Novel il10ra mutant protein and use thereof for treating inflammatory diseases

Abstract

An IL10RA mutant protein according to one aspect of the present invention solves the issue of commercial IL10RA proteins being impossible to purify, and is a protein with improved purification efficiency. The IL10RA mutant protein is expressed at a similar level similar to the wild-type IL10RA protein, is normally located in the cell membrane, and exhibits the same level of anti-inflammatory signaling activity as the wild-type protein upon IL-10 stimulation, and thus can be effectively used as a protein replacement therapy for the treatment of inflammatory diseases.

Description

Novel IL10RA variant protein and its use in the treatment of inflammatory diseases

[0001] The present invention relates to a novel IL10RA variant protein and its use in the treatment of inflammatory diseases.

[0002] Interleukin-10 (IL-10) is a representative anti-inflammatory cytokine that is secreted by activated immune cells to suppress inflammatory responses and maintain tissue homeostasis. The biological action of IL-10 primarily begins by binding to a heterodimeric receptor complex composed of IL10RA (Interleukin-10 receptor subunit alpha) and IL10RB (Interleukin-10 receptor subunit beta).

[0003] IL10RA is a cell membrane protein directly involved in the ligand binding of IL-10, and consists of an extracellular ligand-binding domain, a transmembrane domain that penetrates the cell membrane, and a cytoplasmic signal-transducing domain that mediates the JAK1 and STAT3 pathways. When IL-10 binds to IL10RA, a receptor complex is formed, and JAK1 and TYK2 kinases are activated, inducing STAT3 phosphorylation and thereby promoting the expression of anti-inflammatory genes.

[0004] However, due to the membrane protein nature of IL10RA, it has strong hydrophobic regions and is structurally unstable, which presents a problem in that it is difficult to purify it into a functional form outside the cell.

[0005] Accordingly, commercial IL10RA proteins reported to date are primarily produced and purified in the form of truncated proteins, in which a portion of the extracellular domain is truncated. This has limitations in that it cannot sufficiently reproduce the structural integrity and physiological signaling functions of the full-length protein. In particular, truncated IL10RA proteins are unable to accurately localize to the cell membrane or exhibit incomplete JAK / STAT signaling after IL-10 binding, which has limited their commercialization as therapeutic proteins for inflammatory diseases. Therefore, there is a need to develop a structurally stable IL10RA protein that is easy to purify while maintaining the original function of the wild-type IL10RA protein.

[0006] The inventors constructed a gene vector capable of expressing a mutant IL10RA protein that maintains anti-inflammatory signaling activity equivalent to that of wild-type IL10RA, while improving protein expression and purification efficiency by substituting specific amino acid residues within the signaling domain of the IL10RA protein.

[0007] The IL10RA variant protein according to the present invention maintains the functional activity of the wild-type protein while improving structural stability and productivity, and thereby can be utilized as a protein replacement therapy for the treatment of inflammatory diseases.

[0008] One aspect is to provide an IL10RA variant protein in which the amino acids at the 3rd, 5th, and 6th positions of the wild-type IL10RA (Interleukin-10 receptor subunit alpha) protein are substituted with other amino acids.

[0009] Another aspect is to provide a nucleotide encoding the above IL10RA variant protein and a recombinant vector containing it.

[0010] Another aspect is to provide host cells transformed with the aforementioned recombinant vector.

[0011] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease (IBD) comprising the above-mentioned IL10RA variant protein.

[0012] Another aspect provides a method for preventing or treating an inflammatory disease comprising the step of administering an effective amount of an IL10RA variant protein in which the amino acids at the 3rd, 5th, and 6th positions of the wild-type IL10RA (Interleukin-10 receptor subunit alpha) protein are substituted with other amino acids to an individual in need.

[0013] Another aspect is to provide for the use of an IL10RA variant protein in which the amino acids at the 3rd, 5th, and 6th positions of the wild-type IL10RA (Interleukin-10 receptor subunit alpha) protein are substituted with other amino acids for use in the manufacture of pharmaceutical preparations for the prevention or treatment of inflammatory diseases.

