KR20220045559A - Stabilized Ace2 Mutant, Ace2-Fc Fusion Protein Using Same and Pharmaceutical Composition for Preventing or Treating COVID-19 - Google Patents

Abstract

The present invention relates to a stabilized angiotensin-converting enzyme 2 (Ace2) mutant with increased stability through a disulfide bond, an Ace2-Fc fusion protein using the same, and a pharmaceutical composition for preventing or treating COVID-19. According to the present invention, a disulfide bond is introduced by substituting a residue pair at a predetermined position of an Ace2 protein with cysteine, and thus an Ace2 protein mutant having high binding affinity to a SARS-CoV-2 virus and exhibiting excellent stability even in an aqueous solution can be provided. Accordingly, when the stabilized Ace2 mutant is applied to a treatment for COVID-19, which is an infectious disease of SARS-CoV-2, storage stability, in vivo stability, and therapeutic effect can be improved. In addition, since an amino acid sequence derived from a receptor for SARS-CoV-2 is used, the stabilized Ace2 mutant can effectively act on various variant viruses.

Description

Stabilized Ace2 mutant, Ace2-Fc fusion protein using same, and pharmaceutical composition for preventing or treating COVID-19 {Stabilized Ace2 Mutant, Ace2-Fc Fusion Protein Using Same and Pharmaceutical Composition for Preventing or Treating COVID-19}

The present invention relates to a stabilized Ace2 variant, an Ace2-Fc fusion protein using the same, and a pharmaceutical composition for preventing or treating COVID-19, and more particularly, a variant of the Ace2 protein with improved stability through disulfide bond, Ace2-Fc using the same It relates to a fusion protein and a pharmaceutical composition for preventing or treating COVID-19.

Coronavirus Infectious Disease-19 (COVID-19) has spread rapidly since it occurred in December 2019, and the World Health Organization (WHO) has declared COVID-19 a global pandemic following Hong Kong Flu and H1N1 flu.

The pathogen of COVID-19 is SARS-CoV-2, It is transmitted when droplets of an infected person penetrate the respiratory tract or the mucous membranes of the eyes, nose, and mouth. COVID-19 is highly contagious and can be fatal if infected, such as the elderly or people with low immunity, and in severe cases, death. In addition, with the rapid spread of COVID-19, damage is occurring not only in the health field, but also in various fields such as society and economy. Therefore, there is an urgent need to develop vaccines and therapeutics for COVID-19.

As an example of a therapeutic agent for COVID-19, an antiviral agent using a nucleoside analog is being developed. For example, Korean Patent Publication No. 10-2145197 describes the use of treating a coronavirus infection by administering a drug containing a specific L-nucleoside compound. However, currently approved antiviral drugs have only been shown to be effective in patients with very severe conditions.

Another type of therapeutic agent is a therapeutic agent using an antibody. Antibody therapy is a therapeutic agent with a mechanism that prevents the virus from entering human cells by binding the antibody injected from the outside to the spike protein on the surface of the virus. For example, Korean Patent No. 10-2205028 describes various antibody therapeutics that neutralize viruses by binding to the spike protein on the surface of SARS-CoV-2.

However, in the case of SARS-CoV-2, numerous mutated viruses have been reported since the first virus was discovered, and in particular, mutants such as B.1.1.7 and B.1.351 were found to have higher transmission power compared to existing viruses. However, since the antibody binds only to a specific antigen, there is a problem that the antibody therapeutic agent against SARS-CoV-2 and its variant virus cannot work properly against variants other than the target virus.

Therefore, there is a need to develop a therapeutic agent that has a strong therapeutic effect on SARS-CoV-2 virus-infected diseases and can also exhibit effects on various virus strains.

The inventors of the present invention found that when using a protein derived from Ace2, which is a receptor that induces human cell infection of SARS-CoV-2, the virus can be transmitted to the human body due to the high binding affinity between the spike protein and Ace2 protein on the surface of SARS-CoV-2. It has been found that it can effectively prevent invasion into cells.

However, the wild-type Ace2 protein has a problem in that stability is not high because a part of the structure moves when interacting with the angiotensin peptide. Wild-type Ace2 in the cell membrane protein state can be stabilized through interaction with angiotensin peptides and interactions with other proteins and biomolecules in the cell membrane, but stability may be very low in the water-soluble Ace2 state, and thus applied to therapeutics There was a limit to what it was difficult to do.

Accordingly, in order to develop a therapeutic agent with excellent therapeutic effect using the Ace2 protein, it is necessary to solve the stability problem of the Ace2 protein.

It is an object of the present invention to provide Ace2 variants stabilized through disulfide bonds.

