WO2024112143A1

WO2024112143A1 - Use of stat1-tmd in immune-related diseases

Info

Publication number: WO2024112143A1
Application number: PCT/KR2023/019074
Authority: WO
Inventors: 김정호; 김범석
Original assignee: 주식회사 굳티셀
Priority date: 2022-11-24
Filing date: 2023-11-24
Publication date: 2024-05-30
Also published as: KR20240087555A

Abstract

A fusion protein (ndSTAT1-TMD) provided in the present invention can be efficiently delivered to the nucleus of cells without affecting cytotoxicity and a TcR-mediated signaling event. In addition, the fusion protein provided in the present invention specifically inhibited the transcriptional activity of endogenous STAT1 in Th1 and Th17 cells, and showed similar therapeutic potential to an anti-IL-17A antibody when administered to psoriasis and IBD mouse models. Therefore, the fusion protein provided in the present invention regulates the functions of pathogenic Th1 and Th17 cells together and thus can be a new medicine for Th1/17 cell-mediated autoimmune diseases.

Description

Use of STAT1-TMD in immune-related diseases

The present invention relates to a novel fusion protein containing STAT1 (Signal transducer and activator of transcription 1, STAT1) and a protein transduction domain (PTD) and uses thereof.

CD4 ⁺ T helper (Th) cells play an essential role in various immune responses and inflammation. Homeostatic imbalance in the functional immune network caused by excessively activated CD4 ⁺ effector T cells is associated with the initiation and maintenance of inflammatory responses that lead to serious autoimmune diseases. More specifically, T helper 1 (Th1) cells, which secrete interferon-γ (IFN-γ), and T helper 17 (Th17) cells, which secrete interleukin-17 (IL-17), are involved in long-term diseases, including psoriasis and inflammatory bowel disease. It is known to be related to the pathogenesis of specific inflammatory diseases. In addition, T helper 1 (Th1) cells and T helper 17 (Th17) cells are known to be related to the pathogenesis of various metabolic diseases, including degenerative brain or vascular diseases, obesity, and arteriosclerosis.

Additionally, T helper 1 cells (Th1 cells) and T helper 17 cells (Th17 cells) play a central role in the pathogenesis of various autoimmune diseases, including psoriasis and inflammatory bowel disease (IBD). Additionally, STAT1 (Signal transducer and activator of transcription 1) regulates Th1 and Th17 cell lineage commitment at early stages and maintains immunological functions in vitro and in vivo. Previous strategies to block STAT1 inhibit its ability to treat autoimmune diseases but also cause hyperactivation of Th17 cells.

Meanwhile, the protein transduction domain (PTD) is a short peptide with strong hydrophobicity and is known to effectively deliver bioactive molecules such as proteins, DNA, and RNA fused together into cells. Since the protein transport domain can transport bioactive molecules not only to the cytoplasm but also to the nucleus, it has characteristics suitable for delivering various proteins into the nucleus.

The purpose of the present invention is to provide a fusion protein containing the transcription modulation domain (TMD) and protein transduction domain (PTD) of STAT1.

Another object of the present invention is to obtain a fusion protein containing a plurality of protein transduction domains and a transcription modulation domain by expressing a nucleic acid molecule; and purifying the fusion protein obtained in the above step. To provide a method for producing a fusion protein containing a plurality of protein transduction domains and a transcription modulation domain.

Another object of the present invention is to provide a pharmacological composition containing the fusion protein for the treatment, improvement or prevention of immune-related diseases.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

Various embodiments described in the present invention are described with reference to the drawings. In the following description, various specific details, such as specific forms, compositions, and processes, are set forth in order to provide a thorough understanding of the invention. However, certain embodiments may be practiced without one or more of these specific details or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily or obscure the invention. Reference throughout this specification to one embodiment means that a particular feature, feature, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Accordingly, the contexts of embodiments expressed in various places throughout this specification do not necessarily represent the same embodiment of the invention. Additionally, particular features, shapes, compositions, or properties may be combined in any suitable way in one or more embodiments.

1. STAT1 (Signal transducer and activator of transcription 1)의 전사 조절 도메인 및 단백질 운반 도메인 (Protein transduction domain, PTD)을 포함하는 융합 단백질1. Fusion protein containing the transcriptional regulatory domain and protein transduction domain (PTD) of STAT1 (Signal transducer and activator of transcription 1)

One embodiment of the present invention provides a fusion protein comprising a transcriptional regulatory domain and a protein transduction domain (PTD) of STAT1 (Signal transducer and activator of transcription 1).

T helper 1 cells (Th1 cells) and T helper 17 cells (Th17 cells) play a pivotal role in the pathogenesis of various autoimmune diseases, including psoriasis and inflammatory bowel disease (IBD). In addition, T helper 1 (Th1) cells and T helper 17 (Th17) cells are known to be related to the pathogenesis of various metabolic diseases, including degenerative brain or vascular diseases, various inflammatory diseases, obesity, and arteriosclerosis.

Signal transducer and activator of transcription 1 (STAT1) regulates Th1 and Th17 cell lineage commitment at early stages and maintains immune function in vitro and in vivo. Previous strategies to block STAT1 function to treat autoimmune diseases suppress Th1 cell activity while simultaneously causing hyperactivation of Th17 cells. In the present invention, we generated a nuclear delivery form (ndSTAT1-TMD) of the STAT1 transcriptional regulatory domain that can be delivered to the nucleus of the target to regulate the function of pathogenic Th1 and Th17 cells without genetic modification under normal physiological conditions. The fusion protein provided by the present invention protects cells in a dose- and time-dependent manner without affecting cell viability and T cell activation signaling events. The ndSTAT1-TMD provided by the present invention significantly blocked the differentiation of naïve CD4 ⁺ T cells into Th1 or Th17 cells through competitive inhibition of endogenous STAT1-mediated transcription, and did not affect Th2 and Treg cell differentiation.

