS-CoV-2) has occurred, and with no established treatment, it continues to cause significant damage in various areas, including healthcare and the economy.

[0003] Antibodies that can specifically bind to coronaviruses are thought to be useful for treatment, but there is a problem of resistance. On the other hand, ACE2 (Angiotensin-converting enzyme 2) is known to be present on the cell membrane and to be involved in the entry of coronaviruses into cells by binding to the S protein on the surface of coronaviruses, and its use in treatment has been reported (Non-Patent Document 1). ACE2 is thought to be useful for treatment because it is less likely to develop resistance. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Science 13 Mar 2020: Vol. 367, Issue 6483, pp. 1260-1263. Summary of the Invention [Problem to be solved by the invention]

[0005] Wild-type ACE2 binds less to the coronavirus S protein than antibodies.

[0006] Therefore, an objective of the present invention is to provide a mutant ACE2 protein that has higher binding affinity to coronavirus S protein. [Means for solving the problem]

[0007] As a result of intensive research conducted in view of the above problems, the present inventor has discovered the following: The inventors have found that the above-mentioned problems can be solved by a mutant ACE2 protein comprising an amino acid sequence A shown in SEQ ID NO: 1, or an amino acid sequence C obtained by mutating an amino acid sequence B having 70% or more identity to the amino acid sequence A, wherein the amino acid sequence C comprises a mutation a: a substitution ax of at least two amino acids selected from the group consisting of T20, A25, K26, K31, H34, E35, Q60, N64, S70, T78, L79, N90, T92, and Q101 in the amino acid sequence A, or a substitution ay corresponding to the substitution ax in the amino acid sequence B. Based on this finding, the inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0008] Item 1. A method for producing a nucleic acid sequence comprising: a) an amino acid sequence A shown in SEQ ID NO: 1; or b) an amino acid sequence C obtained by mutating an amino acid sequence B having 70% or more identity to the amino acid sequence A; the amino acid sequence C includes a mutation a: a substitution ax of at least two amino acids selected from the group consisting of T20, A25, K26, K31, H34, E35, Q60, N64, S70, T78, L79, N90, T92, and Q101 in the amino acid sequence A, or a substitution ay corresponding to the substitution ax in the amino acid sequence B; Mutant ACE2 protein.

[0009] Item 2. The mutant ACE2 protein according to Item 1, wherein the substitution ax comprises a substitution of at least two amino acids selected from the group consisting of A25, K26, K31, E35, N64, L79, N90, and T92.

[0010] Item 3. The mutant ACE2 protein according to Item 2, wherein the substitutions ax include substitutions at A25, K31, and E35.

[0011] Item 4. The mutant ACE2 protein according to Item 2 or 3, wherein the substitutions ax are substitutions of at least five amino acids.

[0012] Item 5. The mutant ACE2 protein according to any one of Items 2 to 4, wherein the substitution ax is a substitution of seven types of amino acids.

[0013] Item 6. The mutant ACE2 protein according to any one of Items 2 to 5, wherein, in the mutation a, the amino acid after the mutation at A25 is a bulkier hydrophobic amino acid, the amino acid after the mutation at K26 is a non-basic amino acid, the amino acid after the mutation at K31 is a non-basic amino acid, the amino acid after the mutation at E35 is a non-acidic amino acid, the amino acid after the mutation at N64 is a hydrophobic amino acid, the amino acid after the mutation at L79 is a bulkier hydrophobic amino acid, and / or the amino acid after the mutation at N90 is a basic amino acid or an acidic amino acid.

[0014] Item 7. The mutant ACE2 protein according to any one of Items 2 to 6, wherein, in the mutation a, the amino acid after the mutation at A25 is a branched-chain amino acid, the amino acid after the mutation at K26 is an acidic amino acid, the amino acid after the mutation at K31 is a hydrophilic neutral amino acid, the amino acid after the mutation at E35 is a basic amino acid, the amino acid after the mutation at N64 is a branched-chain amino acid, the amino acid after the mutation at L79 is an aromatic amino acid, and / or the amino acid after the mutation at N90 is a basic amino acid.

[0015] Item 8. The mutant ACE2 protein according to any one of Items 2 to 7, wherein, in the mutation a, the amino acid after the mutation at A25 is valine, the amino acid after the mutation at K26 is glutamic acid, the amino acid after the mutation at K31 is asparagine, the amino acid after the mutation at E35 is lysine, the amino acid after the mutation at N64 is isoleucine, the amino acid after the mutation at L79 is phenylalanine, and / or the amino acid after the mutation at N90 is histidine.

[0016] Item 9. The mutant ACE2 protein according to Item 1, wherein the substitution ax comprises substitution of at least two amino acids selected from the group consisting of K31, E35, Q60, S70, L79, and N90, or substitution of at least two amino acids selected from the group consisting of T20, A25, H34, T78, T92, and Q101.

[0017] Item 10. The mutant ACE2 protein according to Item 1 or 9, wherein the substitutions ax include substitutions of K31, E35, Q60, and L79, or substitutions of T20, H34, T92, and Q101H.

[0018] Item 11. The mutant ACE2 protein according to any one of Items 1 to 10, wherein the amino acid sequence B is the amino acid sequence B1 shown in SEQ ID NO: 2, all or a part of the amino acid sequence B2 shown in SEQ ID NO: 3, or an orthologous amino acid sequence B3 of the amino acid sequence A, B1, or B2.

[0019] Item 12. The mutant ACE2 protein according to any one of Items 1 to 11, further comprising the entire or partial amino acid sequence of an antibody.

[0020] Item 13. A complex comprising a plurality of mutant ACE2 proteins according to any one of Items 1 to 12.

[0021] Item 14. A polynucleotide comprising a coding sequence for the mutant ACE2 protein according to any one of Items 1 to 12.

[0022] Item 15. A cell comprising the polynucleotide according to Item 14.

[0023] Item 16. A pharmaceutical comprising at least one selected from the group consisting of the mutant ACE2 protein according to any one of Items 1 to 12, the complex according to Item 13, the polynucleotide according to Item 14, and the cell according to Item 15.

[0024] Item 17. A reagent comprising at least one selected from the group consisting of the mutant ACE2 protein according to any one of Items 1 to 12, the complex according to Item 13, the polynucleotide according to Item 14, and the cell according to Item 15.

[0025] Item 18. An anti-coronavirus agent comprising at least one selected from the group consisting of the mutant ACE2 protein of any one of Items 1 to 12, the complex of Item 13, the polynucleotide of Item 14, and the cell of Item 15. [Effects of the Invention]