[0014] Another aspect is to provide for the use of an IL10RA variant protein in which the amino acids at the 3rd, 5th, and 6th positions of the wild-type IL10RA (Interleukin-10 receptor subunit alpha) protein are substituted with other amino acids to prevent or treat inflammatory diseases.

[0015] To achieve the above objective, one aspect provides an IL10RA variant protein in which the amino acids at the 3rd, 5th, and 6th positions of the wild-type IL10RA (Interleukin-10 receptor subunit alpha) protein are substituted with other amino acids.

[0016] The terms "IL10RA (Interleukin-10 receptor subunit alpha)" or "IL-10 receptor α-chain" in this specification refer to a protein constituting the α-chain (subunit α) of the IL-10 receptor complex, which specifically binds to the cytokine interleukin-10 (IL-10) in humans to mediate intracellular anti-inflammatory signaling. The IL10RA protein is a type I single-pass transmembrane protein composed of an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic signal-transducing domain, and mediates an anti-inflammatory response by activating the JAK1-STAT3 pathway through binding to IL-10. UniProtKB Accession No. The amino acid sequence registered as Q13651 (standard form, 578 aa) is used as the basic sequence. However, the present invention includes physiologically acceptable isoforms, allelic variants, or functionally equivalent derivatives corresponding to the standard form.

[0017] In this specification, the term “mutant protein” refers to a protein that has an amino acid sequence different from that of a wild-type protein by modifying one or more of the amino acid sequences of the wild-type protein through substitution, deletion, or insertion. The mutant protein may include, for example, a protein in which a specific amino acid residue is substituted with another amino acid (substitution mutant), a protein in which one or more residues are deleted (deletion mutant), or a protein in which one or more residues are added (insertion mutant). In this specification, “mutant protein” is understood to include proteins that are physiologically or functionally equivalent to the wild-type protein, for example, proteins that have undergone conservative substitution, isoforms, or allelic variants.

[0018] In one embodiment, the wild-type IL10RA protein may include a signal-transducing domain.

[0019] In one embodiment, the wild-type IL10RA protein may have the amino acid sequence of SEQ ID NO. 2.

[0020] In one embodiment, the other amino acid is any one selected from the group consisting of arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine ​​(C), selenocysteine ​​(U), glycine (G), proline (P), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), tryptophan (W), and all variants of said amino acids, and may be an amino acid excluding the amino acid that the wild-type IL10RA protein has at the mutation site, but is not limited thereto.

[0021] In one embodiment, the IL10RA variant protein may have amino acids at positions 3, 5, and 6 of the wild-type IL10RA (Interleukin-10 receptor subunit alpha) protein substituted with other amino acids and have at least 80% identity or homology with the amino acid sequence of SEQ ID NO. 4, but is not limited thereto.

[0022] For example, the above IL10RA variant protein may have amino acids at positions 3, 5, and 6 of the wild-type IL10RA (Interleukin-10 receptor subunit alpha) protein substituted with other amino acids, and may have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity or homology with the amino acid sequence of SEQ ID NO. 4.

[0023] In one embodiment, the IL10RA variant protein may have the amino acid sequence of SEQ ID NO. 4.

[0024] In one embodiment, the IL10RA variant protein may have increased purification efficiency compared to the wild-type IL10RA protein.

[0025] The term “purification” in this specification refers to a series of processes for isolating a target protein from cells or cell culture media, etc., and improving the purity of the protein. It encompasses all protein separation and purification processes including the physical and chemical methods described above, and the purpose is to improve the purity and yield of the target protein by removing non-protein impurities or other protein contaminants.

[0026] The term "protein with increased purification efficiency" in this specification refers to a protein from which a higher purity can be obtained from the same expression level compared to a wild-type protein, or in which the same purity can be achieved in fewer steps or in a shorter time.

[0027]

[0028] Another aspect provides a nucleotide encoding the IL10RA mutant protein.