Another object of the present invention is to provide an Ace2-Fc fusion protein using the stabilized Ace2 mutant.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating COVID-19 using the stabilized Ace2 variant.

In order to achieve the above object, the present invention provides a stabilized Ace2 variant comprising a disulfide bond formed by substituting at least one pair of amino acid residues present in Ace2 (Angiotensin-converting enzyme 2)-derived protein with cysteine.

In the present invention, the distance between the central carbon (C-alpha) of the pair of amino acid residues forming the disulfide bond is preferably 4.5 to 7.0 Å.

In the present invention, the amino acid residue pair is N51 / V343, N53 / Q340, I54 / K341, H239 / V604, I21 / E87, M62 / S47, A193 / V107, V364 / V298, T365 / T294 present in Ace2-derived protein. , H401/H378, T445/T276, S502/R169, N508/S124 and A348/H378 may include one or more selected from the group consisting of.

In the present invention, the Ace2-derived protein may be a protein derived from the extracellular domain (ectodomain) of the Ace2 protein.

In the present invention, the Ace2-derived protein may include residues 1 to 615 of the wild-type Ace2 protein.

The present invention also provides an Ace2-Fc fusion protein, wherein the stabilized Ace2 variant is linked to one or more of the two chains constituting the Fc domain derived from immunoglobulin (Ig).

In the present invention, the stabilizing Ace2 variant and the Fc domain may be linked through a linker consisting of 0 to 20 amino acid residues.

In the present invention, the Ace2-Fc fusion protein may be a homodimer or a heterodimer.

In the present invention, the immunoglobulin may be IgG1, IgG2, IgG3 or IgG4.

In the present invention, the chain constituting the Fc domain may include amino acid residues 221 to 447 of an IgG1 heavy chain (based on EU numbering).

In the present invention, at least one amino acid in one chain of the Fc domain is substituted with an amino acid selected from the group consisting of tryptophan (W), arginine (R), phenylalanine (F) and tyrosine (Y), and in the other chain One or more amino acids may be substituted with an amino acid selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V).

In the present invention, the Ace2-Fc fusion protein may be a bispecific or multispecific antibody further comprising a protein binding to an immune cell surface antigen.

In the present invention, the immune cells may be natural killer cells (NK cells) or T cells.

The present invention also provides a pharmaceutical composition for preventing or treating coronavirus infection-19 (COVID-19) comprising the Ace2-Fc fusion protein.

According to the present invention, by introducing a disulfide bond by replacing a pair of residues at a specific position of the Ace2 protein with cysteine, it is possible to provide an Ace2 protein variant that has high binding affinity with SARS-CoV-2 virus and exhibits excellent stability even in aqueous solution. can Accordingly, when the stabilized Ace2 mutant of the present invention is applied to a therapeutic agent for COVID-19, a SARS-CoV-2 infectious disease, it is possible to improve both storage stability, in vivo stability and therapeutic effect. In addition, since the amino acid sequence derived from the receptor for SARS-CoV-2 is used, it can effectively act on various strains of viruses.

1 shows a schematic design of the stabilizing Ace2 variant provided by the present invention.
Figure 2 shows the position of a pair of residues to be subjected to loop stabilization mutation according to an embodiment of the present invention in the three-dimensional structure of the Ace2 protein.
Figure 3 shows the position of a pair of residues to be subjected to helix-helix or strand-helix stabilization mutation according to an embodiment of the present invention in the three-dimensional structure of the Ace2 protein.
4 shows the amino acid sequence encoded by the expression vector of the stabilized Ace2-Fc fusion protein according to an embodiment of the present invention.
5 shows the first derivative melting curves of the stabilized Ace2-Fc fusion protein and the wild-type Ace2-Fc fusion protein according to an embodiment of the present invention.
6 shows the results of ELISA analysis of the stabilized Ace2-Fc fusion protein and the wild-type Ace2-Fc fusion protein according to an embodiment of the present invention.

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.

The present invention relates to Ace2 protein variants stabilized via disulfide bonds.

Ace2 is an abbreviation of Angiotensin-converting enzyme 2, and is a carboxypeptidase related to angiotensin-converting enzyme (ACE). Ace2 is a functional receptor of a coronavirus associated with severe acute respiratory syndrome (SARS). When SARS-CoV-2 enters the human body, Ace2 binds to the spike protein of SARS-CoV-2, resulting in infection.

Ace2 protein consists of 805 amino acids, and is divided into an extracellular domain (ectodomain), a transmembrane domain (membrane spanning domain) and a cytoplasmic domain. Among them, the extracellular domain of Ace2 protein is a water-soluble protein consisting of about 600 amino acids and has high affinity with the SARS-CoV-2 virus spike protein. The inventors of the present invention can prevent the interaction and infection of SARS-CoV-2 virus with target cells by using the characteristic that Ace2 extracellular domain-derived protein strongly binds to SARS-CoV-2 spike protein. found that it can.