[0026] According to the present invention, a mutant ACE2 protein having higher binding affinity to the S protein of coronavirus can be provided. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1 shows the results of the pseudovirus neutralization assay in Test Example 2. In the legend, N-39 indicates the mutant ACE2 protein. [Figure 2] 1 shows the results of the authentic virus neutralization assay (viral RNA measurement results) of Test Example 3. 3N-39 indicates the mutant ACE2 protein. [Figure 3] 1 shows the results of the authentic virus neutralization assay (virus titer measurement results) of Test Example 3. 3N-39 indicates the mutant ACE2 protein. [Figure 4]1 shows the results of the authentic virus neutralization assay (viral RNA measurement results) of Test Example 7. [Figure 5] a: An overview of the method used in Test Example 9. b: The results of analyzing the number of SARS-CoV-2 genomic RNA copies in the culture medium at each passage are shown. At each passage, virus from the indicated well (arrowhead) was added to the well of the next passage of cells. Growth of resistant strains was observed only with the H4 antibody at passage 4. For 3J113v2, no virus growth was observed at passage 2, so the test was discontinued. c: The results of analysis at passage 15 are shown. [Figure 6] a: Schematic diagram of the animal experiment in Test Example 10. b: Calculation results of the weight change rate from day 0 for all hamsters. c: Axial CT images of the thorax 5 days after infection. d: Quantification results of viral titers (plaque-forming units; PFU) and SARS-CoV-2 genomic RNA copy numbers in lung homogenates. The vertical axis indicates the viral load as PFU per gram of lung homogenate and the viral genomic RNA copy number per gram of lung tissue RNA. e and f: (e) H&E staining and (f) SARS-CoV-2 antigen staining of hamster lung lobes. Scale bars, 100 μm (upper panel) and 40 μm (lower panel). g: mRNA expression of inflammatory cytokines or chemotactic cytokines in hamster lung lobes. b, d, and g) Data are means ± SEM for n = 4. P values ​​by two-tailed unpaired t-test. *P < 0.05, **P < 0.01. [Figure 7] 1 shows a comparison of protein yields in Test Example 11. [Figure 8] 1 shows the results of the pseudovirus neutralization assay in Test Example 11. [Figure 9] 1 shows a comparison of protein yields in Test Example 12. [Figure 10] The measurement results of neutralizing activity against pseudo SARS-CoV-2 in Test Example 12 are shown. [Figure 11] 1 shows the measurement results of neutralizing activity against SARS-CoV-2 in Test Example 12. [Figure 12]1 shows the measurement results of ACE2 enzyme activity in Test Example 12. [Figure 13] 1 shows the pharmacokinetic measurement results of Test Example 12. [Figure 14] 1 shows the results of measuring the viral load in lung tissue in Test Example 12. [Figure 15] 1 shows the results of measuring the expression levels of inflammatory cytokines in lung tissue in Test Example 12. DETAILED DESCRIPTION OF THE INVENTION

[0028] 1. Definition etc. In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0029] As used herein, the "identity" of an amino acid sequence refers to the degree of correspondence between two or more comparable amino acid sequences. Therefore, the greater the identity between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity between amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc. Natl. Acad. Sci. USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc. Natl. Acad. Sci. USA. 90:5873-7 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known, and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of a nucleotide sequence is also defined in accordance with the above. As used herein, "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, a conservative substitution occurs when amino acid residues having basic side chains, such as lysine, arginine, and histidine, are substituted with each other.Conservative substitutions also include substitutions of amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with uncharged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with nonpolar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0030] As used herein, nucleotides such as DNA and RNA may be chemically modified as described below. To prevent degradation by hydrolases such as nucleases, the phosphate residue of each nucleotide may be substituted with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, for example by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include, but are not limited to, those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. Also preferably used are BNA (LNA), in which the conformation of the sugar moiety is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety of the nucleotide.

[0031] As used herein, amino acid mutation specifically refers to amino acid deletion, substitution, insertion, or addition.

[0032] Herein, the position of an amino acid in an amino acid sequence may be indicated by the single-letter amino acid code plus the amino acid number counted from the N-terminal amino acid. For example, "A25" indicates alanine, the 25th amino acid from the N-terminus. Note that, herein, the amino acid number is the amino acid number counted from the N-terminus of human ACE2 (1-615), the amino acid sequence shown in SEQ ID NO: 1. Thus, for example, A25 in human ACE2 (18-615: SEQ ID NO: 2) is the alanine in SEQ ID NO: 2, which corresponds to the alanine that is the 25th amino acid from the N-terminus of SEQ ID NO: 1, and is the 8th amino acid from the N-terminus of SEQ ID NO: 2.

[0033] 2. Mutant ACE2 protein In one aspect, the present invention relates to a mutant ACE2 protein (sometimes referred to herein as the "mutant ACE2 protein of the present invention") that comprises amino acid sequence A shown in SEQ ID NO: 1 or amino acid sequence C obtained by mutating amino acid sequence B having 70% or more identity to amino acid sequence A, wherein amino acid sequence C comprises mutation a: substitution ax of at least two amino acids selected from the group consisting of T20, A25, K26, K31, H34, E35, Q60, N64, S70, T78, L79, N90, T92, and Q101 in amino acid sequence A, or substitution ay corresponding to substitution ax in amino acid sequence B. This will be explained below.

[0034] The amino acid sequence C is not particularly limited as long as it is an amino acid sequence obtained by mutating the amino acid sequence A shown in SEQ ID NO: 1 or the amino acid sequence B having 70% or more identity to the amino acid sequence A, and contains the mutation a.

[0035] Amino acid sequence A (SEQ ID NO: 1) is a sequence consisting of the 1st amino acid to the 615th amino acid from the N-terminus of the amino acid sequence (SEQ ID NO: 3) of the ACE2 (Angiotensin-converting enzyme 2) protein (NCBI Accession Numbers: NP_001358344 and NP_068576) endogenously expressed (intrinsically possessed) by humans (Homo sapiens).

[0036] The amino acid sequence B is a sequence obtained by introducing mutation a into the sequence (protein B), which is a protein having a K activity against coronavirus S protein. D The value of K is the value of the protein (protein A) consisting of a sequence in which mutation a is introduced into amino acid sequence A. D There are no particular limitations as long as it is not significantly higher than the value of the K value of protein B. D The K value of protein A D The identity of amino acid sequence B to amino acid sequence A is, for example, 10 times or less, preferably 5 times or less, more preferably 3 times or less, even more preferably 2 times or less, still more preferably 1.5 times or less, and particularly preferably 1.2 times or less, relative to the value. From this viewpoint, the identity of amino acid sequence B to amino acid sequence A is preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more, and is less than 100%.

[0037] K against coronavirus S protein D The value can be measured according to the binding assay method described in the Examples below.

[0038] Examples of amino acid sequence B include amino acid sequence B1 (SEQ ID NO: 2), which is obtained by removing the signal peptide sequence (the sequence consisting of the first to 17th amino acids from the N-terminus) from amino acid sequence A; amino acid sequence B2, which is all or a part of the amino acid sequence of human ACE2 protein (SEQ ID NO: 3) (for example, a sequence consisting of any of amino acids 1 to 20 from the N-terminus to 520 to 805 (preferably 550 to 805, more preferably 580 to 805, and even more preferably 600 to 805) of SEQ ID NO: 3, preferably a sequence consisting of amino acids 18 to 740 from the N-terminus of SEQ ID NO: 3 (SEQ ID NO: 12)); and orthologous amino acid sequence B3 of amino acid sequence A, B1, or B2 (for example, the amino acid sequence of ACE2 protein in animals other than humans (e.g., mammals such as monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer), or a part thereof), and intraspecies variants thereof. The amino acid sequence of the ACE2 protein of animals other than humans is publicly known, or can be predicted from genomic information of a known amino acid sequence, or can be identified by cloning based on a known amino acid sequence.

[0039] Mutation a is a substitution ax of at least two amino acids selected from the group consisting of T20, A25, K26, K31, H34, E35, Q60, N64, S70, T78, L79, N90, T92, and Q101 in amino acid sequence A, or a substitution ay corresponding to the substitution ax in amino acid sequence B.