[0029] The above polynucleotide may be a single-stranded or double-stranded having a base sequence, may be DNA or RNA, and the above polynucleotide may be isolated from nature or prepared by chemical synthesis.

[0030] In the present invention, the nucleotide capable of encoding the IL10RA variant protein can be artificially synthesized using a DNA synthesizer or the like according to known methods, or can be prepared by performing a polymerase chain reaction (PCR) using genomic DNA of a gene encoding the IL10RA protein as a template and oligonucleotides having base sequences complementary to both ends of the gene capable of encoding the target IL10RA variant protein as primers. The prepared nucleotide can subsequently be used in a recombinant molecular biological system capable of expressing the target IL10RA variant protein through cloning, sequence verification, and expression vector insertion processes.

[0031] Meanwhile, the gene capable of encoding the IL10RA variant protein of the present invention may be implemented in various nucleotide sequences according to codon optimization, and all nucleotide sequence variants capable of encoding said variant protein are also included within the scope of the present invention. Specifically, the gene capable of encoding said IL10RA variant protein may include a nucleotide sequence having sequence homology or sequence identity of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with the nucleotide sequence described in SEQ ID NO. 3. As such, it is understood that the gene capable of encoding the variant protein of the present invention includes all sequences that encode the same amino acid sequence or express a functionally equivalent protein regardless of codon differences.

[0032] The “% of sequence homology” for the above polynucleotide is determined by comparing two optimally arranged sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal arrangement of the two sequences.

[0033]

[0034] Another aspect provides a recombinant vector containing a nucleotide encoding the above-mentioned IL10RA mutant protein.

[0035] In the present invention, the recombinant vector can be any type of recombinant vector, provided that a polynucleotide capable of encoding a mutant IL10RA protein is inserted into an appropriate vector.

[0036] The term "vector" in this specification refers to a self-replicating DNA molecule used to carry a clonal gene (or other fragment of clonal DNA). The vector may be, but is not limited to, a plasmid, a phage particle, or simply a potential genomic insert. When the vector is transformed into a suitable host, it may replicate and function independently of the host genome, or parts of it may be incorporated into the genome itself.

[0037] Specifically, it refers to a plasmid, virus, or other medium into which the polynucleotide sequence encoding the IL10RA variant protein can be inserted or introduced. For example, the plasmids include plasmids derived from E. coli (pBR322, pBR325, pUC118 and pUC119, pET-22b(+)), plasmids derived from Bacillus subtilis (pUB110 and pTP5), and plasmids derived from yeast (YEp13, YEp24, and YCp50), and the viruses may include animal viruses such as retroviruses, adenoviruses, or vaccinia viruses, or insect viruses such as baculoviruses. Specifically, a vector suitable for introducing the polynucleotide that carcinogenizes the IL10RA variant protein of the present invention into a host cell may be used, and preferably, an expression vector designed to facilitate the induction of protein expression and the isolation of the expressed protein may be used.

[0038] The term "expression vector" in this specification refers to a recombinant DNA molecule comprising a nucleic acid sequence essential for expressing a target sequence and an coding sequence linked so as to be operable in a specific host organism. Specifically, the polynucleotide sequence encoding the IL10RA variant protein according to the present invention may be operablely linked to an expression regulatory sequence, and the operablely linked gene sequence and the expression regulatory sequence may be included in a single expression vector that also includes a selection marker and a replication origin.

[0039] The expression vector may preferably include one or more selectable markers. The marker is a nucleic acid sequence having characteristics that can typically be selected by chemical means, and includes any gene capable of distinguishing transformed cells from non-transformed cells. Examples include, but are not limited to, antibiotic resistance genes such as ampicillin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol, and can be appropriately selected by a person skilled in the art.

[0040] The term “operably linked gene sequence” in this specification may be a gene and an expression control sequence linked in such a manner that gene expression is enabled when a suitable molecule is bound to the expression control sequence. The “expression control sequence” means a DNA sequence that controls the expression of an operably linked polynucleotide sequence in a specific host cell. Such a control sequence includes a promoter for carrying out transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0041]

[0042] Another aspect provides a host cell transformed with the above-mentioned recombinant vector.