However, it is known that the extracellular domain of Ace2 protein has poor stability because a part of the protein structure moves when it interacts with the angiotensin peptide for its natural function. Wild-type Ace2 in the state of cell membrane protein can be stabilized through interaction with angiotensin peptides and interactions with other proteins and biomolecules of cell membranes, but stability is very low in soluble Ace2 (soluble Ace2) state, making it suitable for application to therapeutic agents There was a difficult problem.

The present invention is to solve the problem of instability of the Ace2 protein, and the stabilized Ace2 mutant of the present invention has a mutation for introducing a disulfide bond into the Ace2-derived protein as shown in FIG. 1 .

The inventors of the present invention have developed a structurally stable disulfide bond by substituting cysteine for at least one pair of specific amino acid residues in the Ace2 protein, thereby improving the thermal stability and structural stability of the Ace2 protein to be stable even in a water-soluble Ace2 state, and to form a disulfide bond. A mutant capable of maintaining high binding affinity with the virus by minimizing structural modification caused by the virus was developed.

In the present invention, the term "disulfide bond" refers to a state in which a sulfide (-SH) group in cysteine among protein amino acids meets with a sulfide group of another cysteine to form a covalent bond, providing great stability to the protein structure do.

In the present invention, the term "water-soluble Ace2" means that the extracellular domain of Ace2 is genetically engineered to form a protein in a stable state in aqueous solution, not a wild-type membrane protein. As described above, the soluble receptor made from the virus receptor can be used to prevent the virus from entering the cell.

In the present invention, the term “variant” is meant to include substitution, insertion and/or deletion of amino acid residues, and preferably, the variant of the present invention includes substitution of amino acid residues. Substitution of amino acid residues may be indicated in the order of the residues in the parent amino acid sequence, the number of amino acid residues, and the amino acid residues substituted.

In the present invention, the mutation for introducing the disulfide bond may include a mutation in which at least one of the pair of amino acid residues present in the Ace2-derived protein is substituted with cysteine. In this case, the amino acid residue pair is a pair of two amino acids composed of amino acids other than cysteine, and in order to effectively stabilize the structure by forming a disulfide bond through cysteine substitution, the central carbon (C -alpha) is preferably 4.5 to 7.0 Å.

In the present invention, the pair of amino acid residues to be mutated are N51 / V343, N53 / Q340, I54 / K341, H239 / V604, I21 / E87, M62 / S47, A193 / V107, V364 / V298, It may include one or more pairs of residues selected from the group consisting of T365/T294, H401/H378, T445/T276, S502/R169, N508/S124 and A348/H378.

In an embodiment of the present invention, the distance between the C-alpha of the residue pairs is in the range of 4.5 to 7.0 Å through three-dimensional structural analysis of the Ace2 protein, and the loop 331-347 region and loop 599-614 region of the Ace2 protein , it was confirmed that the pair of residues involved in the stabilization of the helix-helix region and the strand-helix region. In addition, when a disulfide bond is formed on the pair of amino acid residues, the melting temperature of the protein is significantly increased compared to the wild-type, but experimental results with little effect on the binding force with the virus were obtained. Accordingly, according to the mutant of the present invention, structural distortion caused by the formation of disulfide bonds is minimized, and it was confirmed that improved structural stability compared to the wild-type Ace2 protein can be implemented while maintaining excellent binding affinity with the virus, which is a characteristic of Ace2. .

In the present invention, the Ace2-derived protein, which is the parent of the mutation, may include the amino acid sequence of SEQ ID NO: 1 corresponding to 1 to 615 of the Ace2 protein.

[SEQ ID NO: 1] 1 to 615 of Ace2

MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD

Specifically, in the Ace2-derived protein, through one or more mutations selected from the group consisting of N51C / V343C, N53C / Q340C and I54C / K341C, the loop 331-347 region, which is a fluid loop of the Ace2 protein, can be stabilized, and , it is possible to stabilize the loop 599-614 region through the H239C/V604C mutation.

In addition, through one or more mutations selected from the group consisting of 21C / E87C, M62C / S47C, A193C / V107C, V364C / V298C, T365C / T294C, H401C / H378C, T445C / T276C, S502C / R169C and N508C / S124C, Ace2 It can be stabilized by linking the helix of the protein, and the strand and the helix can be stably linked through the A348C/H378C mutation.

As described above, the Ace2 mutant stabilized by forming a disulfide bond by substituting a target residue pair with cysteine has a high binding affinity to SARS-CoV-2 virus like wild-type Ace2, thereby exhibiting an excellent therapeutic effect. In addition, since it is stable even in a water-soluble Ace2 state, it maintains a structure effective for disease treatment in the human body for a long period of time, thereby exhibiting long-term efficacy even at a low dose.