In addition, as a result of analyzing the gene expression profile of Th1 or Th17 cells after treatment with ndSTAT1-TMD provided by the present invention by mRNA sequencing, the expression of genes involved in the differentiation capacity and immunological function of Th1 or Th17 cells was substantially reduced. . The therapeutic potential of ndSTAT1-TMD provided by the present invention has been tested in animal models of psoriasis and colitis, the pathogenesis of which is mainly caused by Th1 and/or Th17 cells. The symptoms and progression of psoriasis and colitis were significantly alleviated by the ndSTAT1-TMD treatment provided by the present invention, similar to anti-IL-17A antibody treatment. In conclusion, the present invention shows that ndSTAT1-TMD can be a new therapeutic agent for Th1/17 cell-mediated autoimmune diseases by jointly regulating the functions of pathogenic Th1 and Th17 cells.

Th1 cells are known to protect our bodies from invading intracellular pathogens and viruses by secreting IFN-γ to activate both macrophages and dendritic cells (DC). Secreted IFN-γ also acts as a Th1 differentiation inducer by activating the STAT1 signaling pathway in the early stages. Th17 cells secrete IL-17 and IL-23, which are mainly involved in eliminating extracellular bacteria and fungi. Moreover, the presence of IFN-γ during Th17 cell lineage development undermines the full commitment of Th17 cell differentiation. In addition to all these unique properties of Th1 and Th17 cells, hyperactivated Th1 and Th17 cells play an important role in autoimmune disorders by disrupting the immunological balance between helper and regulatory T cells and with subsequent cytokine dysregulation. Do it.

STAT1, a member of the STAT family, regulates the axes of Th1/Th2 and Th1/17 cell differentiation by controlling the expression pattern of T helper subset-specific genes during the early stages of CD4 ⁺ T cell differentiation. STAT1 has an N-terminal domain (NTD), coiled-coil domain (CCD), DNA binding domain (DBD), linker domain (LD), and SCR2 homology. It consists of six domains, including a SCR2 homology domain (SH2) and a transactivation domain (TAD). When IFN-γ and IL-27 bind to their respective receptors on the surface of naïve CD4 ⁺ T cells, intracellular signaling causes activation and homodimerization of STAT1. STAT1 homodimers translocate to the nucleus and induce and maintain the expression of T-bet, a transcription factor involved in Th1 responses. STAT1 has also been shown to inhibit Th17 cell differentiation by directly binding to the Rorc or Il17a loci, the corresponding chromosomal loci of ROR-γt and IL-17A expression, respectively, which are critically involved in the regulation of Th17 responses. STAT1-knockout (KO) studies reported that loss of endogenous STAT1 impairs Th17 cell lineage commitment. Functional inhibition of STAT1 using genetic methods has been consistently proposed as a treatment strategy for Th1 or Th17 cell-mediated systemic autoimmune diseases and has shown substantial effects in limiting Th1 cells. However, no significant functional regulation of Th17 cells was detected, and Th17 cells lacking or with low levels of STAT1 displayed a hyperactivated phenotype.

As used herein, the term “transcription modulation domain” (TMD) refers to a domain that constitutes a transcription factor and consists of only a DNA binding site without a transactivation domain. As demonstrated in the following examples, the fusion protein of the present invention does not have a transactivation domain but has a DNA binding site, so it can bind to the desired promoter (e.g. Foxp3 and CD25) but cannot promote transcription. . Therefore, since the fusion protein of the present invention is a dominant negative mutant for the Foxp3 and CD25 genes, it can act as a competitive inhibitor for wild-type STAT1 in cells and inhibit the transcription and activity of STAT1.

In the present invention, the “protein transport domain (PTD)” is a short, highly hydrophobic peptide consisting of 7 to 50 amino acids, and refers to a domain capable of delivering not only proteins with a molecular weight of 120 kDa or more, but also DNA or RNA into cells. .

In the present invention, the protein transport domains include Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (antennapedia), Pep-1 (peptide-1), PTD-5 (protein transduction domain-5), and 11R. , 7R, and CTP (cytoplamic transduction peptide), or a macromolecule transduction domain may be used, but is not limited thereto, and is commonly used or commercially available in the art. Any protein transport domain can be used without limitation. Preferably CTP represented by SEQ ID NO: 1, Hph-1 represented by SEQ ID NO: 2, or Tat represented by SEQ ID NO: 3, Sim-2 represented by SEQ ID NO: 4, Mph-1 represented by SEQ ID NO: 5, VP22 represented by SEQ ID NO: 6, Antp represented by SEQ ID NO: 7, Pep-1 represented by SEQ ID NO: 8, PTD-5 represented by SEQ ID NO: 9, 7R represented by SEQ ID NO: 10, and SEQ ID NO: 11. It may be any one of 11R and CTP represented by SEQ ID NO: 12.

In the present invention, the protein transport domains include Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (antennapedia), Pep-1 (peptide-1), PTD-5 (protein transduction domain-5), and 11R. , 7R and CTP (cytoplamic transduction peptide), Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (antennapedia), Pep-1 (peptide-1), PTD One or more domains selected from the group consisting of -5 (protein transduction domain-5), 11R, 7R, and CTP (cytoplamic transduction peptide) are repeated, for example, 2, 3, 4 or more times. can be included. In the present invention, the number of protein transport domains is not particularly limited, but may be 2 or more and 10 or less, 2 or more and 5 or less, and 2 or more and 3 or less.

2. 상기 융합 단백질을 암호화하는 핵산 분자, 지질 나노입자, 발현 벡터, 숙주 세포2. Nucleic acid molecules, lipid nanoparticles, expression vectors, host cells encoding the fusion protein

According to another embodiment of the present invention, a nucleic acid molecule encoding the fusion protein provided by the present invention is provided.

The nucleic acid molecule of the present invention includes all nucleic acid molecules in which the amino acid sequence of the fusion protein provided by the present invention has been translated into a polynucleotide sequence as known to those skilled in the art. Therefore, various polynucleotide sequences can be prepared by ORF (open reading frame), and all of these are also included in the nucleic acid molecule of the present invention.

According to another embodiment of the present invention, an expression vector is provided into which the isolated nucleic acid molecule provided by the present invention is inserted.

In the present invention, the “vector” is a nucleic acid molecule capable of transporting another nucleic acid to which a nucleic acid molecule is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors are episomal mammalian vectors that have a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) can enter a host cell and become integrated into the host cell's genome, thereby replicating along with the host genome. In addition, some vectors can direct the expression of genes to which they are operationally linked. Such vectors are referred to herein as “recombinant expression vectors” or simply “expression vectors”. In general, expression vectors useful in recombinant DNA techniques often exist in the form of plasmids. In this specification, “plasmid” and “vector” may be used interchangeably since plasmid is the most commonly used form of vector.