[0043] The recombinant vector comprising a polynucleotide encoding the IL10RA variant protein of the present invention can be introduced into a host cell using methods known in the art. For example, methods for introducing the recombinant vector according to the present invention into a host cell may include, but are not limited to, calcium chloride (CaCl2) and heat shock methods, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, and precipitation with PEG (polyethylenglycol).

[0044] In one embodiment, the host cell may be a prokaryotic cell or a eukaryotic cell depending on the type of recombinant vector.

[0045]

[0046] Another aspect provides a method for producing an IL10RA variant protein with improved purification efficiency, comprising the step of culturing a transformed host cell in a medium to express the IL10RA variant protein.

[0047] The above method comprises culturing the transformed host cells under appropriate media and conditions so that a polynucleotide encoding the IL10RA variant protein of the present invention is expressed within the transformed cells. A method for culturing the transformed cells to express a protein is known in the art; for example, protein expression can be induced by inoculating the transformed cells into a suitable medium for growth and performing a seed culture, and then inoculating the cells into a medium for main culture and culturing them under suitable conditions.

[0048] The term "culture" in this specification refers to growing microorganisms under appropriately artificially controlled environmental conditions. The recombinant microorganisms can be grown in ordinary media, and, for example, can be cultured in a nutrient broth medium. The medium contains nutrients required by the microorganisms to be cultured, i.e., the microorganisms serving as the culture medium, in order to cultivate specific microorganisms, and may contain substances for special purposes that are additionally added and mixed. The medium is also referred to as a culture medium or culture solution, and is a concept that encompasses natural media, synthetic media, and selective media. The recombinant microorganisms can be cultured according to ordinary culture methods.

[0049] Specifically, the medium used for the above culture must satisfy the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc., within a conventional medium containing suitable carbon sources, nitrogen sources, amino acids, vitamins, etc. Carbon sources that can be used include a mixed sugar of glucose and xylose as the primary carbon source, and additionally include sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances may be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products may be used. These nitrogen sources may be used alone or in combination. The medium may contain monopotassium phosphate, dipotassium phosphate, and corresponding sodium-containing salts as phosphorus. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts may be used as phosphorus. Additionally, inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate may be used. Finally, essential growth substances such as amino acids and vitamins may be used in addition to the above materials.

[0050] In addition, suitable precursors may be used in the culture medium. The aforementioned raw materials may be added to the culture in a batch, fed-batch, or continuous manner in a manner suitable for the culture process, but are not particularly limited thereto. The pH of the culture may be controlled by using basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia, or acid compounds such as phosphoric acid or sulfuric acid in a suitable manner.

[0051] For details regarding the genetic engineering technology used in the present invention, reference can be made to the literature of Sambrook et al. (Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (2001)) and the literature of Frederick et al. (Frederick M. Ausubel et al., Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994)).

[0052] Another aspect provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the above-mentioned IL10RA variant protein.

[0053] In one embodiment, the inflammatory disease is psoriasis, psoriatic arthritis, rheumatoid arthritis (RA), hepatitis, asthma, inflammatory bowel disease (IBD), vasculitis, pneumonia, dermatitis, mouth ulcers, uveitis, temporal arteritis, atopic dermatitis, systemic lupus, multiple sclerosis or sarcoidosis, synovitis, meningitis, encephalitis, Bickerstaff's encephalitis, encephalomyelitis, spondylitis, osteomyelitis, Guillain-Barré syndrome, It may be any one selected from the group consisting of neuromyelitis optica, cystitis, nephritis, and glomerulonephritis, but is not limited thereto.

[0054] The term "Inflammatory Bowel Disease (IBD)" in this specification means an autoimmune or immune-mediated disease in which chronic inflammation occurs in the mucosa and submucosa of the gastrointestinal tract or intestinal tract.