In one embodiment of the present invention, an Ace2-Fc fusion protein can be formed using the stabilized Ace2 mutant.

In the present invention, the Ace2-Fc fusion protein refers to a protein produced by linking a water-soluble Ace2 protein to an Fc domain derived from immunoglobulin.

In the present invention, the immunoglobulin-derived Fc domain refers to a C-terminal region including CH2 and CH3 domains (or CH2, CH3 and CH4 domains) among the heavy chain constant regions of immunoglobulin, wild-type It is used in the sense of encompassing the Fc domain and its variants. The immunoglobulin serving as the parent of the Fc domain may be IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

In the present invention, each chain of the Fc domain may include a region extending from residue 221 to the C-terminus of a human IgG1 heavy chain, or a region further comprising a hinge in the region. The numbering of amino acid residues in the Fc region is according to EU numbering (see Kabat et al. et al.), which defines the number of residues in human immunoglobulin heavy chains.

In the present invention, each chain of the Fc domain may include sequences 221 to 447 of the heavy chain of IgG1. The heavy chain sequences 221 to 447 of the IgG1 may be represented by the amino acid sequence of SEQ ID NO: 2 below.

[SEQ ID NO: 2] heavy chains 221 to 447 of IgG1

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQKVSLTCLDSDNGQPQSDIKVYTLPPSRDELTKKNQVSLTCLDWSDNGQFQSDIKFSKVVSHEDGSGFFNWYTKSKSPVWSDNGGFN

In the Ace2-Fc fusion protein of the present invention, the stabilizing Ace2 variant may be linked to one or more of the two chains constituting the Fc domain.

In this case, the Ace2 variant and the Fc domain may be linked by 0 to 20 amino acid residues. That is, the Ace2 variant and the Fc domain may be directly linked or linked through a linker consisting of 1 to 20 amino acids. In addition, the Ace2 variant may be linked to the N-terminus or C-terminus of the Fc domain.

The Ace2-Fc fusion protein of the present invention may be in the form of a homodimer or heterodimer, and the Ace2 variant may be linked to both chains of the Fc domain, or may be linked to only one chain.

In one embodiment of the present invention, when the Ace2-Fc fusion protein forms a heterodimer, for example, a bispecific (or multispecific) antibody, a recombinant variant for forming a dimer in the Fc domain may be formed. there is.

For example, a knob-into-hole technique may be used as the recombinant mutant for forming the dimer.

The knob-into-hole technology is a technology designed to allow only heterodimers to be formed between the heavy chains of antibody fragments. Here, the knob is designed to have a side chain protruding to the opposite side chain, and is inserted into the hole of the opposite side domain. Due to this, heavy chains cannot be homodimerized due to side chain collision, and only heterodimerization is possible. In the present invention, a structure in which a knob or a hole is formed in each chain of the Fc domain may be referred to as an Fc-knob or an Fc-hole, respectively.

The Fc-knob may be formed by substituting one or more amino acids in the chain constituting the Fc domain with a large amino acid selected from the group consisting of tryptophan (W), arginine (R), phenylalanine (F) and tyrosine (Y). . For example, the Fc-knob may be one in which the T366W mutation is formed in sequences 221 to 447 of the IgG1-Fc heavy chain.

The Fc-hole may be formed by substituting one or more amino acids in the chain constituting the Fc domain with small amino acids selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V). . For example, Fc-holes are T366S, L368A and Y407V in the sequences 221 to 447 of the IgG1-Fc heavy chain. A mutation may be formed.

In this way, when the stabilizing Ace2 variant of the present invention is fused with the Fc domain, the Ace2 variant can not only strongly bind to the virus to inhibit infection, but also induce an immune response to eliminate the virus, and interact with the Fc receptor. It is possible to enhance the drug effect by extending the period of residence in the body. In addition, by producing a bispecific or multispecific antibody using this, NK cells or immune cells are induced into infected cells, and virus-derived diseases can be treated through active killing of infected cells and activation of immune responses.

As used herein, the term "bispecific (or multispecific) antibody" refers to an antibody capable of binding to two different (or more) antigens, and includes a form produced by genetic engineering. . In the present invention, the bispecific (or multispecific) antibody may refer to an antibody that simultaneously binds to a target antigen and an immune cell surface antigen, thereby inducing apoptosis of the target antigen by antibody-dependent cytotoxicity.