Specific examples of the expression vector in the present invention include widely used commercially pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, and Ti vectors; cosmid; Phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμμ, T-even, T2, T3, T7; It may be selected from the group consisting of plant viruses, but is not limited thereto, and all expression vectors known to those skilled in the art as expression vectors can be used in the present invention, and when selecting an expression vector, it depends on the properties of the target host cell. Introduction of vectors into host cells may be performed by calcium phosphate transfection, viral infection, DEAE-dextran control transfection, lipofectamine transfection, or electroporation, but is not limited thereto, and those skilled in the art will use the method. An introduction method suitable for the expression vector and host cell can be selected and used. Preferably, the vector contains one or more selection markers, but is not limited to this, and selection can be made depending on whether or not the product is produced using a vector that does not contain a selection marker. The selection of the selection marker is selected based on the target host cell, and since this method is already known to those skilled in the art, the present invention is not limited thereto.

To facilitate purification of the nucleic acid molecule of the present invention, a tag sequence can be inserted and fused into an expression vector. The tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag that facilitates purification known to those skilled in the art can be used in the present invention.

According to another embodiment of the present invention, a host cell line transformed with the expression vector provided by the present invention is provided.

In the present invention, the “host cell” includes an individual cell or cell culture that can be or has been a recipient of vector(s) for incorporation of a polypeptide insert. A host cell includes the progeny of a single host cell, which progeny may not necessarily be completely identical (morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental or intentional mutations. Host cells include cells transfected in vivo with the polypeptide(s) herein.

In the present invention, the host cells may include cells of mammalian, plant, insect, fungal, or cellular origin, for example, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella Typhimurium; Fungal cells such as yeast cells and Pichia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells), SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cells, HT-1080, BHK Animal cells of (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells) or PERC.6 (Human Retinal Cells); Or it may be a plant cell, but is not limited thereto, and any cell that can be used as a host cell line known to those skilled in the art can be used.

According to another embodiment of the present invention, lipid nanoparticles (LNPs) containing the nucleic acid molecules are provided. In the present invention, “nanoparticles” are particles containing one or more lipids and one or more therapeutic agents. Nanoparticles are typically on the order of micrometers or smaller and may include a lipid layer. The lipid nanoparticle (LNP) refers to an endoplasmic reticulum with an adjacent lipid bilayer, such as a spherical endoplasmic reticulum. Lipid nanoparticles can be used in methods to deliver pharmaceutical therapies to targeted locations. Non-limiting examples of LNPs include liposomes, amphiphiles, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and monolayer membrane structures (e.g., archaeosomes and micelles).

Methods for producing lipid nanoparticles using lipids are well known in the art. For the production of lipid nanoparticles with various formulations, in addition to lipopeptides, auxiliary lipids selected from positively charged lipids, neutral lipids, and negatively charged lipids can be additionally used. For example, for the production of cationic liposomes, positively charged lipids and neutral lipids may be used, for the production of neutral liposomes, neutral lipids may be used, and for the production of anionic liposomes, negatively charged lipids and neutral lipids may be additionally used. Positively charged lipids, neutral lipids, and negatively charged lipids that can be used for the preparation of lipid nanoparticles are known in the art.

The lipid component of the nanoparticle may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids can be assembled into one or more lipid layers. Generally, phospholipids may include a phospholipid moiety and one or more fatty acid moieties. Phospholipids useful in the compositions and methods include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine ( DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2 -Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl- sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn- Glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl- sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero- 3-phosphocholine, 1,2-didocosahexaenoylsn-glycero-3-phosphocholine, 1,2-dipitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE) , 1,2-distearoyl sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl -sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-snglycero-3- Can be selected from the non-limiting group consisting of phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) and sphingomyelin. there is.

Additionally, the lipid component of the nanoparticle may include one or more structural lipids. Structural lipids may be selected from, but are limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. It doesn't work.

The lipid component of the nanoparticle may also include one or more PEG or PEG-modified lipids. These lipids may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and these. It may be selected from the non-limiting group consisting of mixtures of. For example, PEG lipids include PEG-c-DOMG, PEG-DMG (1,2-dimyristoyl-OT-glycerol methoxypolyethylene glycol, available from Avanti Polar Lipid, Elabaster, AL), PEG- It may be DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid.

3. 상기 융합 단백질, 핵산분자, 지질나노입자(LNP), 발현 벡터, 숙주 세포 중 어느 하나를 유효 성분으로 포함하는 면역 관련 질환의 예방 또는 치료용 약학 조성물.3. A pharmaceutical composition for the prevention or treatment of immune-related diseases containing any one of the above fusion proteins, nucleic acid molecules, lipid nanoparticles (LNPs), expression vectors, and host cells as an active ingredient.

The “immune-related disease” of the present invention may be at least one selected from the group consisting of autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease, but is not limited thereto. no.

The autoimmune diseases of the present invention include psoriasis, acanthosis, parakeratosis, neo-angiogenesis, rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, and alopecia areata. , asthma, Crohn's disease, Behce's disease, Sjögren's syndrome, Guillain-Barre syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyositis, and polyarteritis nodosa, but limited thereto. It doesn't work.

The present invention relates to the acute or chronic inflammatory diseases such as colitis, sepsis, septic shock, inflammatory bowel disease (IBD), psoriasis, peritonitis, nephritis, acute bronchitis, chronic bronchitis, osteoarthritis, and intestinal spondylitis. , chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, acute lung injury, and broncho-pulmonary dysplasia. However, it is not limited to this.