[0040] Note that "corresponding substitution" refers to a substitution of an amino acid at the same position in the aligned sequences when two sequences are compared using BLAST (default settings). For example, a substitution corresponding to the substitution of A25 in amino acid sequence A refers to a substitution of an amino acid at the same position in amino acid sequence B as A25 in amino acid sequence A in the aligned sequences obtained by comparing the sequences of amino acid sequence A and amino acid sequence B. Furthermore, the amino acid after substitution in amino acid sequence A and the amino acid after substitution in amino acid sequence B are the same.

[0041] The substitution ax preferably comprises at least three amino acid substitutions, i.e., at least three amino acids selected from the group consisting of T20, A25, K26, K31, H34, E35, Q60, N64, S70, T78, L79, N90, T92, and Q101 in the amino acid sequence A. Similarly, the substitution ax more preferably comprises at least four amino acid substitutions, even more preferably at least five amino acid substitutions, even more preferably at least six amino acid substitutions, and especially preferably at least seven amino acid substitutions.

[0042] The substitution ax preferably includes a substitution of at least two amino acids (substitution ax1) selected from the group consisting of A25, K26, K31, E35, N64, L79, N90, and T92.

[0043] It is particularly preferred that substitution ax1 includes substitutions at A25, K31, and E35. Among these, it is particularly preferred that substitution ax1 includes substitutions at A25, K31, E35, and T92, or substitutions at A25, K31, E35, and L79.

[0044] The substitution ax1 is preferably a substitution of at least three amino acids, i.e., a substitution of at least three amino acids selected from the group consisting of A25, K26, K31, E35, N64, L79, N90, and T92 in the amino acid sequence A. Similarly, the substitution ax1 is more preferably a substitution of at least four amino acids, even more preferably a substitution of at least five amino acids, even more preferably a substitution of at least six amino acids, and particularly preferably a substitution of seven amino acids (A25, K26, K31, E35, N64, L79, and N90).

[0045] The substitution ax preferably includes substitution of at least two amino acids (substitution ax2) selected from the group consisting of K31, E35, Q60, S70, L79, and N90.

[0046] Preferably, substitution ax2 includes substitutions of K31, E35, and Q60. It is especially preferred that substitution ax2 includes substitutions of K31, E35, Q60, and L79.

[0047] The substitution ax2 is preferably a substitution of at least three amino acids, i.e., a substitution of at least three amino acids selected from the group consisting of K31, E35, Q60, S70, L79, and N90 in the amino acid sequence A. Similarly, the substitution ax2 is more preferably a substitution of at least four amino acids, even more preferably a substitution of at least five amino acids, and even more preferably a substitution of six amino acids.

[0048] The substitution ax preferably includes substitutions of at least two amino acids selected from the group consisting of T20, A25, H34, T78, T92, and Q101 (substitution ax3). It is particularly preferred that the substitution ax3 includes substitutions of T20, H34, T92, and Q101H. Furthermore, the substitution ax3 preferably includes substitutions of A25, H34, T78, and T92.

[0049] The substitution ax3 is preferably a substitution of at least three amino acids, i.e., a substitution of at least three amino acids selected from the group consisting of T20, A25, H34, T78, T92, and Q101 in the amino acid sequence A. Similarly, the substitution ax3 is more preferably a substitution of at least four amino acids, even more preferably a substitution of at least five amino acids, and even more preferably a substitution of six amino acids.

[0050] The amino acid after mutation of T20 or a corresponding amino acid in mutation a is preferably a hydrophobic amino acid. Examples of hydrophobic amino acids include valine, leucine, isoleucine, glycine, alanine, proline, tryptophan, phenylalanine, and methionine. Among these, branched-chain amino acids such as valine, leucine, and isoleucine are preferred, and isoleucine is more preferred.

[0051] The amino acid after mutation of A25 or a corresponding amino acid in mutation a is preferably a bulkier hydrophobic amino acid. In this specification, "bulkier" refers to a larger molecular weight of the side chain (for example, addition or substitution of an alkyl group (methyl group, ethyl group, etc.) in the side chain, substitution of a chain structure of the side chain (for example, an alkyl group) with an aromatic group, an increase in the number of rings in the aromatic group in the side chain, etc.), and is not particularly limited thereto. Examples of bulkier hydrophobic amino acids include valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine, etc. Among these, branched-chain amino acids such as valine, leucine, and isoleucine are preferred, and valine is more preferred.

[0052] The amino acid after mutation of K26 or a corresponding amino acid in mutation a is preferably a non-basic amino acid. Examples of non-basic amino acids include glutamic acid, aspartic acid, serine, threonine, glutamine, asparagine, tyrosine, cysteine, glycine, alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, and methionine. Among these, preferred are non-basic hydrophilic amino acids such as glutamic acid, aspartic acid, serine, threonine, glutamine, asparagine, tyrosine, and cysteine, more preferred are acidic amino acids such as glutamic acid and aspartic acid, and even more preferred is glutamic acid.

[0053] The amino acid after the mutation of K31 or a corresponding amino acid in mutation a is preferably a non-basic amino acid. Examples of non-basic amino acids include glutamic acid, aspartic acid, serine, threonine, glutamine, asparagine, tyrosine, cysteine, glycine, alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, and methionine. In one embodiment of the present invention (particularly in substitution ax1), among these non-basic amino acids, preferred are non-basic hydrophilic amino acids such as glutamic acid, aspartic acid, serine, threonine, glutamine, asparagine, tyrosine, and cysteine, more preferred are hydrophilic neutral amino acids such as serine, threonine, glutamine, asparagine, tyrosine, and cysteine, even more preferred are asparagine and glutamine, and even more preferred is asparagine. In one embodiment of the present invention (particularly in substitution ax2), among the above non-basic amino acids, preferred are hydrophobic amino acids such as valine, leucine, isoleucine, glycine, alanine, proline, tryptophan, phenylalanine, and methionine, more preferably methionine.

[0054] The amino acid after mutation of H34 or the amino acid corresponding thereto in mutation a is preferably a non-basic amino acid. Examples of non-basic amino acids include glutamic acid, aspartic acid, serine, threonine, glutamine, asparagine, tyrosine, cysteine, glycine, alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, and methionine. Among these, preferred are hydrophobic amino acids such as glycine, alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, and methionine, more preferred are glycine, alanine, valine, leucine, and isoleucine, and even more preferred is alanine.

[0055] The amino acid after mutation of E35 or a corresponding amino acid in mutation a is preferably a non-acidic amino acid. Examples of non-acidic amino acids include lysine, arginine, histidine, serine, threonine, glutamine, asparagine, tyrosine, cysteine, glycine, alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, and methionine. Among these, preferred are non-acidic hydrophilic amino acids such as lysine, arginine, histidine, serine, threonine, glutamine, asparagine, tyrosine, and cysteine, more preferred are basic amino acids such as lysine, arginine, and histidine, and even more preferred is lysine.

[0056] The amino acid after mutation of Q60 or a corresponding amino acid in mutation a is preferably a basic amino acid, such as lysine, arginine, histidine, etc., preferably arginine.

[0057] The amino acid after mutation of N64 or a corresponding amino acid in mutation a is preferably a hydrophobic amino acid. Examples of hydrophobic amino acids include valine, leucine, isoleucine, glycine, alanine, proline, tryptophan, phenylalanine, and methionine. Among these, branched-chain amino acids such as valine, leucine, and isoleucine are preferred, and isoleucine is more preferred.

[0058] The amino acid after mutation of S70 or the corresponding amino acid in mutation a is preferably a hydrophobic amino acid. Examples of hydrophobic amino acids include valine, leucine, isoleucine, glycine, alanine, proline, tryptophan, phenylalanine, and methionine. Among these, aromatic amino acids such as tryptophan and phenylalanine are preferred, and phenylalanine is more preferred.