In the present invention, the bispecific (or multispecific) antibody is Fab, Fab', F(ab')2, Fv fragment, rIgG, single chain Fv fragment (scFv), tandem single chain Fv fragment (scFv)2 , bispecific T-cell participant (BiTE), diabody (Diabody), tandem diabody (TandAb), triabody (Triabody) or tetrabody (Tetrabody) form.

In the present invention, the term "immune cell" refers to a cell that recognizes an antigen and attacks it directly or indirectly, preferably a natural killer cell (NK cell) or T cell.

In the present invention, the term "immune cell surface antigen" is a concept including target molecules and antigens present on the surface of immune cells. For example, the immune cell surface antigen is IL-1 alpha, IL-1 beta, IL-1R, IL-4, IL-5, IL-6R, IL-9, IL-12, IL-13, IL- 18, IL-18R, IL-25, TARC, MDC, MEF, TGF-β, LHR agonist, TWEAK, CL25, SPRR2a, SPRR2b, ADAM8, PED2, TNF alpha, TGF beta, VEGF, MIF, ICAM-1, PGE4 , PEG2, RANK ligand, Te38, BAFF, CTLA-4, GP130, HER1, HER2, HER3, HER4, VEGF-A, PDGF, VEGF-A, VEGF-C, VEGF-D, DR5, MET, EGFR, MAPG, CSPGs, CTLA-4, IGF1, IGF2, Erb2B, MAG, RGM A, NogoA, NgR, OMGp, PDL-I, NRP1, NRP2, 2B4, CD2, CD3 zeta, CD3, CD8, CD16, CD19, CD20, CD22, CD27, CD28, CD40, CD56, CD57, CD62L, CD64, CD94, CD96, CD100, CD160, CD244, CEACAM1, CRACC, CRTAM, CS1, DAP10, DAP12, DNAM-1 (CD226), FcR gamma, KIRs (KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR2DL4, KIR3DL2, KIR3DS1), LFA-1, NCRs, NKG2A, NKG2C, NKG2D, NKG2E, NKG2H, NKp30a, NKp30b, NKp40, PSGL1, se NK horp80, NKp It may be selected from the group consisting of 4D, SLAMF4, SLAMF6, SLAMF7, TIGIT, TLR and TRAIL. Alternatively, it is also possible to use in the form of a multispecific antibody by binding to one or more additional target molecules.

In the Ace2-Fc fusion protein of the present invention, a bispecific antibody can be prepared by linking a stabilizing Ace2 variant and a binding protein binding to an immune cell surface antigen to the chain of the Fc domain, respectively. This bispecific antibody can simultaneously bind to SARS-CoV-2 virus and immune cell surface antigen. Alternatively, multispecific antibodies can be prepared by linking two or more types of binding proteins that bind to immune cell surface antigens.

When such a bispecific (or multispecific) antibody is used, immune cells can recognize SARS-CoV-2 by antibody dependent cellular cytotoxicity (ADCC) and induce virus neutralization.

By using the stabilized Ace2 mutant of the present invention, immune cells can effectively recognize and kill SARS-CoV-2 due to the high binding affinity between SARS-CoV-2 and Ace2, and because Ace2 is structurally stable, it is applied to therapeutic agents. In this case, it has excellent stability against storage and environmental changes, and maintains a structure effective for disease treatment in the human body for a long period of time, so that it can exhibit long-term efficacy even at low doses. In addition, even if a virus is mutated, since the site for recognizing cell receptors on the surface antigen does not change, there is an advantage in that the virus killing effect appears for various mutants.

Accordingly, the present invention relates to a pharmaceutical composition for preventing or treating a SARS-CoV-2 virus infection disease comprising a stabilized Ace2 mutant or Ace2-Fc fusion protein.

Since the stabilized Ace2 mutant of the present invention contains a protein derived from Ace2, which is a human cell receptor for SARS-CoV-2, it can strongly bind to SARS-CoV-2. Therefore, it may interfere with the interaction between SARS-CoV-2 and the Ace2 receptor present in human cells. In addition, the Ace2-Fc fusion protein introduced with the stabilized Ace2 mutant can effectively neutralize SARS-CoV-2 virus through an immune response.

According to the experimental results described in the present inventor's prior patent application No. 10-2021-0090947, as a result of treating lung cells transfected with the SARS-CoV-2 spike expression vector with a wild-type Ace2-Fc fusion protein, SARS-CoV- 2 It was confirmed that the spike protein expression can kill lung cells. In addition, when Ace2-Fc fusion protein was treated in the abdominal cavity of transgenic mice infected with SARS-CoV-2 virus, it was confirmed that the viral titer was significantly reduced compared to the control group.

Accordingly, it was confirmed that the Ace2-Fc fusion protein exhibits a therapeutic effect on SARS-CoV-2 infection diseases through the study of the present inventor's prior patent application. It was confirmed that the binding affinity of wild-type Ace2 protein and SARS-CoV-2 spike protein was similar and stability was better. know it will do.