In the present invention, “psoriasis” is a recurrent inflammatory skin disease characterized by thick, non-pruritic plaques covered with silvery scales of the skin. Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative disease (UC), is characterized by dysregulation of the intestinal immune response to intestinal microorganisms, followed by gastrointestinal inflammation, with persistent inflammation. Typical symptoms include diarrhea, rectal bleeding, or weight loss. The exact mechanism of how psoriasis and inflammatory bowel disease (IBD) develop is still unclear, but overly activated Th1 and Th17 cells are believed to be the main cause of irregular immune responses in the skin and intestinal tract. Additionally, recent studies have shown that IFN-γ and IL-17A, secreted by Th1 and Th17 cells, respectively, are important mediators that induce and accelerate the pathological progression of psoriasis and IBD. However, therapeutic strategies targeting IFN-γ or IL-17A for psoriasis and IBD still face several limitations that need to be overcome.

In the present invention, nuclear delivery of the transcription modulation domain of STAT1 (ndSTAT1-TMD), which is a fusion protein between the transcription modulation domain (TMD) of STAT1 and the human protein transduction domain (PTD) Hph-1, is possible. created a shape. The ndSTAT1-TMD provided by the present invention can be efficiently delivered to the nucleus of cells without cytotoxicity and without affecting TcR-mediated signaling events. The ndSTAT1-TMD provided by the present invention specifically inhibited the transcriptional activity of endogenous STAT1 in Th1 and Th17 cells and showed therapeutic potential similar to that of anti-IL-17A antibody when administered to psoriasis and IBD mouse models. This suggests that ndSTAT1-TMD provided by the present invention can be a new and effective treatment reagent for Th1/17 cell-mediated autoimmune diseases.

In the present invention, 'metabolic disease' means a condition or disease closely related to or caused by obesity, and specifically selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, and arteriosclerosis. There may be more than one.

In the present invention, fatty liver refers to a condition or disease in which fat accumulates in excessive amounts in liver cells due to a disorder of liver fat metabolism.

In the present invention, hyperlipidemia refers to a condition or disease in which the concentration of fat components in the blood, especially cholesterol and triglyceride, is higher than normal, and includes all conditions requiring lowering the lipid concentration in the blood. It is used in a broad sense.

In this specification, arteriosclerosis refers to a condition or disease in which blood circulation to organs and tissues in the body is reduced due to thickening of the artery wall and reduction in elasticity, and the deposition of fat, cholesterol, and other substances on the inner wall of the artery, forming plaque ( It has a meaning that includes “atherosclerosis,” which refers to a condition or disease in which blood circulation is reduced by forming plaques and narrowing the lumen. Atherosclerosis can occur anywhere in the body. If it occurs in blood vessels within the heart, it can cause coronary artery disease such as angina pectoris or myocardial infarction. If it occurs in the brain, it can cause cerebral infarction. If it occurs in the kidneys, it can cause renal failure. It can be triggered.

In the present invention, the “neurodegenerative disease” or “degenerative brain or vascular disease” may refer to a disease caused by decreased or lost function of nerve cells, and the “neuroinflammatory disease” may refer to a disease caused by excessive inflammatory response in the nervous system. It may mean a disease that occurs. Specific examples of the neurodegenerative disease or neuroinflammatory disease in the present invention include stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and Niemann-Pick disease. , multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration. , multiple system atrophy disease, progressive supranuclear palsy, nervous system autoimmune disease, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy. It may be selected from the group, but is not limited thereto.

As used herein, “cancer” refers to or refers to a physiological condition typically characterized by uncontrolled cell growth in mammals. The cancer subject to prevention, improvement, or treatment in the present invention may be a solid tumor consisting of a lump generated by abnormal cell growth in a solid organ, for example, depending on the part of the solid organ. It may be stomach cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer. For example, it may be melanoma. It is not limited.

In the present invention, the “object of interest” refers to an individual who has developed, or is likely to develop, an immune-related disease.

Meanwhile, in the present invention, “prevention” may include without limitation any act of blocking, suppressing or delaying the symptoms of a disease using the pharmaceutical composition of the present invention.

Additionally, in the present invention, “treatment” may include, without limitation, any action that improves the symptoms of a disease or provides benefits using the pharmaceutical composition of the present invention.

Meanwhile, although not limited thereto, the method for preventing or treating the disease may be a combination therapy that further includes administering a compound or substance having therapeutic activity for one or more diseases.

In the present invention, the “combined use” should be understood to indicate simultaneous, separate or sequential administration. If the administration is sequential or separate, the interval between the administrations of the second components should be such that the beneficial effects of the combination are not lost.

In the present invention, the pharmaceutical composition may be in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be intended for human subjects.

In the present invention, the pharmaceutical composition is not limited to these, but can be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods. You can. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. for oral administration, and buffers, preservatives, and analgesics for injections. Topics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing it with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be manufactured in the form of unit dose ampoules or multiple doses. there is. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc.

Meanwhile, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose. , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

The route of administration of the pharmaceutical composition according to the present invention is not limited to these, but may be administered directly to the eye, orally, intravenously, intramuscularly, intraarterially, intramedullary, intrathecally, intracardiacally, transdermally, subcutaneously, intraperitoneally, or nasally. Includes internal, intestinal, topical, sublingual or rectal. Oral or parenteral administration is preferred. It is more preferable to administer directly to the eye.

In the present invention, the term “parenteral” includes direct administration to the eye, subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or injection techniques. . The pharmaceutical composition of the present invention can also be administered in the form of a suppository for rectal administration. It is preferable to administer directly to the eye.

The "usage and dosage" of the composition of the present invention includes the activity of the specific compound used, age, weight, general health, gender, diet, administration time, administration route, excretion rate, drug combination, and the severity of the specific disease to be prevented or treated. It may vary depending on various factors, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by a person skilled in the art, and is 0.0001 per day. It can be administered at 0.001 to 50 mg/kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

The appropriate total daily amount can be determined by the treating physician within the scope of sound medical judgment, and can be administered once or in several divided doses. However, for the purposes of the present invention, the specific therapeutically effective amount for a specific patient is determined by the type and degree of response to be achieved, the composition containing the active ingredient, the patient's age, and whether other agents are used as the case may be. Various factors well known in the medical field, including body weight, general health condition, gender and diet, administration time, administration route and secretion rate of the composition containing the active ingredient, treatment period, and drugs used together or simultaneously with the specific composition. It is desirable to apply it differently depending on similar factors.

In another embodiment of the present invention, STAT1 provides a fusion protein represented by the amino acid sequence of SEQ ID NO: 13.