[0055] In one embodiment, the inflammatory bowel disease may be one or more selected from the group consisting of Crohn's disease, ulcerative colitis, indeterminate colitis, intestinal Behcet's disease, autoimmune enteropathy, and monogenic inflammatory bowel disease related to IL10 / IL10RA, but is not limited thereto. It is understood that the "inflammatory bowel disease" of the present invention includes the acute or chronic phase of the above diseases and prodromal or variant forms of inflammatory bowel disease caused by immune abnormalities, intestinal epithelial damage, or dysbiosis.

[0056] The term "treatment" in this specification may be used to mean both therapeutic treatment and preventive treatment. In this context, prevention may be used to mean alleviating or reducing a pathological condition or disease of an individual. In one embodiment, the term "treatment" includes all applications or any form of medication for treating a disease in mammals, including humans. Furthermore, the term includes inhibiting or slowing the progression of a disease or disease; restoring or repairing damaged or deficient functions to partially or completely alleviate the disease; or stimulating inefficient processes; or alleviating a severe disease.

[0057] The term "pharmaceutical composition" in this specification means a preparation comprising an active agent or active ingredient for treatment, prevention, or diagnosis, a formulation configured to allow the active agent to be administered in an appropriate form, and auxiliary components (excipients, carriers, diluents, etc.).

[0058] A pharmaceutical composition according to one aspect may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, according to conventional methods, and may include a suitable carrier, excipient, or diluent that is conventionally used in the manufacture of pharmaceutical compositions for formulation. The carrier, excipient, or diluent may include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, it can be manufactured using diluents or excipients such as commonly used fillers, weights, binders, wetting agents, disintegrants, and surfactants.

[0059] Solid dosage forms for oral administration can be prepared by mixing at least one excipient, such as starch, calcium bonate, sucrose or lactose, gelatin, etc., with the above-mentioned legume extract. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin.

[0060] Preparations for parenteral administration include sterile aqueous solutions, water-insoluble preparations, suspensions, emulsions, lyophilized preparations, and suppositories. As water-insoluble solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, Witepsol, Macrogol, Tween 20, cocoa gel, laurin gel, glycerol gelatin, etc. may be used.

[0061] A pharmaceutical composition according to one aspect of the present invention may be administered to mammals, including rats, mice, livestock, and humans, by various routes. The route of administration of the pharmaceutical composition is not particularly limited and may be administered, for example, orally, rectally, intravenously, intramuscularly, or subcutaneously. Additionally, the composition may be administered by other pharmaceutically acceptable routes, such as intradermal, intraperitoneal, nasal, ocular, or topical administration, as needed.

[0062] The preferred route of administration, dosage, and frequency of administration of a pharmaceutical composition according to one aspect may be administered to a subject in various ways and amounts depending on the patient's condition and the presence or absence of side effects, and the optimal method of administration, dosage, and frequency of administration can be selected within an appropriate range by a person skilled in the art.

[0063] An IL10RA variant protein according to one aspect of the present invention is a protein with improved purification efficiency that resolves the problem of impossibility of purifying commercial IL10RA proteins. It is expressed at a level similar to that of the wild-type IL10RA protein, is normally located on the cell membrane, and exhibits anti-inflammatory signaling activity equivalent to that of the wild-type protein upon IL-10 stimulation, and can be usefully utilized as a protein replacement therapy for the treatment of inflammatory diseases.

[0064] Figure 1 is a graph showing the results of measuring the cell surface expression levels of wild-type (WT) IL10RA protein and IL10RA variant protein using FACS analysis.

[0065] Figure 2 is an image showing the results of observing the cell membrane expression locations of GFP-tagged wild-type (WT) IL10RA protein and IL10RA variant protein using a confocal microscope.

[0066] Figure 3 shows Western blot band images confirming the expression of p-STAT3, STAT3, and IL10RA proteins (left), and a graph of the quantitative analysis of the p-STAT3 / STAT3 ratio (right).

