KR20230166166A - Method for regulating the activity of immune cells through neutralization control of the Inlterleukin2 (IL2) receptor using ACE2 protein-overexpressed inbred mouse(SARS-CoV2, simulatory model) - Google Patents

Abstract

The present invention relates to a method of controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice induced to overexpress the ACE2 protein. Specifically, the Th1 immune response in which the ACE2 protein is overexpressed Interleukin-2 (IL-2) receptor, which can control the activity of immune cells against infectious respiratory diseases through IL-2 neutralization control in an experimental animal model with SARS-CoV2 reproducibility by using mice of the dominant inbred lineage It relates to a method of controlling the activity of immune cells through neutralization control.

Description

Method for regulating the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice (SARS-CoV2 clinical reproducibility model) induced to overexpress the ACE2 protein {Method for regulating the activity of immune cells through neutralization control of the Interleukin2 (IL2) receptor using ACE2 protein-overexpressed inbred mouse(SARS-CoV2, simulatory model)}

The present invention relates to a method of controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice (SARS-CoV2 clinical reproducibility model) induced to overexpress the ACE2 protein. Specifically, ACE2 Interleukin that can control the activity of immune cells against infectious respiratory diseases through IL-2 neutralization control in experimental animal models with SARS-CoV2 reproducibility by using inbred mice with a dominant Th1 immune response in which the protein is overexpressed It relates to a method of controlling the activity of immune cells through neutralization control of the -2 (IL-2) receptor.

Research models using experimental animals play a very important role in the evaluation of various medical approaches, such as drug suitability, toxicity evaluation, and application of new medical technologies. In particular, inbred mice are genetically stable and have consistent phenotypes and clear background information that can be confirmed, so they are used in most biomedical research.

The ACE2 gene stands for Angiotensin-converting enzyme2 and plays an important role in regulating body moisture and blood pressure. The ACE2 protein is expressed in the heart, lungs, kidneys, vascular endothelium, and digestive system, and is a receptor used by various coronaviruses, especially SARS-CoV-2, when they invade cells. In other words, if a protein derived from ACE2, a receptor that induces infection of human cells by SARS-CoV-2, is used, the virus can invade human cells due to the high binding affinity of the spike protein on the surface of SARS-CoV-2 and the ACE2 protein. This can be effectively prevented.

In the case of experimental animals in which the ACE2 protein is overexpressed, they have an organic environment of organisms that can mimic coronavirus disease, and it is easy to track and observe the activity/inactivity of various immune cells in the body and evaluate dynamic changes in immune cells. It has the advantage of being there.

Therefore, there is a request for a technology to control the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice induced to overexpress the ACE2 protein.

Republic of Korea Patent No. 10-2205028

The present invention is intended to solve these problems of the prior art, and the purpose of the present invention is to neutralize the interleukin-2 (IL-2) receptor using inbred mice (SARS-CoV2 clinical reproducibility model) induced to overexpress the ACE2 protein. The aim is to provide a method of regulating the activity of immune cells through control.

In order to achieve the above-described object, the present invention includes the steps of producing an inbred mouse induced to overexpress ACE2 (Angiotensin-converting enzyme2) protein; And controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice induced to overexpress the ACE2 (Angiotensin-converting enzyme2) protein. Overexpression of the ACE2 protein comprising a. A method of controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using induced inbred mice is provided.

In the method of controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice induced to overexpress the ACE2 protein according to the present invention, the ACE2 protein is an extracellular domain of the ACE2 protein ( It is characterized as being a protein derived from ectodomain.

In the method of controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice induced to overexpress the ACE2 protein according to the present invention, the immune cells are Natural Killer cells. , NK cell) or T cell.

In the method of controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice induced to overexpress the ACE2 protein according to the present invention, the antibody of the interleukin-2 (IL-2) is characterized by increasing the activity of regulatory T cells (Treg).

In the method of controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice induced to overexpress the ACE2 protein according to the present invention, the inbred mice contain recombinant SARS-CoV-2 It is characterized by realizing SARS-CoV-2 reproducibility using proteins.

The method of controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice (SARS-CoV2 clinical reproducibility model) induced to overexpress the ACE2 protein according to the present invention expresses human ACE2. In mice, the activity of the IL-2 receptor can be inhibited through IL-2 neutralizing antibodies.