In another embodiment of the present invention, a fusion protein is provided in which the transcriptional regulatory domain of STAT1 is represented by the amino acid sequence of SEQ ID NO: 14.

In another embodiment of the present invention, the protein transport domain is Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (antennapedia), Pep-1 (peptide-1), PTD-5 (protein transduction domain) -5), 11R, 7R, and CTP (cytoplamic transduction peptide) are selected from the group consisting of fusion proteins.

Another embodiment of the present invention provides a fusion protein, wherein the protein transport domain is represented by any one of the amino acid sequences of SEQ ID NOs: 1 to 12.

Another embodiment of the present invention provides a fusion protein that competitively inhibits STAT1.

Another embodiment of the present invention provides a fusion protein that inhibits differentiation or function of Th1 or Th17.

One embodiment of the present invention provides a nucleic acid molecule encoding the fusion protein.

One embodiment of the present invention provides lipid nanoparticles (LNPs) containing the nucleic acid molecules.

One embodiment of the present invention provides an expression vector into which the nucleic acid molecule is inserted.

One embodiment of the present invention provides a host cell transfected with the expression vector.

In one embodiment of the present invention, a pharmaceutical composition for preventing or treating immune-related diseases comprising any one of the fusion protein, the nucleic acid molecule, the lipid nanoparticle (LNP), the expression vector, and the host cell as an active ingredient. to provide.

Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating immune-related diseases, wherein the immune-related diseases occur due to activation or dysfunction of Th1 cells or Th17 cells.

In another embodiment of the present invention, the immune-related disease is at least one selected from the group consisting of autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease, A pharmaceutical composition for preventing or treating immune-related diseases is provided.

In another embodiment of the present invention, the autoimmune disease includes psoriasis, acanthosis, parakeratosis, neo-angiogenesis, rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, and atopic dermatitis. , alopecia areata, asthma, Crohn's disease, Behce's disease, Sjögren's syndrome, Guillain-Barre syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyositis, and polyarteritis nodosa. Provides a pharmaceutical composition for preventing or treating immune-related diseases.

In another embodiment of the present invention, the acute or chronic inflammatory disease includes colitis, sepsis, septic shock, inflammatory bowel disease (IBD), psoriasis, peritonitis, nephritis, acute bronchitis, chronic bronchitis, In the group consisting of osteoarthritis, enteropathy spondylitis, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, acute lung injury, and broncho-pulmonary dysplasia. Provided is a pharmaceutical composition for preventing or treating immune-related diseases, which is characterized in that it contains one or more selected substances.

The fusion protein (ndSTAT1-TMD) provided in the present invention can be efficiently delivered to the nucleus of cells without cytotoxicity or affecting TcR-mediated signaling events. Additionally, the fusion protein provided in the present invention specifically inhibited the transcriptional activity of endogenous STAT1 in Th1 and Th17 cells and showed therapeutic potential similar to that of anti-IL-17A antibody when administered to psoriasis and IBD mouse models. Therefore, the fusion protein provided in the present invention can become a new treatment for Th1/17 cell-mediated autoimmune diseases by controlling the functions of pathogenic Th1 and Th17 cells.

Figure 1 shows the production of ndSTAT1-TMD capable of nuclear delivery and verification of its intranuclear delivery kinetics without cytotoxicity.

Specifically, Figure 1A shows the protein structures of Hph-1-PTD with mutated valine 426 and threonine 427, ndSTAT-TMD, and STAT1-TMD without ndSTAT1-TMD (V426D, T427D), PTD and protein transduction domain, respectively. (protein transduction domain); nd, nucleus-transducible; TMD, transcriptional regulatory domain; DBD, DNA binding domain; ND, N-terminal domain; LD, linker domain; SH2, SCR2 homology domain; TAD, stands for transaction activation domain.

Figure 1B shows purified STAT1-TMD, ndSTAT1-TMD, and ndSTAT1-TMD (V426D, T427D) confirmed by Western blot (left panel) or SDS-PAGE (right panel).

Figures 1C and 1D show dose-dependent and time-dependent intranuclear delivery of ndSTAT1-TMD (0.5-2 μM) or STAT1-TMD (2 μM) into mouse splenocytes.

Figure 1E shows the intracellular stability of ndSTAT1-TMD after transduction into mouse splenocytes.

Figure 1F: Subcellular localization of ndSTAT1-TMD in cultured mouse splenocytes. Representative immunofluorescence 63X (left panel) and 189X (right panel) images using confocal microscopy are shown, and the scale bar is 10 μm.

Figure 1G shows cell cytotoxicity evaluation of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) in mouse splenocytes analyzed by CCK-8 assay.

Figure 1H and Figure 1I show ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) analyzed by flow cytometry and ELISA. Graphs are presented as mean ± SEM (n = 3), and statistical analysis was performed using Student's t-test. investigated.

Figure 2 shows the functional impact of ndSTAT1-TMD on the differentiation ability of CD4 ⁺ naive T cells into various T cell subsets.

Specifically, Figure 2A shows flow cytometric analysis of intracellular T-bet and IFN-γ in Th1 cells treated with various concentrations (0.5-2 μM) of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) under Th1-skewing conditions. It represents.

Figure 2B shows the level of IFN-γ secreted in the Th1 cell supernatant of Figure 2A measured by ELISA.

Figure 2C shows flow cytometry analysis of the levels of T-bet ⁺ IRF1 ⁺ cells in the Th1 cells of Figure 2A.

Figure 2D shows ROR-γt and intracellular IL- levels in Th17 cells treated with various concentrations (0.5-2 μM) of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) under Th17-skewing conditions. Flow cytometry analysis of 17A is shown.

Figures 2E and 2F show the levels of IL-17A or IFN-γ secreted from the Th17 cell supernatant of Figure 2D measured by ELISA. All experiments shown here were repeated three times on independent biological samples with similar results. Data are expressed as mean ± SEM (n≥3) and statistical analysis was examined using Student's t-test.

Figure 3 shows that ndSTAT1-TMD suppresses the expression of STAT1 target genes at the transcription level.