Therefore, by using the stabilized Ace2 mutant or Ace2-Fc fusion protein of the present invention, it is possible to provide a pharmaceutical composition that can prevent or treat coronavirus infection-19 (COVID-19), a SARS-CoV-2 virus infection disease. .

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the stabilized Ace2 mutant or stabilized Ace2-Fc fusion protein.

The pharmaceutically acceptable carriers are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and 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 as

The pharmaceutical composition of the present invention may be formulated in the form of a sterile injection solution, a lyophilized formulation, a pre-filled syringe solution, an oral formulation, an external formulation, or a suppository according to a conventional method. there is. 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.

The pharmaceutical composition of the present invention may further comprise at least one other therapeutic or diagnostic agent. For example, interferon, anti-S protein monoclonal antibody, anti-S protein polyclonal antibody, nucleoside analog, DNA polymerase inhibitor, siRNA agent or therapeutic vaccine may be further included as an antiviral drug.

By administering the pharmaceutical composition of the present invention to mammals including humans, it is possible to prevent or treat SARS-CoV-2 infection and diseases caused by SARS-CoV-2 infection. In this case, the dosage depends on the subject to be treated, the severity of the disease or condition, the rate of administration, and the judgment of the prescribing physician.

Example

The present invention will be described in more detail with reference to the following examples. However, these Examples show some experimental methods and compositions for illustrative purposes of the present invention, and the scope of the present invention is not limited to these Examples.

Preparation Example 1: Preparation of stabilized Ace2 variants

A mutant in which a disulfide bond was introduced into a pair of residues causing instability in the Ace2 protein structure was designed and shown in Table 1 below. In addition, in the pair of amino acid residues to be mutated, the C-alpha distance between amino acids and the stabilization mechanism are shown together in Table 1.

No. mutation C-alpha distance (Å) stabilization mechanism One N51C/V343C 5.3 loop 331-347 region stabilization 2 N53C/Q340C 5.9 3 I54C/K341C 6.0 4 H239C/V604C 6.1 loop 599-614 region stabilization 5 I21C/E87C 6.6 helix-helix stabilization 6 M62C/S47C 4.8 7 A193C/V107C 6.5 8 V364C/V298C 6.6 9 T365C/T294C 4.8 10 H401C/H378C 5.7 11 T445C/T276C 5.2 12 S502C/R169C 6.2 13 N508C/S124C 5.6 14 A348C/H378C 6.3 strand-helix stabilization

2 and 3 show the positions of the pairs of residues to be mutated in the three-dimensional structure of the Ace2 protein.

Specifically, (a) and (b) of Figure 2 shows the stabilizing residues of the loop region of the Ace2 protein. In FIG. 2 , the green part represents the Ace2 protein, and the cyan part represents the SARS-CoV-2 spike protein.

As shown in (a) of Figure 2, N51 / V343, N53 / Q340 and I54 / K341 were selected as a residue pair capable of stabilizing the loop 331-347 region by introducing a disulfide bond, and shown in (b) As shown, H239/V604 was selected as a pair of residues capable of stabilizing the loop 599-614 region. The C-alpha distance between the pair of residues was a minimum of 5.2 Å and a maximum of 6.1 Å, and it was confirmed that it was within the range of 4.5 to 7.0 Å.

3 (a) to (i) show the stabilizing residues of the helix-helix region of the Ace2 protein.

As shown in (a) to (i) of Figure 3, as a pair of residues capable of stabilizing the helix region by introducing a disulfide bond, 121/E87, M62/S47, A193/V107, V364C/V298C, T365/T294, H401/H378, T445/T276, S502/R169 and N508/S124 were selected. The C-alpha distance between the pair of residues was a minimum of 4.8 Å and a maximum of 6.6 Å, and it was confirmed that it was within the range of 4.5 to 7.0 Å.

Figure 3 (j) shows the stabilizing residues of the strand-helix region of the Ace2 protein.

As shown in (j) of FIG. 3, A348/H378 was selected as a residue pair capable of stabilizing the strand-helix region by introducing a disulfide bond. The C-alpha distance between the pair of residues was 6.3 Å, which was confirmed to be in the range of 4.5 to 7.0 Å.

Preparation Example 2: Preparation of Ace2-Fc fusion protein

Based on the stabilizing mutant of the Ace2 protein, an Ace2-Fc fusion protein was prepared.

2-1. Expression vector construction

The expression vector was prepared based on pcDNA 3.1 / Myc His-A (Invitrogen).