[0067] The following examples will be explained in more detail. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0068]

[0069] Example 1. Construction of IL10RA variant gene vector

[0070] A gene vector was constructed to produce a mutant IL10RA protein by introducing amino acid substitutions to minimize changes in the structural or physicochemical properties of the protein while maintaining the original physiological function of the wild-type (IL10RA WT) protein.

[0071] Specifically, total RNA (1.5 μg) was isolated from peripheral blood mononuclear cells (PBMCs) collected from healthy donors using QIAzol RNA isolation reagent (Qiagen). The isolated RNA was reverse transcribed into complementary DNA (cDNA) using SuperScript II reverse transcriptase (Invitrogen). Using the synthesized cDNA (50 ng) as a template, the human IL10RA full-length coding sequence was amplified. Primers (IL10RA EcoRI F and IL10RA KpnI R) designed to include an EcoRI restriction enzyme recognition sequence at the 5' end and a KpnI restriction enzyme recognition sequence at the 3' end, respectively, were used, and PCR was performed using Phusion DNA polymerase (NEB).

[0072] The amplified PCR product was cleaved with EcoRI and KpnI restriction enzymes and inserted into the pEGFP-N1 vector (Clontech), which had been cleaved with the same restriction enzymes. The vector expresses the enhanced green fluorescent protein (EGFP) gene and is designed to have a GFP tag attached to the C-terminus of the cloned IL10RA gene. Therefore, the protein expressed using this vector is expressed as C-terminal GFP-labeled IL10RA. The recombinant plasmid was confirmed by sequencing using the pEGFPN1 seq F and pEGFPN1 seq R primers, and the confirmed plasmid was named pEGFP-N1-IL10RA.

[0073] The above IL10RA variant gene was designed and synthesized at the nucleotide level based on the coding sequence of the wild-type IL10RA described in SEQ ID NO. 1, by replacing the codon encoding the 3rd amino acid, Proline, with the codon encoding Glycine, the codon encoding the 5th amino acid, Leucine, with the codon encoding Valine, and the codon encoding the 6th amino acid, Valine, with the codon encoding Leucine, respectively, within the region encoding the signal transduction domain of the coding sequence. The nucleotide sequences encoding the wild-type IL10RA protein and the IL10RA variant protein, as well as the amino acid sequences of the wild-type IL10RA protein and the IL10RA variant protein, are shown in Table 1.

[0074]

[0075] [Table 1]

[0076]

[0077]

[0078] Experimental Example 1. Confirmation of the expression level of the IL10RA variant protein

[0079] The expression level of the IL10RA variant protein of Example 1 was measured using FACS (Fluorescence-Activated Cell Sorting).

[0080] Specifically, HEK 293T cell lines (CRL-3216, ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM; Cytiva, Massachusetts, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 1% penicillin-streptomycin, and 2.05 mM L-glutamine, and maintained at 37°C and 5% CO₂. After transforming HEK 293T cells with the pEGFP-IL10RA expression plasmid, the cells were harvested and washed twice with PBS. Under impermeable conditions, the cells were treated with an APC-conjugated isotype control (RTK2758; BioLegend) and an APC-conjugated IL10RA antibody (3F9; BioLegend), respectively, and washed twice with PBS. Since the GFP fluorescence signal indirectly reflects the transformation and protein biosynthesis of the pEGFP-IL10RA plasmid, it was used as an indicator of the total intracellular IL10RA protein expression level. GFP-positive cells were detected using a FITC channel with a wavelength of 488 nm, and the anti-IL10RA antibody signal was measured via the APC fluorescence channel to confirm the membrane expression of IL10RA protein among these cells. The membrane expression rate was calculated as the percentage of APC-positive cells within the GFP-positive cell population. As a result, it was confirmed that the IL10RA WT ratio was significantly higher in HEK 293T cells expressing the IL10RA variant protein.

[0081] The results are shown in Figure 1.

[0082] Figure 1 is a graph showing the results of measuring the cell surface expression levels of wild-type (WT) IL10RA protein and IL10RA variant protein using FACS analysis.