Specifically, Figure 3A shows that HEK293T cells were co-transfected with a plasmid containing a luciferase reporter gene whose expression is driven by the IL-17A promoter and a wild-type STAT1 gene. That is, the luciferase activity of cells was measured using a luminometer 24 hours after treatment with ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D). Data are expressed as mean ± SEM (n = 5), and statistical significance was achieved when luciferase activity was lower than the positive control (pCMV-STAT1 alone). Statistical analysis was examined using Student's t-test.

Figures 3B and 3C are a representation of the genes upregulated in STAT3 knockout CD4 ⁺ T cells compared to wild-type CD4 ⁺ T cells in ntSTAT1-treated Th1 cells or in ntSTAT1-TMD treated Th17 cells. This shows GSEA-based enrichment plots for .

Figures 3D and 3E show heatmaps of Th1 or Th17 cell signature genes and naive T cell specific genes in Th1 or Th17 cells treated or not with ndSTAT1-TMD. Each row represents the results from one experimental well and the Z-score was calculated from log2(TPM+1).

Figure 4 shows that ndSTAT1-TMD treatment attenuates the disease severity of IMQ-induced psoriasis.

Specifically, Figure 4A shows PBS (IMQ group), anti-IL-17A antibody (60 μg/mouse), and STAT1-TMD (20 μg/mouse or 60 μg/mouse), or ndSTAT1-TMD (V426D, T427D). This shows a treatment method for 5% IMQ-induced psoriasis mice using (60 μg/mouse).

Figure 4B shows a plot of the PASI scores on the back skin of each group in Figure 4A against disease progression.

Figure 4C shows representative images of skin damage on the back of the mouse in Figure 4A on day 5 (top panel) and day 7 (bottom panel).

Figure 4D shows the change in back skin thickness within each group from Figure 4A on the 5th day, calculated based on the thickness on the 1st day. The graph was expressed as mean ± SEM (n = 8), and statistical analysis was performed using Student's t-test.

Figure 4E shows the back skin thickness of mice in each treatment group.

Figure 4F shows the change in body weight of each group.

Figure 4G shows the levels of proinflammatory cytokines (TNF-α, IL-1β, or IL-6) in the serum of mice from each group measured by ELISA. Experiments in the psoriasis model were performed twice independently. Graphs are presented as mean ± SEM (n = 5) and statistical analysis was examined using Student's t-test.

Graphs in Figure 4B, E, and F are presented as mean ± SEM (n = 8) and statistical analysis was also examined using ANOVA analysis and Dunnett's multiple comparison test. In the graph, * IMQ and ndSTAT1-TMD 60 μg, # IMQ and anti-IL-17A antibody 60 μg, + IMQ and ndSTAT1-TMD 20 μg, x IMQ and ndSTAT1-TMD (V426D, T427D) 60 μg, respectively.

Figure 5 shows that ndSTAT1-TMD treatment restores the destroyed functional state of CD4 ⁺ T cells in a psoriasis animal model.

Specifically, Figures 5A and 5B show CD4 ⁺ T-bet ⁺ T cells (left panel of (A) and (B)) or CD4 ⁺ in the spleen (A) and draining lymph node (dLN) (B) of each treatment group. The level of ROR-γt ⁺ T cells (right panel (A) of (A) and (B)) is shown as examined on day 7 by flow cytometry.

Figures 5C and 5D show CD4 ⁺ IFN-γ ⁺ (left panels of (C) and (D)) or CD4 ⁺ IL-17A ⁺ ( (C) and (D) right panel) levels were analyzed on day 7.

Figure 5E shows CD4 ⁺ Foxp3 ⁺ regulatory T cell levels in the spleen (left panel) or draining lymph nodes (dLN) (right panel) for each treatment group. Graphs are presented as mean ± SEM (n = 5) and statistical analysis was examined using Student's t-test.

Figure 6 shows that ndSTAT1-TMD treatment of a psoriasis animal model prevents leukocytes from infiltrating the back skin.

Specifically, Figure 6A shows representative H&E staining (top panel) or Masson's trichrome staining (bottom panel) results on the back skin of mice from each group at day 7. The scale bar represents 200 μm for 100X and 100 μm for 200X.

Figure 6B shows changes in the thickness of the skin epidermis on the back of mice in each treatment group measured using the histological image of Figure 6A. The graph was expressed as mean ± SEM (n = 8), and statistical analysis was performed using Student's t-test.

Figure 6C shows the level of CD45 ⁺ leukocytes infiltrated in the dorsal epidermis (grey) or dermis (blue) of each group on day 7.

Figure 6D shows the levels of infiltrating macrophages expressing CD45, CD11b, and F4/80 in the dorsal epidermis (grey) or dermis (blue) region of each group at day 7.

Figure 6E shows the level of nitric oxide (NO) in the serum of mice in each treatment group on day 7.

Figures 6F and 6G show infiltration of T-bet ⁺ (Figure 6F) or ROR-γt ⁺ (Figure 6G) T cells in total CD45 ⁺ leukocytes in the dorsal epidermis (gray) or dermis (blue) region of each treatment group on day 7. It shows the degree to which it has been done.

Figures 6H and 6I show infiltration of IFN-γ ⁺ (Figure 6H) or IL-17A ⁺ (Figure 6I) T cells in total CD45 ⁺ leukocytes in the dorsal epidermis (gray) or dermis (blue) region of each treatment group on day 7. It shows the degree to which it has been done.

The graphs in Figures 6C to 6I are expressed as mean ± SEM (n = 6), and statistical analysis was performed using Student's t-test, with statistical significance shown when the ratio of the cell group was low compared to the IMQ group.

Figure 7 shows that ndSTAT1-TMD alleviates the disease progression of DSS-induced colitis.

Specifically, Figure 7A shows 100 μg of PBS (DSS group), anti-IL-17A antibody (100 μg/mouse), ndSTAT1-TMD (20 μg/mouse or 100 μg/mouse), or ndSTAT1-TMD (V426D, T427D) (100 μg). /mouse) shows the treatment plan for mice with 2.5% DSS-induced colitis.

Figure 7B shows the change in body weight in each treatment group.

Figure 7C shows the disease activity index (DAI) score for each treatment group. The DAI score was calculated by measuring the clinical scores of stool, stool occult blood, and weight change in each group.

The left panel of Figure 7D shows a comparative analysis of the colon length of mice in each treatment group on day 12, and the right panel shows a representative image of the 7A colon.