DNA encoding the amino acid sequence of the target protein was amplified by chain enzyme polymerization and cloned using restriction enzymes and T4 DNA binding enzymes. Through this, human immunoglobulin G1 (IgG1) Fc tag and histidine tag were expressed at the C terminus of residues 1 to 615 of Ace2 protein.

Primers were prepared according to the Quik Change Site-directed mutagenesis (Stratagene) protocol, and mutations were introduced through a chain enzyme polymerization reaction. After treatment, it was confirmed that the intended sequence change was made to all nucleotides using a DNA sequencing service.

4 (a) to (n) show the amino acid sequence encoded by the stabilized Ace2-Fc expression vector into which Mutations 1 to 14 of Preparation Examples are introduced. In FIG. 4, a portion indicated by a straight underline corresponds to the Ace2-derived protein region, and a portion indicated by a dashed underline corresponds to the IgG1 221 to 447 region of the Fc domain. Residues substituted with cysteine in wild-type Ace2 are marked with an asterisk, and linkers and tags are not indicated separately.

2-2. protein expression

Each protein was transiently expressed by transfection of the expression vector into ExpiCHO™ cells.

In transfection, the cell concentration was diluted to 6 x 10 ⁶ cells/mL, and the expression vector was mixed so that the final concentration of the expression vector was 0.8 μgDNA/mL. In this process, ExpiCHO™ Expression Medium, OptiPRO™ SFM and ExpiFectamine™ transfection kit (Gibco) were used according to the manufacturer's instructions.

After culturing the transfected cells in a carbon dioxide incubator under 8% carbon dioxide conditions for 8 days, the supernatant containing the recombinant protein was separated by centrifugation.

2-3. refine

Affinity chromatography using a Ni-NTA (Qiagen) column was performed to separate target proteins from a culture medium containing each recombinant protein.

Specifically, after filling the Ni-NTA column with a buffer solution containing 50 mM Tris-HCl pH 7.5 and 200 mM NaCl, the culture solution obtained through centrifugation was filtered with a 0.45 μm filter paper and added. After completion of the reaction with the culture solution, the column was sequentially washed with a buffer solution containing 50 mM Tris-HCl pH 7.5 and 500 mM NaCl and a buffer solution containing 50 mM Tris-HCl pH 7.5, 200 mM NaCl and 30 mM imidazole. Finally, using a buffer solution containing 50 mM Tris-HCl pH 7.5, 200 mM NaCl, and 500 mM imidazole, the protein bound to the column was eluted. After elution, the composition of the buffer solution containing the protein was replaced with a composition containing 20 mM Tris-HCl pH 7.5 and 150 mM NaCl using a Hitrap™ Desalting (GE healthcare) column.

Experimental Example 1: Measurement of protein stability through heat denaturation analysis

To measure protein stability, thermal shift assay was performed using Protein Thermal Shift™ Dye Kit (Thermo Fisher Scientific).

The fluorescent dye stock solution and protein were diluted in a buffer so that the final composition of the mixed solution was 0.1M HEPES pH 7.5 and 150mM NaCl, and then mixed. The concentration of the protein was adjusted so that the final concentration was at a level of 0.5 mg/ml. 20 μl of the mixed solution was dispensed onto MicroAmp Optical 8-Tube Strip (Thermo Fisher Scientific). For real-time fluorescence measurement, an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific) instrument was used, and a melting curve was obtained using the melting curve protocol entered into the software.

The first derivative melting curve of the stabilized Ace2(N51C/V343C)-Fc protein through the thermal denaturation analysis is shown in FIG. 5, and for comparison, the same analysis was performed on the wild-type Ace2(WT)-Fc protein and shown together. In FIG. 5, the melting curve of the stabilized Ace2(N51C/V343C)-Fc protein is indicated by a dashed red line, and the melting curve of the wild-type Ace2(WT)-Fc protein is indicated by a black straight line. In addition, the dissolution temperature of the protein was calculated through the curve and shown in Table 2.

protein Tm(℃) wild-type Ace2-Fc 53.9 Stabilizing Ace2-Fc 58.8

As can be seen in FIG. 5 and Table 2, it was confirmed that the dissolution temperature of the protein using the stabilized Ace2 was increased by about 5° C. than that of the wild type. Through such excellent thermal stability results, it was confirmed that the stabilized Ace2 mutant of the present invention was structurally very stable compared to the wild type.

Experimental Example 2: Measurement of binding affinity to SARS-CoV-2 spike protein through enzyme-linked immunosorbent assay (ELISA)

SARS-CoV-2 spike protein (residues 319 to 541) was diluted to a concentration of 2.5 μg/ml in a buffer of 0.1 M sodium carbohydrate pH 9.0, and coated on a 96-well plate.