[0083] As shown in Figure 1, when comparing the cell surface expression levels of IL10RA protein through FACS analysis, the positive signal rate in wild-type (WT) IL10RA protein-expressing cells was approximately 9.9%, whereas it increased to approximately 14.5% in IL10RA-positive variant protein-expressing cells.

[0084] This means that the expression level of the IL10RA variant protein according to one aspect of the present invention is equivalent to or even improved compared to the wild-type protein, and that the cell membrane expression ability or structural stability of the protein is not reduced by the introduction of the variant.

[0085]

[0086] Experimental Example 2. Confirmation of the cell membrane expression site of the IL10RA mutant protein

[0087] To confirm whether the IL10RA variant protein of Example 1 is properly located on the cell membrane, imaging analysis of GFP-IL10RA on the membrane was performed using a confocal microscope.

[0088] Specifically, to express GFP-tagged IL10RA WT or IL10RA variants in HEK 293T cells, 50,000 cells were seeded into each well of a 12-well plate, and 2 μL of Lipofectamine 2000 (ThermoFisher, Waltham, MA) was mixed with 1 μg each of pEGFP, pEGFP-IL10RA-WT, and pEGFP-IL10RA mutant plasmids. Transfection was performed for 6 hours. Afterward, the culture medium was replaced with normal medium (DMEM containing 10% FBS). After 24 hours, imaging was performed using a confocal microscope for analysis.

[0089] The results are shown in Figure 2.

[0090] Figure 2 is an image showing the results of observing the cell membrane expression sites of GFP-tagged wild-type (WT) IL10RA protein and IL10RA mutant protein (P3G / L5V / V6L) using a confocal microscope. In Figure 2, the green fluorescence signal represents the IL10RA protein, the red fluorescence represents the cell membrane, and the blue fluorescence represents the nucleus.

[0091] As shown in Figure 2, when cells expressing the wild-type IL10RA and IL10RA variant proteins, respectively, were stained with a fluorescently labeled antibody, a strong green fluorescent signal was observed in the cell membrane region for both proteins. In particular, the fluorescent signal of the IL10RA variant protein exhibited a distribution pattern similar to that of the wild-type (WT) protein, and it was confirmed that it was expressed uniformly along the cell membrane. This means that the IL10RA variant protein according to one aspect of the present invention maintains the cell membrane targeting and expression site at a substantially identical or even improved level compared to the wild-type protein.

[0092] These results indicate that the IL10RA variant protein of the present invention substantially preserves the cell membrane localization characteristics of the wild-type protein, and that the membrane targeting or structural stability of the protein is not compromised by the introduction of the variant.

[0093]

[0094] Experimental Example 3. Analysis of the Anti-inflammatory Signaling Function of IL10RA Variant Protein

[0095] To determine whether the IL10RA variant protein of Example 1 could perform the same anti-inflammatory signaling function as the wild-type (WT) IL10RA protein in response to IL10 stimulation, STAT3 phosphorylation (p-STAT3) levels were compared.

[0096] Specifically, HEK293T cells were transfected with the recombinant expression vectors IL10RA (WT) and IL10RA (P3G / L5V / V6 gene) using Lipofectamine 2000 for 6 hours. Subsequently, the medium was replaced with standard culture medium. Twenty-four hours after transfection, the cells were treated with IL10 (80 ng / ml) protein for 2 hours prior to harvesting. The cells were then lysed using lysis buffer. The cell lysates were centrifuged at 15,000 rpm for 20 minutes at 4°C. After quantifying an equal amount of protein in the supernatant using BCA analysis,

[0097] Proteins were separated by molecular weight using SDS-PAGE (Sodium Dodecyl sulfate-polyacylamide gel electrophoresis). The eluted proteins were analyzed by immunoblotting using anti-pSTAT3, anti-STAT3, anti-IL10RA, or anti-β-actin antibodies, and the results are shown in Figure 3.