[0059] The amino acid after mutation of T78 or a corresponding amino acid in mutation a is preferably a basic amino acid, such as lysine, arginine, histidine, etc., preferably arginine.

[0060] The amino acid after mutation of L79 in mutation a or a corresponding amino acid thereto is preferably a bulkier hydrophobic amino acid. Examples of bulkier hydrophobic amino acids include isoleucine, proline, tryptophan, and phenylalanine. Among these, aromatic amino acids such as tryptophan and phenylalanine are preferred, and phenylalanine is more preferred.

[0061] The amino acid after mutation of N90 or the amino acid corresponding thereto in mutation a is preferably a charged amino acid. Examples of charged amino acids include lysine, arginine, histidine, aspartic acid, and glutamic acid. In one embodiment of the present invention (particularly in substitution ax1), among these charged amino acids, preferred are basic amino acids such as lysine, arginine, and histidine, more preferred are histidine. In one embodiment of the present invention (particularly in substitution ax2), among the above charged amino acids, preferred are acidic amino acids such as aspartic acid and glutamic acid, more preferred are aspartic acid.

[0062] The amino acid after mutation of T92 in mutation a or a corresponding amino acid is preferably an amino acid having a carbamoyl group in the side chain, such as glutamine or asparagine. Of these, glutamine is more preferred.

[0063] The amino acid after mutation of Q101 or a corresponding amino acid in mutation a is preferably a basic amino acid. Examples of basic amino acids include lysine, arginine, and histidine. Among these, histidine is preferred.

[0064] The amino acid sequence C may contain other amino acid mutations (e.g., substitutions, conservative substitutions) as long as the binding to the coronavirus S protein is not significantly impaired. The number of other amino acid mutations is, for example, 1 to 50, 1 to 20, 1 to 10, or 1 to 5.

[0065] The amino acid sequence C preferably contains mutations to fix the amino acid sequence in a closed conformation. The mutations are typically substitutions of two non-cysteine ​​amino acids with cysteines. The mutation positions can be appropriately determined based on the crystal structure of ACE2. Examples of such mutations include a combination of S128C and V343C, or a combination of V59C and N121C.

[0066] Specific examples of amino acid sequence C include amino acid sequence C1 shown in SEQ ID NO: 4 to 6, and amino acid sequence C2 having 80% or more identity to amino acid sequence C1 (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, particularly preferably 99% or more, and less than 100%).

[0067] The mutant ACE2 protein of the present invention may have an amino acid sequence other than amino acid sequence C added thereto, such as a protein tag, a fluorescent protein, a luminescent protein, a secretory signal sequence (e.g., Igκ signal sequence), a protease recognition sequence (e.g., TEV protease recognition sequence), or other signal sequence, as long as its binding ability to coronavirus S protein is not significantly impaired. Examples of protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, and PA tag.

[0068] In a preferred embodiment, the mutant ACE2 protein of the present invention comprises the entire or partial amino acid sequence of an antibody. Examples of antibodies include, but are not limited to, IgA, IgD, IgE, IgG, and IgM, as well as subclasses thereof. The origin of the antibody is also not particularly limited, and may be, for example, a human-derived antibody, a mouse-derived antibody, a rat-derived antibody, a rabbit-derived antibody, a monkey-derived antibody, or a chimpanzee-derived antibody. When using a partial amino acid sequence of an antibody, the amino acid sequence preferably comprises an Fc region, which allows the effector function to be exerted. Furthermore, the mutant ACE2 protein of the present invention can be linked via the Fc region to form a multimer. From this perspective, in one embodiment, the present invention relates to a complex (sometimes referred to herein as the "complex of the present invention") comprising multiple (e.g., two, three, four, or five) mutant ACE2 proteins of the present invention.

[0069] The mutant ACE2 protein of the present invention may be linked to a drug, as long as its binding ability to coronavirus S protein is not significantly impaired.

[0070] The mutant ACE2 protein of the present invention may be chemically modified as long as its binding ability to the coronavirus S protein is not significantly impaired.

[0071] The mutant ACE2 protein of the present invention has a C-terminal carboxyl group (-COOH), a carboxylate group (-COO - ), amide (-CONH2) or ester (-COOR).

[0072] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1-6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3-8 Cycloalkyl groups such as phenyl and α-naphthyl 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2Alkyl groups; α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0073] In the mutant ACE2 protein of the present invention, a carboxyl group (or carboxylate) other than that at the C-terminus may be amidated or esterified, such as the C-terminal esters described above.

[0074] Furthermore, in the mutant ACE2 protein of the present invention, the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). 1-6 C such as alkanoyl 1-6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); 1-6 C such as alkanoyl group 1-6 Also included are those protected by an acyl group or other suitable glycan, or conjugated proteins such as glycoproteins to which sugar chains are attached.

[0075] The mutant ACE2 protein of the present invention may be in the form of a salt with an acid or a base. The salt is not particularly limited, and both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0076] The mutant ACE2 protein of the present invention may be in the form of a solvate. The solvent is not particularly limited, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0077] The mutant ACE2 protein of the present invention can be easily produced using known genetic engineering techniques, such as PCR, restriction enzyme digestion, DNA ligation, in vitro transcription / translation, and recombinant protein production techniques. Recombinant protein production techniques include, for example, methods using cultured cells as hosts, as well as methods using plants such as tobacco.

[0078] 3. Polynucleotides, cells In one aspect, the present invention relates to a polynucleotide comprising a coding sequence for a mutant ACE2 protein of the present invention (also referred to herein as a "polynucleotide of the present invention") and a cell comprising a polynucleotide of the present invention (also referred to herein as a "cell of the present invention"), which are described below.

[0079] The coding sequence of the mutant ACE2 protein of the present invention is not particularly limited, as long as it is a polynucleotide consisting of a base sequence that encodes the mutant ACE2 protein of the present invention.

[0080] In one embodiment, the polynucleotide of the present invention comprises an expression cassette for the mutant ACE2 protein of the present invention.

[0081] The expression cassette for the mutant ACE2 protein of the present invention is not particularly limited as long as it is a polynucleotide that can express the mutant ACE2 protein of the present invention in cells. Typical examples of the expression cassette for the mutant ACE2 protein of the present invention include a polynucleotide comprising a promoter and a coding sequence for the mutant ACE2 protein of the present invention placed under the control of the promoter.

[0082] The promoter contained in the expression cassette of the mutant ACE2 protein of the present invention is not particularly limited and can be appropriately selected depending on the target cell. For example, various Pol II promoters can be used. Pol II promoters are not particularly limited, but examples include the CMV promoter, EF1 promoter, SV40 promoter, and MSCV promoter. Other examples of promoters include tryptophan promoters such as trc and tac, lac promoters, T7 promoters, T5 promoters, T3 promoters, SP6 promoters, arabinose-inducible promoters, cold shock promoters, and tetracycline-inducible promoters.

[0083] The expression cassette for the mutant ACE2 protein of the present invention may contain other elements (e.g., a multiple cloning site (MCS), a drug resistance gene, an origin of replication, an enhancer sequence, a repressor sequence, an insulator sequence, a reporter protein (e.g., a fluorescent protein), a drug resistance gene coding sequence, etc.) as needed.