After coating, the plate was blocked using 5% (w/v) skim milk. Ace2(N51C/V343C)-Fc protein and wild-type Ace2(WT)-Fc protein were diluted to each concentration in 5% (w/v) skim milk powder and added to each coated well, followed by reaction at room temperature for 2 hours.

The reaction solution was discarded, and an HRP-binding secondary antibody (GW Vitek) capable of binding to a human Fc antibody was diluted in 5% (w/v) skim milk powder and added, followed by reaction at room temperature for 2 hours. At the end of each procedure, the wells were washed three times with phosphate buffer.

After the reaction was completed, a solution of tetramethylbenzidine (Goma Biotech) was added to each well and left for color development to appear, and then 0.2 M sulfuric acid solution was added to stop the reaction. The absorbance of the reaction solution was measured at 450 nm, and the results are shown in FIG. 6 . In FIG. 6 , the left bar shows the results for the wild-type Ace2(WT)-Fc protein at each concentration, and the right bar shows the results for the stabilized Ace2(N51C/V343C)-Fc protein at each concentration.

According to the results of FIG. 6 , it was confirmed that the protein into which the stabilizing Ace2 according to the present invention was introduced had the SARS-CoV-2 spike protein binding ability very similar to that of the protein into which the wild-type Ace2 was introduced.

Accordingly, it was confirmed that the stabilized Ace2 mutant of the present invention could effectively improve only the stability of the Ace2 protein without significantly affecting the binding ability with the SARS-CoV-2 spike protein.

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.

<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> Stabilized Ace2 Mutant, Ace2-Fc Fusion Protein Using Same and Pharmaceutical Composition for Preventing or Treating COVID-19 <130> HPN21055 <150> KR 10-2020-0128402 <151> 2020-10-05 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 615 <212> PRT <213> Homo sapiens <400> 1 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp 610 615 <210> 2 <211> 227 <212> PRT <213> Homo sapiens <400> 2 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225

Claims (14)

Ace2 (Angiotensin-converting enzyme 2) stabilizing Ace2 mutant comprising a disulfide bond formed by substituting cysteine for at least one of the pair of amino acid residues present in the derived protein. The method of claim 1,
The distance between the central carbons (C-alpha) of the pair of amino acid residues forming the disulfide bond is 4.5 to 7.0 Å, the stabilized Ace2 variant. The method of claim 1,
N51/V343, N53/Q340, I54/K341, H239/V604, I21/E87, M62/S47, A193/V107, V364/V298, T365/T294, H401/H378, A stabilizing Ace2 variant comprising at least one selected from the group consisting of T445/T276, S502/R169, N508/S124 and A348/H378. The method of claim 1,
The Ace2 derived protein is a protein derived from the extracellular domain (ectodomain) of the Ace2 protein, the stabilized Ace2 mutant. The method of claim 1,
The stabilizing Ace2 variant, wherein the Ace2 derived protein comprises residues 1 to 615 of the wild-type Ace2 protein. An Ace2-Fc fusion protein wherein the stabilizing Ace2 variant according to any one of claims 1 to 5 is linked to at least one of the two chains constituting the Fc domain derived from immunoglobulin (Ig). 7. The method of claim 6,
The stabilizing Ace2 variant and the Fc domain are linked through a linker (linker) consisting of 0 to 20 amino acid residues, Ace2-Fc fusion protein. 7. The method of claim 6,
The Ace2-Fc fusion protein is a homodimer or a heterodimer, Ace2-Fc fusion protein. 7. The method of claim 6,
The immunoglobulin is IgG1, IgG2, IgG3 or IgG4, Ace2-Fc fusion protein. 7. The method of claim 6,
The chain constituting the Fc domain comprises amino acid residues 221 to 447 of an IgG1 heavy chain (based on EU numbering), Ace2-Fc fusion protein. 7. The method of claim 6,
In one chain of said Fc domain, at least one amino acid is substituted with an amino acid selected from the group consisting of tryptophan (W), arginine (R), phenylalanine (F) and tyrosine (Y);
An Ace2-Fc fusion protein, wherein in another chain, one or more amino acids are substituted with an amino acid selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V). 7. The method of claim 6,
The Ace2-Fc fusion protein is a bispecific or multispecific antibody further comprising a protein binding to an immune cell surface antigen, Ace2-Fc fusion protein. 13. The method of claim 12,
The immune cells are natural killer cells (Natural Killer cells, NK cells) or T cells, Ace2-Fc fusion protein. A coronavirus comprising an Ace2-Fc fusion protein in which the stabilizing Ace2 variant according to any one of claims 1 to 5 is linked to at least one of the two chains constituting the Fc domain derived from immunoglobulin (Ig). A pharmaceutical composition for preventing or treating Infectious Disease-19 (COVID-19).

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