Figure 1 shows a schematic design of the stabilized Ace2 variant provided by the present invention.
Figure 2 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.

Since the present invention can be modified in various ways and have various embodiments, the preferred embodiments will be described in detail. It is obvious to those skilled in the art that the present invention can be embodied in other specific forms without departing from the spirit and essential features of the present invention.

Unless otherwise defined, all technical and chemical terms used in this specification have the same meaning as commonly understood by an expert skilled in the technical field to which the present invention pertains. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

The present invention will be described in detail below with reference to the drawings.

The method of controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice induced to overexpress the ACE2 protein according to the present invention is to control the activity of immune cells using inbred mice induced to overexpress the ACE2 (Angiotensin-converting enzyme2) protein. Steps of manufacturing a mouse; and controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice induced to overexpress the ACE2 (Angiotensin-converting enzyme2) protein.

The present invention utilizes inbred mice with a dominant Th1 immune response overexpressing the ACE2 protein to target experimental animal models with SARS-CoV2 reproducibility, and uses IL-2 neutralization control as a response technology to infectious respiratory diseases of immune cells. It is about a technology that controls activity by regulating it.

Inbred mice are inbred mice that have continued sibling or parent-breeding for more than 20 generations, and the relatedness coefficient (R) is 99.6%, so they are almost identical to identical twins and the individuals are genetically uniform. Inbred mouse strains include C57BL/6, C58, 129, DBA, and CBA, and each strain has specific diseases.

The inbred mouse of the present invention is preferably a K18-hACE2 mouse (hACE2 mouse (B6.Cg-Tg(K18-ACE2)2Prlmn/J)).

The inbred mouse is characterized by realizing SARS-CoV-2 reproducibility using recombinant SARS-CoV-2 protein.

The present invention created a reproduction model using Recombinant SARS-CoV-2 to reproduce SARS-CoV-2, and measured the immune response caused by IL-2 neutralizing antibodies in inbred mice using flow cytometry.

Recombinant SARS-CoV-2 (B.1.617.2 S Alexa Fluor® 647 Protein (Covid-19 spike protein)) was used as a similar protein for clinical reproduction of SARS-CoV-2.

In the present invention, the ACE2 protein is characterized as a protein derived from the extracellular domain (ectodomain) of the ACE2 protein.

In the present invention, the immune cells may be Natural Killer cells (NK cells) or T cells, but are not limited thereto.

Interleukin-2 (IL-2) antibodies increase the activity of regulatory T cells (Treg).

Ace2 stands for angiotensin-converting enzyme 2, and is a carboxypeptidase related to angiotensin-converting enzyme (ACE). Ace2 is a functional receptor for the coronavirus related to severe acute respiratory syndrome (SARS). When SARS-CoV-2 enters the human body, infection occurs by binding to Ace2 to the spike protein of SARS-CoV-2.

The Ace2 protein consists of 805 amino acids and is divided into an ectodomain, a membrane spanning domain, and a cytoplasmic domain. Among these, the extracellular domain of the Ace2 protein is a water-soluble protein consisting of about 600 amino acids and has high affinity with the spike protein of the SARS-CoV-2 virus. The inventors of the present invention took advantage of the characteristic of the protein derived from the extracellular domain of Ace2 to strongly bind to the spike protein of SARS-CoV-2 to prevent the interaction and infection of the SARS-CoV-2 virus with target cells. It was discovered that it was possible.

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

The present invention is intended to solve the instability problem of the Ace2 protein, and the stabilized Ace2 variant of the present invention is a mutation to introduce a disulfide bond into the Ace2-derived protein, as shown in Figure 1.

The inventors of the present invention improved the thermal and structural stability of the Ace2 protein by substituting one or more specific amino acid residue pairs of the Ace2 protein with cysteine to form a structurally stable disulfide bond, making it stable even in the water-soluble Ace2 state and preventing the formation of a disulfide bond. We developed a variant that can maintain high binding affinity to the virus by minimizing structural modifications.

In the present invention, the term "disulfide bond" refers to a state in which the sulfide (-SH) group of cysteine among protein amino acids meets the 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 cut out through genetic manipulation to form a protein that is stable in aqueous solution rather than a wild-type membrane protein. In this way, soluble receptors made from virus receptors can be used to prevent virus invasion into cells.