[0084] The polynucleotide of the present invention may be in the form of a vector. An appropriate vector is selected depending on the intended use (cloning, protein expression) and the type of host cell. Examples of vectors using E. coli as a host include M13 phage or modified versions thereof, λ phage or modified versions thereof, and pBR322 or modified versions thereof (pB325, pAT153, pUC8, etc.); examples of vectors using yeast as a host include pYepSec1, pMFa, pYES2, and pPIC3.5K; examples of vectors using insect cells as a host include pAc and pVL; and examples of vectors using mammalian cells as a host include pcDNA, pCDM8, and pMT2PC.

[0085] The cells of the present invention are not particularly limited as long as they contain the polynucleotide of the present invention. Examples of cells include Escherichia coli such as Escherichia coli K12, Bacillus bacteria such as Bacillus subtilis MI114, yeast such as Saccharomyces cerevisiae AH22, the Sf cell line derived from Spodoptera frugiperda or the HighFive cell line derived from Trichoplusia ni, insect cells such as olfactory nerve cells, and animal cells such as COS7 cells. Preferred animal cells include cultured cells derived from mammals, specifically COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, Hela cells, PC12 cells, N1E-115 cells, SH-SY5Y cells, etc.

[0086] In one embodiment, the cell of the present invention expresses the mutant ACE2 protein of the present invention, for example, the cell of the present invention secretes the mutant ACE2 protein of the present invention or has the mutant ACE2 protein of the present invention on the cell surface.

[0087] 4.Applications The mutant ACE2 protein of the present invention can bind to the S protein of coronaviruses. Therefore, at least one selected from the group consisting of the mutant ACE2 protein of the present invention, the complex of the present invention, the polynucleotide of the present invention, and the cell of the present invention can be used as an active ingredient of medicines, reagents, etc. (sometimes referred to herein as "drugs of the present invention"), more specifically, as an active ingredient of antiviral agents, etc.

[0088] The coronaviruses targeted by the agents of the present invention are not particularly limited as long as they belong to the Orthocoronavirus subfamily. Examples of coronaviruses include the Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus genera, with Betacoronavirus being preferred. Examples of Betacoronavirus include SARS-related coronavirus (SARSr-CoV), coronavirus HKU1, and MERS coronavirus, with SARSr-CoV being preferred. Examples of SARSr-CoV include SARS-CoV-2 and SARS-CoV-1, with SARS-CoV-2 being preferred. The agents of the present invention are particularly suitable for use against SARS-CoV-2. Furthermore, they can be used against various strains of SARS-CoV-2, including the Wuhan strain, alpha strain, delta strain, and lambda strain.

[0089] The agent of the present invention can exert an antiviral effect against coronaviruses. Specific examples of the antiviral effect include the effect of suppressing viral infection, the effect of suppressing viral cell death, the effect of inactivating viruses, the effect of suppressing viral proliferation, the effect of suppressing viral budding, and the effect of inducing viral resistance. Furthermore, due to this effect, the agent can also be used as a preventive or therapeutic agent for coronavirus infections (particularly COVID-19).

[0090] The pharmaceutical preparation of the present invention is not particularly limited as long as it contains the above-mentioned active ingredient, and may further contain other ingredients as necessary. The other ingredients are not particularly limited as long as they are pharmaceutically acceptable. The other ingredients include not only ingredients with pharmacological effects but also additives. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0091] The mode of use of the agent of the present invention is not particularly limited, and an appropriate mode of use can be adopted depending on the type of agent. Depending on the intended use, the agent of the present invention can be used, for example, in vitro (e.g., added to the medium of cultured cells) or in vivo (e.g., administered to animals).

[0092] The agents of the present invention may be applied to, but are not limited to, mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Examples of cells include animal cells. The types of cells are also not particularly limited, and examples include blood cells, hematopoietic stem cells and progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0093] The agents of the present invention may take any dosage form, for example, oral preparations such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jellies; and parenteral preparations such as injectable preparations (for example, drip injections (e.g., intravenous drip preparations), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (for example, ointments, poultices, and lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, and liposomes.

[0094] The route of administration of the drug of the present invention is not particularly limited as long as the desired effect can be obtained, and examples include enteral administration such as oral administration, tube feeding, and enema administration; and parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

[0095] The content of the active ingredient in the drug of the present invention depends on the mode of use, the subject to which it is applied, the condition of the subject to which it is applied, etc., and is not limited thereto, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0096] The dosage of the agent of the present invention when administered to an animal is not particularly limited as long as it is an effective amount that exerts a pharmacological effect, and is usually 0.1 to 1000 mg / kg body weight, preferably 0.5 to 500 mg / kg body weight per day in terms of the weight of the active ingredient, in the case of oral administration, and 0.01 to 100 mg / kg body weight, preferably 0.05 to 50 mg / kg body weight per day in the case of parenteral administration. The dosage can be increased or decreased as appropriate depending on the age, pathological condition, symptoms, etc. [Example]

[0097] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0098] Example 1. Preparation of expression vector for mutant ACE2 protein 1 An expression vector (derived from p3xFLAG-CMV-14: mutant ACE2 expression vector 1) containing the coding sequence (sequence number 9) of a mutant ACE2 protein (fusion protein) consisting of, from the N-terminus, an Igκ signal sequence (sequence number 7), a sequence (sequence number 5) in which seven mutations (A25V, K26E, K31N, E35K, N64I, L79F, N90H) have been introduced into human ACE2 (18-615), an amino acid sequence (SR) encoded by an XbaI recognition sequence, and the sequence of the human IgG1 Fc region (sequence number 8) was constructed using known subcloning techniques.

[0099] Example 2. Preparation of expression vector for mutant ACE2 protein 2 An expression vector (derived from pcDNA3.1: mutant ACE2 expression vector 2) containing the coding sequence (sequence number 11) of a mutant ACE2 protein (fusion protein) consisting of, from the N-terminus, an Igκ signal sequence (sequence number 7), a sequence (sequence number 5) in which seven mutations have been introduced into human ACE2 (18-615), an amino acid sequence (SR) encoded by the XbaI recognition sequence, and a group of tag sequences (TEV protease recognition sequence-G-His tag-Myc tag-His tag: sequence number 10) was constructed using known subcloning techniques.

[0100] Comparative Example 1. Preparation of wild-type ACE2 protein expression vector 1 Wild-type ACE2 expression vector 1 was obtained in the same manner as in Example 1, except that the sequence of SEQ ID NO: 2 (wild-type human ACE2 (18-615)) was used instead of SEQ ID NO: 5.

[0101] Comparative Example 2. Preparation of wild-type ACE2 protein expression vector 2 Wild-type ACE2 expression vector 2 was obtained in the same manner as in Example 2, except that the sequence of SEQ ID NO: 2 (wild-type human ACE2 (18-615)) was used instead of SEQ ID NO: 5.

[0102] Example 3. Expression and purification of ACE2 protein 1 ACE2 protein was expressed and purified using mutant ACE2 expression vector 1 (Example 1) and wild-type ACE2 expression vector 1 (Comparative Example 1). Specifically, the procedure was as follows. Protein synthesis was performed using the Expi29™ Expression System (manufacturer: Thermo Fisher Scientific, catalog number: A14635). First, the vector plasmid was transfected into Expi293F cells using Expifectamine. The enhancer was added the following day, and the culture supernatant was collected 5 days later. For purification, 20 mL of the culture supernatant was centrifuged at 4°C and 4000 g for 10 minutes and passed through a 0.45 μm filter. 2 mL of Protein A Sepharose (manufacturer: GE Healthcare, catalog number: 17-1279-03) and 200 μL of 1 M Tris-HCl pH 9.0 were added, and the mixture was mixed on a rotator at room temperature for 2 hours. The eluate was then loaded into a mini-column, washed four times with 10 mL of PBS, eluted with 1 mL of IgG Elute Buffer (manufacturer: Thermo Fisher Scientific, catalog number: 21004), and neutralized with 0.1 mL of 1 M Tris-HCl pH 9.0. The eluate was dialyzed against PBS and concentrated to 1-2 mg / mL using an ultrafiltration device, Amicon Ultra-4 (30K MWCO) (manufacturer: Merck Millipore, catalog number: UFC803024), and then frozen and stored at -80°C.