Figure 7E shows representative vertical (above panel) and horizontal (below panel) colon sections from Figure 7D stained with H&E or PAS. The scale bar represents 500 μm for 40X and 100 μm for 200X.

Figure 7F shows the thickness of the colonic muscle layer of each treatment group measured from the image in Figure 7E.

Figure 7G shows the histopathological score, which is calculated by inflammatory cell infiltration, crypt damage, and goblet cell depletion. Graphs are presented as mean ± SEM (n = 6).

Figure 7H shows the levels of pro-inflammatory cytokines (TNF-α, IL-1β or IL-6) in the serum of each treatment group measured by ELISA. Graphs are presented as mean ± SEM (n = 5). Experiments in the IBD model were performed twice independently.

The graphs in Figures 7B-7D and 7F are presented as mean ± SEM (n=7). Statistical analysis was conducted using Student's t-test or ANOVA analysis and Dunnett's multiple comparison test, and in Figure 7G, statistical significance was shown when the histopathological score was lower than that of the DSS group.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

[준비예][Preparation example]

1. 재조합 단백질 생성을 위한 DNA 클로닝1. DNA cloning for recombinant protein production

The plasmid of mouse STAT1 (BC004808) was purchased from Origene Technologies, Inc., USA. The sequence coding for amino acids 317 to 488 of the wild-type STAT1 DNA binding domain (DBD) was amplified by PCR and then inserted into the pET28a(+) expression vector containing Hph-1-PTD, 6x His tag, and FLAG tag. introduced. Point mutation forms of DBD (V426D, T427D) were generated using pfu DNA polymerase (Agilent). After cloning, all generated cloned DNA structures were sequenced to confirm the fidelity of the open reading frame (ORF).

2. STAT1-TMD, ndSTAT1-TMD (V426D, T427D) 및 ndSTAT1-TMD의 발현 및 정제2. Expression and purification of STAT1-TMD, ndSTAT1-TMD (V426D, T427D) and ndSTAT1-TMD

All recombinant proteins were expressed in the BL21 Codon Plus (DE3) RIPL strain of Escherichia coli (Invitrogen, Waltham, MA, USA) and purified through affinity chromatography. Cells expressing STAT1-TMD, ndSTAT1-TMD, or ndSTAT1-TMD (V426D, T427D) were collected in native lysis buffer (10mM imidazole, 50mM NaH ₂ PO ₄ , 300mM NaCl, pH 8.0) and sonicated. Inclusion bodies were pelleted by centrifugation of the cell lysate and further sonicated in native lysis buffer containing 3M urea (10mM imidazole, 50mM NaH ₂ PO ₄ , 300mM NaCl, 3M Urea, pH 8.0). did. Cell lysates were pelleted, and the supernatant was mixed with Ni-NTA resin (Qiagen). The recombinant protein was washed with a buffer containing 30mM imidazole, 50mM NaH ₂ PO ₄ , 300mM NaCl, pH 8.0, and eluted with a buffer containing 500mM imidazole, 50mM NaH ₂ PO ₄ , 300mM NaCl, pH 8.0. The eluted recombinant protein was further purified to remove endotoxin through ion exchange chromatography and desalted using a PD-10 Sephadex G-25 column (GE Healthcare). The purified recombinant protein contained a safe level of endotoxin of approximately 6 EU/ml. In subsequent animal experiments, none of the animals showed signs of an immune response to endotoxin, such as anaphylactic shock.

3. 웨스턴 블랏3. Western blot

The recombinant protein purified in step 2 above was separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF, Bio-Rad, Hercules, CA, USA) membrane. . Membranes were blocked with 5% bovine serum albumin in 0.1% Tween-20 in Tris-buffered saline. Recombinant proteins were incubated with anti-6x His-tag antibody (27E8 Clone, Cell Signaling, Danvers, MA, USA) and anti-mouse IgG-HRP antibody (Abcam, Cambridge, MA, USA). Then, ECL reagent (Bio-Rad) was used to visualize the protein in ChemiDoc (Bio-Rad).

4. 세포배양4. Cell culture

HEK293T cells were incubated with 10% heat-inactivated fetal bovine serum (FBS, Hyclone), 2mM L-glutamate (Lonza), 100 μg/ml penicillin-streptomycin (Lonza), 1mM sodium pyruvate (Lonza), and NEAA (Gibco, Waltham). , MA, USA) was cultured in DMEM medium (Lonza, Basel, Switzerland) supplemented with .

All primary cells were incubated with 7.5% heat-activated fetal bovine serum (FBS, Hyclone), 2mM L-glutamate (Lonza), 100 μg/ml penicillin-streptomycin (Lonza), and 50 μM β-Mercaptoethanol (Sigma Aldrich, St. Louis; MO, USA) was maintained in RPMI 1640 (Lonza) medium supplemented with All cells were cultured at 37°C in a humidified atmosphere of 5% CO ₂ .

5. 단백질 처리 (Protein Treatment)5. Protein Treatment

^2x105 mouse splenocytes were cultured with recombinant proteins in 96-well cell culture plates (Eppendorf) in a dose-dependent manner (0.5-2 μM, 1 h) or time-dependent manner (2 μM, 0-72 h) for flow cytometric analysis. was used in ^2x106 mouse splenocytes were seeded on coverslips placed on the bottom of a 12-well cell culture plate (SPL, Pocheon, South Korea) and incubated with 1 μM of ndSTAT1-TMD or STAT1-TMD for 1 h before analysis, followed by confocal analysis. Observation was made using a microscope (LSM 980, Carl Zeiss, Jena, Germany). ^2x105 mouse naïve CD4 ⁺ T cells were cultured in 96-well cell culture plates (Eppendorf) under appropriate T cell subset-polarizing conditions with the addition of recombinant proteins in a dose-dependent manner (0.5-2 μM).