In the present invention, the term “mutation” 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 can be indicated by the residue of the parent amino acid sequence, the number of the amino acid residue, and the order of the substituted amino acid residue.

In the present invention, the mutation for introducing the disulfide bond may include a mutation in which one or more amino acid residue pairs present in the Ace2-derived protein are replaced with cysteine. At this time, 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 It is preferable that the distance between -alpha) is 45 to 70 Å.

In an embodiment of the present invention, through three-dimensional structural analysis of the Ace2 protein, the distance between the C-alpha residue pairs is within the range of 45 to 70 Å, and the loop 331-347 region, loop 599-614 region of the Ace2 protein, It was confirmed that it was a pair of residues involved in stabilizing the helix-helix region and the strand-helix region. In addition, when a disulfide bond was formed in the above amino acid residue pair, the melting temperature of the protein was significantly increased compared to the wild type, but the binding force with the virus was almost unaffected. Accordingly, it was confirmed that, according to the variant of the present invention, structural distortion due to disulfide bond formation is minimized and improved structural stability can be achieved compared to the wild-type Ace2 protein while maintaining excellent binding affinity to the virus, which is a characteristic of Ace2. .

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

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

In the present invention, the Fc domain derived from the immunoglobulin refers to the C-terminal region including the CH2 and CH3 domains (or CH2, CH3, and CH4 domains) of the heavy chain constant region of the immunoglobulin, and is a wild-type domain. It is used to encompass the Fc domain and its variants. The immunoglobulin that serves 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 of the human IgG1 heavy chain to the C-terminus, or a region further including a hinge in this region. The numbering of amino acid residues in the Fc region is according to the EU numbering (see Kabat et al, etc.), which defines the numbering of residues in human immunoglobulin heavy chains.

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

At this time, the Ace2 variant and the Fc domain may be connected by 0 to 20 amino acid residues. That is, the Ace2 variant and the Fc domain may be connected directly or may be connected through a linker consisting of 1 to 20 amino acids. Additionally, 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 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 may be formed to form a dimer in the Fc domain. there is.

For example, knobs-into-hole technology can be used as a recombinant variant to form the dimer.

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

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

In this way, when the stabilized Ace2 variant of the present invention is fused with the Fc domain, the Ace2 variant not only binds strongly to the virus and can inhibit infection, but also induces an immune response to eliminate the virus, and interacts with the Fc receptor. The efficacy of the drug can be enhanced by extending its retention period in the body. In addition, by using this to produce bispecific or multispecific antibodies, NK cells or immune cells can be induced to infected cells, and virus-derived diseases can be treated through active killing of infected cells and activation of immune response.

In the present invention, the term "bispecific (or multispecific) antibody" refers to an antibody that can bind to two (or more) different types of antigens, and includes forms produced through 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 death of the target antigen through 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 engager (BiTE), 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 directly or indirectly, and may preferably be a Natural Killer cell (NK cell) or T cell.

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

Using such a bispecific (or multispecific) antibody, immune cells can recognize SARS-CoV-2 and induce neutralization of the virus through antibody dependent cellular cytotoxicity (ADCC).

Using the stabilized Ace2 variant of the present invention, immune cells can effectively recognize and kill SARSCoV-2 due to the high binding affinity between SARS-CoV-2 and Ace2, and because Ace2 is structurally stable, it can be stored when applied to a therapeutic agent. It has excellent stability against environmental changes and maintains a structure effective in treating diseases in the human body for a long period of time, allowing long-term efficacy even at low doses. In addition, even if a virus mutates, the region that recognizes the cell receptor on the surface antigen does not change, so it has the advantage of being effective in killing viruses against a variety of strains.

IL-2Rγ

Interleukin-2 receptor subunit gamma also has CD132; Common cytokine receptor γc-chain; IL-2RG; IL-2Rg; IL2Rgamma; IL-2Rγ, IMD4; P64:SCIDX; or known as SCIDX1. IL2Rγ is a subunit common to several interleukin receptors, including IL-2R, IL-4R, IL-7R, IL-9R, IL-15R, and IL21R.

antigen-binding protein

The present invention relates to antigen-binding proteins, such as antibodies (e.g., human antibodies, monoclonal antibodies and recombinant antibodies) that specifically bind to the IL2Rγ protein or antigen fragments thereof (e.g., the extracellular domain of IL2Rγ). Antibodies) and antigen-binding fragments thereof are provided. Antigen-binding proteins that bind to the same epitope on IL2Rγ or that compete for the binding of IL2Rγ with any of the antigen-binding proteins presented herein are also part of the invention.