[0103] Example 4. Expression and purification of ACE2 protein 2 ACE2 protein was expressed and purified using mutant ACE2 expression vector 2 (Example 2) and wild-type ACE2 expression vector 2 (Comparative Example 2). Specifically, the procedure was as follows. Protein synthesis was performed using the Expi293™ Expression System (manufacturer: Thermo Fisher Scientific, catalog number: A14635). First, the vector plasmid was transfected into Expi293F cells using Expifectamine. The enhancer was added the following day, and the culture supernatant was collected five days later. For purification, 20 mL of the culture supernatant was centrifuged at 4°C and 4000 g for 10 minutes and passed through a 0.45 μm filter. 1 mL of Ni-NTA agarose (manufacturer: Qiagen, catalog number: 30230) was added, and the mixture was mixed on a rotator at room temperature for 2 hours. The mixture was then packed into a mini-column and washed four times with 5 mL of wash buffer (20 mM Tris-HCl, pH 7.5, 0.3 M NaCl, 20 mM imidazole). The eluate was then eluted with 1 mL of elution buffer (20 mM Tris-HCl, pH 7.5, 0.3 M NaCl, 250 mM imidazole). The eluate was dialyzed against PBS and concentrated to 1–2 mg / mL using an Amicon Ultra-4 (30K MWCO) ultrafiltration device (manufacturer: Merck Millipore, catalog number: UFC803024). The concentrate was then frozen and stored at −80°C.

[0104] Test example 1. Binging assay The ACE2 protein obtained in Example 4 was used to analyze its binding to SARS-CoV-2 S protein Spike RBD-SD1-Fc (a purified protein in which human IgG1 Fc is fused to the C-terminus of the 319-591 region of the SARS-CoV-2 spike protein) using a molecular interaction analyzer (Biacore T200). The analysis conditions were as follows: RBD-SD1-Fc was immobilized on a Sensor Chip CM5 (manufacturer: GE Healthcare, catalog number: BR100530) using a Human Antibody Capture Kit (manufacturer: GE Healthcare, catalog number: BR100838). An equal amount of human Fc alone was immobilized on a control flow cell. The ACE2 protein obtained in Example 4 was diluted with Running Buffer (PBS containing 0.05% Tween 20) to concentrations of 0.1 to 300 nM, and this was passed over the flow cell at a flow rate of 30 μL / min. The association time was set to 120 seconds per concentration and analyzed in Single-Cycle kinetics mode. Binding kinetics were calculated using Biacore T200 evaluation software version 4.1. As a result, the dissociation constant (K D ) is 41.4 nM, whereas the dissociation constant (K D ) was 0.37 nM.

[0105] Test Example 2: Pseudovirus neutralization assay The ACE2 protein obtained in Example 3 was used to measure the neutralizing activity against pseudo-SARS-CoV-2 expressing the SARS-CoV-2 S protein. Specifically, the procedure was as follows. A total of three pseudovirus plasmids, pLenti Firefly, psPAX2, and pcDNA4 SARS2-Spike, were transfected into Lenti-X 293 cells (manufacturer: Clontech, catalog number: 632180) using FuGENE HD (manufacturer: Promega, catalog number: E2311). The virus solution secreted into the culture supernatant after 48 hours was used. VeroE6 / TMPRSS2 cells (manufacturer: National Institute of Infectious Diseases, registration number: JCRB1819) were used as infected cells. 50 μL of the virus solution was mixed with 50 μL of the ACE2 protein obtained in Example 3, which had been serially diluted 1 / 3 starting from 10 μg / mL, and the mixture was allowed to stand for 1 hour. The mixture was then added to VeroE6 / TMPRSS2 cells seeded in a 96-well plate. After 1 hour of incubation, the medium was replaced and luciferase activity was measured 48 hours later. Luciferase activity was measured using the Dual-Glo Luciferase Assay System (manufacturer: Promega, catalog number: E2920) on a plate reader (Tecan Infinite F200). Cells incubated with virus alone were used as the 100% infection control, and cells alone were used as the 0% infection control. The inhibition rate was calculated from the signal ratio.

[0106] The results are shown in Figure 1. As shown in Figure 1, the mutant ACE2 protein exhibited much higher neutralizing activity than the wild-type ACE2 protein.

[0107] Test Example 3.Authentic virus neutralization assay 1 The neutralizing activity against SARS-CoV-2 was measured using the ACE2 protein obtained in Example 3. Specifically, the procedure was as follows: 80,000 VeroE6 / TMPRSS2 cells (manufacturer: National Institute of Infectious Diseases, registration number: JCRB1819) were seeded onto a 24-well plate, and the cells were infected with SARS-CoV-2 at an moi of 0.1 together with the modified ACE2 protein. After 2 hours, the cells were washed with PBS and the medium was replaced. After 22 hours, the viral RNA of the cultured cells was measured by qPCR, and the viral titer in the supernatant was measured by plaque assay.

[0108] The results are shown in Figures 2 and 3. As shown in Figures 2 and 3, the mutant ACE2 protein exhibited much higher neutralizing activity than the wild-type ACE2 protein.

[0109] Test Example 4: Identification of essential amino acids A mutant ACE2 protein containing a sequence (SEQ ID NO: 5) in which seven mutations were introduced into human ACE2 (18-615) was created by reverting some of the mutations to the wild type, and its binding to the SARS-CoV-2 spike protein was examined. Four mutations, A25V, K31N, E35K, and L79F, were found to be important. The dissociation constant (K D ) (measured using the same method as in Test Example 1) was 0.64 nM, and the IC 50 (Measurement method was the same as in Test Example 2) was 0.082 μg / ml.

[0110] Test Example 5. Analysis of other mutant ACE2 proteins 1 The binding of a mutant ACE2 protein containing a sequence in which six mutations (K31M, E35K, Q60R, S70F, L79F, N90D) have been introduced into human ACE2 (18-615) to the SARS-CoV-2 spike protein was examined, and it was found to exhibit high binding. Furthermore, a mutant ACE2 protein was created in which some of the mutations were reverted to the wild type, and its binding to the SARS-CoV-2 spike protein was examined, revealing that the three mutations, K31M, E35K, and Q60R (more preferably, four mutations, including L79F) are important. The dissociation constant (K D ) (measured in the same manner as in Test Example 1) was 1.14 nM, and the IC 50 (Measurement method was the same as in Test Example 2) was 0.33 μg / ml.

[0111] Test Example 6. Analysis of other mutant ACE2 proteins 2 The binding of a mutant ACE2 protein containing a sequence in which six mutations (T20I, A25V, H34A, T78R, T92Q, Q101H) were introduced into human ACE2 (18-615) to the SARS-CoV-2 spike protein was examined, and it was found to exhibit high binding. Furthermore, a mutant ACE2 protein was created in which some of the mutations were reverted to the wild type, and its binding to the SARS-CoV-2 spike protein was examined, revealing that the four mutations, T20I, H34A, T92Q, and Q101H, are important. The dissociation constant (K D ) (measured in the same manner as in Test Example 1) was 3.98 nM, and the IC 50 (Measurement method was the same as in Test Example 2) was 0.068 μg / ml.