[0098] Figure 3 shows Western blot band images (left panel) confirming the expression of p-STAT3, STAT3, and IL10RA proteins, and a graph (right panel) quantifying the p-STAT3 / STAT3 ratio.

[0099] As shown in Figure 3, p-STAT3 signaling was significantly increased in wild-type IL10RA-expressing cells upon IL10 treatment, and substantially the same level of p-STAT3 signaling increase as in WT was observed in cells expressing IL10RA variant proteins (P3G / L5V / V6L).

[0100] This means that the IL10RA mutant protein according to one aspect of the present invention can normally perform IL10-dependent STAT3 phosphorylation signaling (anti-inflammatory response pathway), which is the original function of wild-type IL10RA.

[0101] Therefore, the IL10RA variant protein of the present invention is an IL10RA receptor protein in a form with preserved physiological function, and can be usefully utilized as a protein agent for the prevention or treatment of inflammatory diseases caused by abnormalities in the IL10 pathway.

Claims

1. An IL10RA mutant protein in which the amino acids at the 3rd, 5th, and 6th positions of the wild-type IL10RA (Interleukin-10 receptor subunit alpha) protein are substituted with other amino acids.

2. An IL10RA variant protein according to claim 1, wherein the wild-type IL10RA protein comprises a signal transduction domain.

3. The IL10RA variant protein of Claim 1, wherein the wild-type IL10RA protein has the amino acid sequence of SEQ ID NO.

2.

4. An IL10RA variant protein according to claim 1, wherein the other amino acid is any one selected from the group consisting of arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine ​​(C), selenocysteine ​​(U), glycine (G), proline (P), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), tryptophan (W), and all variants of said amino acids, wherein the other amino acid is an amino acid excluding the amino acid that the wild-type IL10RA protein has at the mutation site.

5. The IL10RA variant protein of Claim 1, wherein the IL10RA variant protein has the amino acid sequence of SEQ ID NO.

4.

6. The IL10RA variant protein of Claim 1, wherein the IL10RA variant protein has increased purification efficiency compared to the wild-type IL10RA protein.

7. A nucleotide encoding the IL10RA variant protein of Claim 1.

8. A recombinant vector comprising the nucleotide of Claim 7.

9. A host cell transformed with the recombinant vector of claim 8.

10. A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising an IL10RA variant protein in which the amino acids at the 3rd, 5th, and 6th positions of the wild-type IL10RA (Interleukin-10 receptor subunit alpha) protein are substituted with other amino acids.

11. In claim 10, the inflammatory disease is psoriasis, psoriatic arthritis, rheumatoid arthritis (RA), hepatitis, asthma, inflammatory bowel disease (IBD), vasculitis, pneumonia, dermatitis, mouth ulcers, uveitis, temporal arteritis, atopic dermatitis, systemic lupus, multiple sclerosis or sarcoidosis, synovitis, meningitis, encephalitis, Bickerstaff's encephalitis, encephalomyelitis, spondylitis, osteomyelitis, Guillain-Barré syndrome, A pharmaceutical composition selected from the group consisting of neuromyelitis optica, cystitis, nephritis, and glomerulonephritis.

12. A method for the prevention or treatment of an inflammatory disease comprising the step of administering an effective amount of an IL10RA variant protein in which the amino acids at the 3rd, 5th, and 6th positions of the wild-type IL10RA (Interleukin-10 receptor subunit alpha) protein are substituted with other amino acids to an individual in need thereof.

13. Use of an IL10RA variant protein in which the amino acids at the 3rd, 5th, and 6th positions of the wild-type IL10RA (Interleukin-10 receptor subunit alpha) protein are substituted with other amino acids for use in the manufacture of pharmaceutical preparations for the prevention or treatment of inflammatory diseases.

14. Use of an IL10RA variant protein in which the amino acids at the 3rd, 5th, and 6th positions of the wild-type IL10RA (Interleukin-10 receptor subunit alpha) protein are substituted with other amino acids for the prevention or treatment of inflammatory diseases.

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