As used herein, the term “antibody” refers to four polypeptide chains (i.e., “complete antibody molecules”), two heavy (HC) and two light (LC) chains interconnected by disulfide bonds (e.g. refers to immunoglobulin molecules, including IgG).

In an embodiment of the invention, an anti-IL2Rγ antigen-binding protein, e.g., an antibody or antigen-binding fragment, is e.g., type IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g. For example, the heavy chain constant domain of IgG1, IgG2, IgG3 and IgG4 (e.g., comprising S228P and/or S108P mutations) or IgM. In an embodiment of the invention, an antigen-binding protein, e.g. The antibody or antigen-binding fragment comprises, for example, a light chain constant domain of kappa or lambda.

As used herein, the term "human" antigen-binding protein, such as antibody or antigen-binding fragment, refers to an immune system of the human germline, whether in human cells or transplanted into non-human cells, e.g., mouse cells. Includes antibodies and fragments having variable and constant regions derived from globulin sequences, see, e.g., US8502018,

See US6596541 or US5789215). In embodiments of the invention, human antibodies and antigen-binding fragments of the invention may contain, for example, amino acid residues in the CDRs and especially CDR3 that are not encoded by human germline immunoglobulin sequences (e.g., in vitro random or having mutations caused by site-directed mutagenesis or by somatic mutations in vivo. However, the term “human antibody” as used herein is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted with human FR sequences. The term includes antibodies produced recombinantly in non-human mammals, or cells of non-human mammals. The term is not intended to include antibodies isolated from or produced in human subjects.

The term "recombinant" antigen-binding protein, as in antibody or antigen-binding fragment thereof, is produced by techniques or methods known in the art by recombinant DNA techniques, including DNA splicing and transgene expression, or Refers to the molecule expressed, isolated or obtained.The term refers to a non-human mammal (including a non-human transgenic mammal, e.g., a transgenic mouse), or a host cell (e.g., a Chinese hamster). antibodies expressed in ovarian (CHO) cells) or cell expression systems or isolated from recombinant combinatorial human antibody libraries.

The antigen-binding fragment of an antibody in embodiments of the invention comprises at least one variable domain. The variable domain may be a domain of any size or amino acid composition and will generally include at least one (e.g., three) CDRs adjacent to or framed by one or more framework sequences. In an antigen-binding fragment having a VH domain associated with a VL domain, the VH and VL domains may be positioned in any suitable alignment relative to each other.

For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain non-covalently linked monomeric VH and/or VL domains.

The present invention includes antigen-binding proteins that specifically bind to IL2Rγ protein. In one embodiment of the invention, the anti-IL2Rγ antigen-binding protein is selected from Table 3- for binding to human and/or mouse and/or Macaca fascicularis and/or rat IL2Rγ or domains thereof. Includes any KD values given in 1 to 3-12. “Anti-IL2Rgamma” refers to an antigen-binding protein (or other molecule), e.g., an antibody or antigen-binding fragment thereof that specifically binds IL2Rgamma.

“Isolated” antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof), polypeptides, polynucleotides and vectors are, at least in part, free from other biological molecules of cell or cell culture origin from which they are produced. Biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other substances, such as cell debris and growth media.Isolated antigen-binding proteins may also be used as biological molecules from host cells. Expression system components, such as molecules or their growth medium, may be at least partially absent.Generally, the term "isolated" refers to the complete absence of said biological molecule (e.g., small or trace impurities may remain) or It refers to a component of a pharmaceutical formulation that contains an antigen-binding protein (e.g., an antibody or antigen-binding fragment) or in the absence of water, buffer or salt.

An antigen is, for example, a molecule to which an antibody binds, e.g., a peptide (e.g., IL2R gamma or a fragment thereof (antigenic fragment)). The specific region on an antigen that an antibody recognizes and binds to is called an epitope. Antigen-binding proteins (e.g., antibodies) of the invention that specifically bind to the antigen are part of the invention.