[0112] Test Example 7.Authentic virus neutralization assay 2 The mutant ACE2 protein (3N39) contains a sequence in which seven mutations (A25V, K26E, K31N, E35K, N64I, L79F, N90H) have been introduced into human ACE2 (18-615), the mutant ACE2 protein (3N39v2) contains a sequence in which four mutations (A25V, K31N, E35K, L79F) have been introduced into human ACE2 (18-615), the mutant ACE2 protein (3J113v2) contains a sequence in which four mutations (K31M, E35K, Q60R, L79F) have been introduced into human ACE2 (18-615), and the mutant ACE2 protein (3J113v2) contains a sequence in which four mutations (T20I, H34A, T92Q, The neutralizing activity against SARS-CoV-2 of a mutant ACE2 protein (3J120v2) containing a sequence in which Q101H was introduced was measured in the same manner as in Test Example 3.

[0113] The results are shown in Figure 4. As shown in Figure 4, the mutant ACE2 protein exhibited much higher neutralizing activity than the wild-type ACE2 protein.

[0114] Test Example 8. Improving the stability of mutant ACE2 protein We prepared mutant ACE2 proteins (3N39, 3N39v2-SS) containing mutations (S128C and V343C) to lock them into a closed conformation. The mutant ACE2 proteins were then subjected to thermal shift assays. The specific method is as follows: 3N39 contains a mutant ACE2 protein (3N39v2) containing a sequence in which seven mutations (A25V, K26E, K31N, E35K, N64I, L79F, and N90H) were introduced into human ACE2 (18-615), and 3N39v2 contains a sequence in which four mutations (A25V, K31N, E35K, and L79F) were introduced into human ACE2 (18-615).

[0115] Purified mutant ACE2 protein was diluted to 200 μg / ml in PBS and placed in 0.2 mL white PCR tubes (Bio-Rad, TLS0851) at 20 μl per tube. After adding 1 μl of SYPR™ Orange protein gel stain (Invitrogen, S6651) diluted 1:150 in water per tube, the tubes were placed in a Bio-Rad CFX96 thermal cycler real-time detection system. Thermal denaturation curves from 25°C to 95°C (ramp rate of 1.27°C / min at 0.5°C intervals with 5 seconds of equilibration at each temperature before measurement) were acquired by measuring fluorescence intensity using the FRET channel with excitation from 450 to 490 nm and detection from 560 to 580 nm. All data were exported and plotted in Microsoft Excel, and Tm was calculated using the first derivative method.

[0116] As a result, the Tm of wild-type human ACE2 (18-615) increased from 44.5°C to 57°C by introducing mutations (S128C and V343C) to lock it into a closed conformation. Furthermore, the Tm of 3N39 increased from 44.5°C to 51.5°C by introducing mutations (S128C and V343C) to lock it into a closed conformation. Furthermore, the Tm of 3N39v2 increased from 44.0°C to 55.5°C by introducing mutations (S128C and V343C) to lock it into a closed conformation. Furthermore, the introduction of mutations (S128C and V343C) did not result in a decrease in neutralizing activity.

[0117] Test Example 9. Evaluation of the occurrence of resistant strains In antiviral therapy, a common concern is that pathogenic viruses may frequently mutate and acquire drug resistance. In fact, it has been reported that SARS-CoV-2 rapidly develops resistant strains when cultured with neutralizing antibodies. Therefore, we evaluated the development of resistant strains using SARS-CoV-2 virus.

[0118] The method is outlined in Figure 5a. In the first passage, a 0.1 MOI of virus was added to the culture medium of a dilution series of mutant ACE2-Fc or a recombinant monoclonal antibody (clone H4) isolated from a convalescent patient. A total of 3 x 10 viruses were amplified from partially neutralized wells. 5 The copies were transferred to the next passage (Fig. 5a). Supernatants were collected from each well and analyzed for viral RNA copy number by quantitative PCR.

[0119] Consistent with previous reports, the virus pool after treatment with a single antibody became insensitive to the highest concentration of the same antibody within four passages (Fig. 5b), and sequencing of the resistant isolates revealed that F490V inhibited H4 antibody binding. On the other hand, the mutant ACE2-Fc maintained a very high neutralizing activity against the virus pool even after 15 passages, indicating that no resistant isolates had emerged (Fig. 5c).

[0120] Test Example 10. Evaluation of therapeutic effect Four-week-old male Syrian hamsters were anesthetized with an intraperitoneal injection of 0.75 mg kg-1 medetomidine (Meiji Seika), 2 mg kg-1 midazolam (Sandoz), and 2.5 mg kg-1 butorphanol tartrate (Meiji Seika). 6 PFU of SARS-CoV-2 (in 60 μL) was administered intranasally. Two hours after infection, control-Fc (20 mg kg-1) or ACE2-Fc (3N39v2, 20 mg kg-1) was administered intraperitoneally. Body weight was monitored daily for 5 days. On day 5 after infection, all animals were euthanized, and lungs were collected for histopathological examination, virus titration, and qRT-PCR. Lung structure was observed using a micro-computed tomography (μCT) system (ScanXmate-RB080SS110, Comscantecno Co., Ltd.). μCT images were reconstructed and visualized using ImageJ software.

[0121] Hamsters administered control-Fc lost 4.3% of their body weight, while those administered 3N39v2-Fc gained 7.3% of their body weight 5 days after infection, similar to the control group (Figure 6b). Pre-necropsy micro-CT scans revealed multilobular ground-glass opacities, primarily on the cranial side, in the control group, whereas lung abnormalities were limited in the treatment group (Figure 6c). The SARS-CoV-2 content in the lungs was assessed by functional viral particles and genomic RNA copy number, revealing a significant reduction in both in the 3N39v2-Fc-treated group (Figure 6d). Histopathological analysis of infected hamsters was also performed. Control groups exhibited severe interstitial pneumonia characterized by inflammatory cell infiltration, alveolar septal thickening, and alveolar hemorrhage, whereas the 3N39v2-Fc-treated group showed a clear reduction in lung pathology and viral antigens (Figures 6e-f). Consistent with these results, the expression of inflammatory or chemotactic cytokines was reduced in the treatment group (Fig. 6g).

[0122] Test Example 11. Analysis of other mutant ACE2 proteins 3 The following six ACE2 proteins were produced: Human ACE2(18-615) ACE2 protein (615 WT), A mutant ACE2 protein (615 WT-S128C / V343C) containing a sequence in which mutations (S128C and V343C) have been introduced into human ACE2 (18-615) to fix it in a closed conformation, A mutant ACE2 protein (615 3N39v2) containing a sequence in which four mutations (A25V, K31N, E35K, L79F) have been introduced into human ACE2 (18-615), A mutant ACE2 protein (615 3N39v2-S128C / V343C) containing a sequence in which mutations (S128C and V343C) for fixing the ACE2 protein in a closed conformation have been introduced into 3N39v2. Human ACE2 (18-740) ACE2 protein (740 WT), and A mutant ACE2 protein (740 3N39v2-S128C / V343C) containing a sequence in which four mutations (A25V, K31N, E35K, L79F) and mutations (S128C and V343C) that fix the protein in a closed conformation have been introduced into human ACE2 (18-740).

[0123] The yields of these proteins were compared, and the results are shown in Figure 7.

[0124] In addition, the neutralizing activity against a pseudo-SARS-CoV-2 (SARS-CoV-2 pseudovirus) expressing the SARS-CoV-2 S protein was measured in the same manner as in Test Example 2. The results are shown in Figure 8. 50 is 0.064 μg / mL, IC of 615 3N39v-S128C / V343C 50 is 0.056 μg / mL, IC of 740 3N39v-S128C / V343C 50 was 0.033 μg / mL.

[0125] Test Example 12. Analysis of other mutant ACE2 proteins 4 As a result of immunogenicity testing of ACE2 amino acid mutations, it was found that elimination of the L79F mutation resulted in lower immunogenicity. Therefore, a mutant ACE2 protein (740 3N39v2-S128C / V343C (also referred to as 3N39v2-SS, 740-SS 3N39v2, or v2 in this test example), prepared in Test Example 11), containing a sequence in which four mutations (A25V, K31N, E35K, L79F) and mutations (S128C and V343C) for fixing the protein in a closed conformation were introduced into human ACE2 (18-740), was used to prepare a mutant ACE2 protein (A25V, K31N, E35K, S128C, V343C) (also referred to as 3N39v3-SS, 740-SS 3N39v3, or v3 in this test example) in which the F79F mutation was removed, and a mutant ACE2 protein (A25V, K31N, E35K, T92Q, S128C, V343C) (in this example, also referred to as 3N39v4-SS, 740-SS 3N39v4, v4, 3N39v4-SS(740)-Fc, 3N39v4-SS(740), or 3N39v4-Fc) was produced.

[0126] The yields of the above proteins were compared, and the results are shown in Figure 9.

[0127] Furthermore, the neutralizing activity of the above proteins against SARS-CoV-2 pseudoviruses expressing the SARS-CoV-2 (Wuhan strain, alpha strain, delta strain, or lambda strain) S protein was measured in the same manner as in Test Example 2. The results are shown in Figure 10.

[0128] Next, the neutralizing activity of the above proteins against SARS-CoV-2 (Wuhan strain, alpha strain, delta strain, or lambda strain) was measured in the same manner as in Test Example 3. The results are shown in Figure 11.

[0129] Subsequently, the thermal structural stability of the above proteins was measured in the same manner as in Test Example 8. As a result, the Tm of v2 was 56.5°C, the Tm of v3 was 58.0°C, and the Tm of v4 was 58.5°C.

[0130] Next, the ACE2 enzyme activity of the above proteins was measured. Specifically, this was done as follows. After reacting various mutant ACE2s with FRET peptides that emit fluorescence when cleaved by ACE2, the fluorescence intensity was measured and the enzyme activity was calculated. The results are shown in Figure 12.

[0131] Next, the pharmacokinetics of the above protein was measured. Specifically, the procedure was as follows: 10 mg / kg body of the protein was administered intravenously to mice, and blood samples were taken at various time intervals. The protein concentration of the plasma samples was measured by ELISA. The results are shown in Figure 13.

[0132] Subsequently, the therapeutic effect of the above protein was evaluated in the same manner as in Test Example 10. The measurement results of the viral load in the lung tissue are shown in Figure 14, and the measurement results of the expression level of inflammatory cytokines in the lung tissue are shown in Figure 15.

Claims

1. The present invention comprises an amino acid sequence C obtained by mutating an amino acid sequence A shown in SEQ ID NO: 1 or an amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence B having 90% or more identity with the amino acid sequence A or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 12, the amino acid sequence C comprises a mutation a: a substitution ax of at least four amino acids selected from the group consisting of T20, A25, K26, K31, H34, E35, Q60, N64, S70, T78, L79, N90, T92, and Q101 in the amino acid sequence A, or a substitution corresponding to the substitution ax in the amino acid sequence shown in SEQ ID NO: 12, or a substitution ay corresponding to the substitution ax in the amino acid sequence B, or a substitution corresponding to the substitution ax in an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 12, the substitution ax comprises the substitutions A25, K31, E35 and L79, or the substitutions A25, K31, E35 and T92, or the substitutions K31, E35, Q60 and L79, or the substitutions T20, H34, T92 and Q101; In the mutation a, the amino acid after mutation at T20 is a hydrophobic amino acid, the amino acid after mutation at A25 is a bulkier hydrophobic amino acid, the amino acid after mutation at K26 is a non-basic amino acid, the amino acid after mutation at K31 is a non-basic amino acid, the amino acid after mutation at H34 is a non-acidic amino acid, the amino acid after mutation at Q60 is a basic amino acid, the amino acid after mutation at N64 is a hydrophobic amino acid, the amino acid after mutation at S70 is a hydrophobic amino acid, the amino acid after mutation at T78 is a basic amino acid, the amino acid after mutation at L79 is a bulkier hydrophobic amino acid, the amino acid after mutation at N90 is a basic amino acid or an acidic amino acid, the amino acid after mutation at T92 is an amino acid having a carbamoyl group in the side chain, and / or the amino acid after mutation at Q101 is a basic amino acid. Mutant ACE2 protein.

2. The mutant ACE2 protein according to claim 1, wherein the substitution ax is a substitution of seven amino acids.

3. 3. The mutant ACE2 protein of claim 1, wherein, in the mutation a, the amino acid after the mutation at A25 is a branched-chain amino acid, the amino acid after the mutation at K26 is an acidic amino acid, the amino acid after the mutation at K31 is a hydrophilic neutral amino acid, the amino acid after the mutation at E35 is a basic amino acid, the amino acid after the mutation at N64 is a branched-chain amino acid, the amino acid after the mutation at L79 is an aromatic amino acid, and / or the amino acid after the mutation at N90 is a basic amino acid.

4. The mutant ACE2 protein of any one of claims 1 to 3, wherein, in the mutation a, the amino acid after the mutation at A25 is valine, the amino acid after the mutation at K26 is glutamic acid, the amino acid after the mutation at K31 is asparagine, the amino acid after the mutation at E35 is lysine, the amino acid after the mutation at N64 is isoleucine, the amino acid after the mutation at L79 is phenylalanine, and / or the amino acid after the mutation at N90 is histidine.

5. 2. The mutant ACE2 protein of claim 1, wherein the substitutions ax include substitutions of at least two amino acids selected from the group consisting of K31, E35, Q60, S70, L79, and N90, or substitutions of at least two amino acids selected from the group consisting of T20, A25, H34, T78, T92, and Q101.

6. The mutant ACE2 protein according to any one of claims 1 to 5, further comprising all or part of the amino acid sequence of an antibody.

7. A complex comprising a plurality of mutant ACE2 proteins according to any one of claims 1 to 6.

8. A polynucleotide comprising a coding sequence for the mutant ACE2 protein of any one of claims 1 to 6.

9. A cell comprising the polynucleotide of claim 8.

10. A pharmaceutical comprising at least one selected from the group consisting of a mutant ACE2 protein according to any one of claims 1 to 6, a complex according to claim 7, a polynucleotide according to claim 8, and a cell according to claim 9.

11. A reagent comprising at least one selected from the group consisting of a mutant ACE2 protein according to any one of claims 1 to 6, a complex according to claim 7, a polynucleotide according to claim 8, and a cell according to claim 9.

12. An anti-coronavirus agent comprising at least one selected from the group consisting of a mutant ACE2 protein according to any one of claims 1 to 6, a complex according to claim 7, a polynucleotide according to claim 8, and a cell according to claim 9.

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