The term “epitope” refers to a specific antigen-binding site of an antigen-binding protein, known as a paratope, e.g., an antigenic determinant that interacts with the variable region of an antibody molecule (e.g., on IL2Rγ). Refers to. A single antigen may have more than one epitope.Therefore, different antibodies may bind to different regions on the antigen and may have different biological effects.The term "epitope" is also used to refer to B and/or T cells. It may refer to a region on an antigen that reacts and/or a region of the antigen bound by an antibody.An epitope may also be structurally or functionally defined.

Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of interaction. Epitopes may have a linear or three-dimensional structure, i.e., may be composed of non-linear amino acids.

In certain embodiments, an epitope may comprise a crystalline group that is a chemically active surface group of a molecule, such as an amino acid, sugar side chain, phosphoryl group, or sulfonyl group, and, in certain embodiments, specific three-dimensional structural properties and/or Alternatively, it may have specific charge characteristics. The epitope to which the antigen-binding protein of the present invention binds may be included in IL2Rγ, for example, a fragment of human IL2Rγ, for example, the ectodomain, domain 1 or domain 2 thereof. Antigen-binding proteins (e.g., antibodies) of the invention that bind to the above epitopes are part of the invention.

The present invention relates to the activity of IL2Rγ to any detectable degree (e.g., from binding to cytokines such as IL-2, IL-4, IL-7, IL-9, IL-15 and/or IL-21). “Neutralizing” or “antagonist” anti-IL2Rγ antigen-binding proteins, including molecules that inhibit the binding of a hybrid receptor containing IL2Rγ complexed with a specific receptor subunit (e.g., an antibody or antigen-binding protein) includes fragments).

Eukaryotic and prokaryotic host cells, including mammalian cells, can be used as hosts for expression of anti-IL2Rγ antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof). These host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells.

Several methods for producing recombinant antibodies are known in the art. One example of a method for recombinant production of antibodies is described in US4816567.

Transformation may be by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, polynucleotides in liposomes ( s), biolistic injection, and microinjection of DNA into the nucleus. Additionally, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods for transforming cells are well known in the art.

The method of controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice induced to overexpress the ACE2 protein according to the present invention is to use IL-2 neutralizing antibodies in mice expressing human ACE2. It can inhibit the activity of IL-2 receptor.

Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the following examples and experimental examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

<Example>

Preparation of stabilizing Ace2 variants

A variant in which a disulfide bond was introduced into the residue pair causing instability in the Ace2 protein structure was designed and is shown in Table 1 below. In addition, in the amino acid residue pairs subject to mutation, the C-alpha distance and stabilization mechanism between amino acids are shown in Table 1.

[Table 1]

<Experimental Example> Measurement of binding force 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 25 μg/ml in a buffer solution containing 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 powder. Ace2(N51C/V343C)-Fc protein and wild-type Ace2(WT)-Fc protein were diluted to respective concentrations 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 human Fc antibodies was added diluted in 5% (w/v) skim milk powder and reacted at room temperature for 2 hours. At the end of each process, the wells were washed three times with phosphate buffer solution.

After completing the reaction, tetramethylbenzidine (Goma Biotech) solution was added to each well and allowed to develop color. Then, 0.2 M sulfuric acid solution was added to stop the reaction. The absorbance of the reacted solution was measured at 450 nm, and the results are shown in Figure 2. In Figure 2, at each concentration, the left bar represents the results for wild-type Ace2(WT)-Fc protein, and the right bar represents the results for stabilized Ace2(N51C/V343C)-Fc protein.

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

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

Meanwhile, the above detailed description should not be construed as restrictive in all respects and should be considered illustrative. The scope of the present invention should be determined by reasonable interpretation of the appended claims, and all changes within the equivalent scope of the present invention are included in the scope of the present invention.

Claims (1)

Creating an inbred mouse in which overexpression of ACE2 (Angiotensin-converting enzyme2) protein is induced; and
Controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice induced to overexpress the ACE2 (Angiotensin-converting enzyme2) protein;
A method of controlling the activity of immune cells through neutralization control of the interleukin-2 (IL-2) receptor using inbred mice induced to overexpress the ACE2 protein, including.