6. 면역세포화학 (Immunocytochemistry)6. Immunocytochemistry

2x10 ⁶ mouse splenocytes were seeded on a round microscope cover glass with a diameter of 18 mm placed on the bottom of a 12-well cell culture plate (SPL). Cells were cultured in 12-well cell culture plates (SPL) in medium containing PBS, 1 μM STAT1-TMD, or 1 μM ndSTAT1-TMD for 1 hour. After recombinant protein treatment, cells were washed with PBS, fixed with 10% formalin solution (Sigma-Aldrich), and permeabilized with 0.5% Triton X-100 solution (Sigma-Aldrich). Samples were blocked with 1% BSA solution. The delivered protein was captured with anti-FLAG Alexa 488 antibody (Invitrogen). After washing with PBS, nuclei were counterstained with 4',6-diamino-2-phenylindole (DAPI), and cells were visualized using a confocal microscope (LSM 980, Carl Zeiss).

7. 세포 독성 분석 (Cytotoxicity assay)7. Cytotoxicity assay

Cell viability after treatment with ndSTAT1-TMD and ndSTAT1-TMD (V426D, T427D) was tested using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). 2x10 ⁵ mouse splenocytes were seeded in a 96-well culture plate (SPL) and incubated with various doses of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) for 1 hour. After protein treatment, CCK-8 reagent was added to the cultured cells and cultured for an additional 4 hours. Cell viability was measured at absorbance at a wavelength of 450 nm using a microplate reader (Bio-Rad).

8. CD4+ T 세포 분화8. CD4+ T cell differentiation

CD4 ⁺ CD62L ⁺ naive T cells were purified from RBC-cleared spleen cells extracted from the spleens of 8-week-old female C57BL/6N (B6) mice using a CD4 ⁺ CD62L ⁺ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated CD4 ⁺ naive T cells were grown in 96-well cell culture plates (Eppendorf) coated with 1 μg/ml anti-CD3ε (BD Bioscience, San Diego, CA, USA) and anti-CD28 (BD Bioscience) for 72 h. activated. To differentiate CD4 ⁺ naive T cells into Th1, Th2, Th17 or Treg cells, various cytokine mixtures and antibodies were added to the culture plates: Th1 (25 ng/ml of recombinant mouse IL-12 and 2 μg/ml of anti- IL-4 antibody), Th2 (200 ng/ml recombinant mouse IL-4 and 2 μg/ml anti-IFN-γ antibody), Th17 (1 ng/ml recombinant mouse TGF-β1, 25 ng/ml recombinant mouse IL) -6, 2 μg/ml anti-IFN-γ antibody and 2 μg/ml anti-IL-4 antibody), iTreg (5 ng/ml recombinant mouse TGF-β and 20 ng/ml recombinant mouse IL-2) . Cytokines and antibodies were purchased from PeproTech (Rocky Hill, NJ, USA), BioLegend (San Diego, CA, USA), and R&D Systems (Minneapolis, MN, USA). All cells were cultured at 37°C in humid conditions with 5% CO ₂ .

9. ELISA9.ELISA

Supernatants of activated or differentiated CD4 ⁺ effector T cells were collected and cytokine levels were measured. Levels of IFN-γ, IL-4, IL-17A, and IL-2 were measured by ELISA according to the manufacturer's protocol (Invitrogen).

Mouse serum samples were obtained by centrifugation of blood samples obtained from IMQ-induced psoriatic mice on day 7 of disease induction or from dextran sulfate sodium (DSS)-induced colitis mice on day 12 of disease induction. TNF-α, IL-1β, and IL-6 levels in blood were measured by ELISA according to the manufacturer's protocol (Invitrogen).

The concentration of cytokines was measured at absorbance at a wavelength of 450 nm using a microplate reader (Bio-Rad).

10. 리포터 유전자 분석 (Reporter Gene Analysis)10. Reporter Gene Analysis

ndSTAT1-TMD and ndSTAT1-TMD (V426D, T427D) were tested for their binding ability to target gene promoters. ^5x10 HEK293T cells were seeded in 6-well culture plates (SPL) and incubated with 1 μg of pCMV6 vector containing mouse wild-type STAT1 and 1 μg of IL using Lipofectamine Reagents (Invitrogen) diluted in Opti-MEM (Gibco). -17A promoter-luciferase vector was co-transfected. After incubation for 4 hours at 37°C with 5% CO ₂ , cells were treated with ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) in a dose-dependent manner (0.5-2 μM). Cells were then cultured overnight, washed with PBS, and lysed using Cell Culture Lysis 5X Reagent (Promega, Madison, WI, USA). Cell lysates were mixed with Luciferase Assay Substrate (Promega), and luciferase activity was measured with a Luminometer (Promega).

11. mRNA 서열 분석 및 유전자 세트 농축 분석 (mRNA-Sequencing and Gene Set Enrichment Analysis)11. mRNA-Sequencing and Gene Set Enrichment Analysis

Mouse CD4 ⁺ T cells differentiated under Th1- or Th17-polarizing conditions with or without the addition of ndSTAT1-TMD were harvested after 72 h. RNA extraction and sequencing were performed by Macrogen (Seoul, South Korea). The Trimmomatic 0.38 program was used to remove low-quality data, adapters, and DNA contamination from the first reads. The truncated data were then mapped to the genomic reference (10 mm) using the HISAT2 program. Transcriptome assembly was performed using the reference-based aligned reads information, and TPM (transcript per million) counts were also obtained from the assembled transcriptome using StringTie.

Gene set enrichment analysis (GSEA) was performed on a group of ndSTAT1-TMD treated samples using GSEA v4.1.0. MSigDB was used as the gene set database, including Hallmark gene set and ImmuneSigDB. The number of permutations was set to 1000 and the permutation type was set to 'gene set'. The chip platform was set to 'Mouse_Gene_Symbol_Remapping_Human_Orthologs_MSigDB.v7.4. TPM values for 17358 genes from four samples of ndSTAT1-TMD treated and untreated Th1 cells and 17201 genes from four samples of ndSTAT1-TMD treated and untreated Th17 cells were used for analysis.

12. 실험 동물12. Laboratory animals

8- to 9-week-old female and male C57BL/6N (B6) mice (8 to 9 weeks) and 9-week-old female C57BL/6J (B6) mice were purchased from Orient Bio (Korea). All mice were bred and maintained in the semi-specific-pathogen-free (SPF) facility at the Yonsei Animal Research Center (YLARC). All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the Yonsei University Laboratory Animal Research Center (YLARC) and were performed in accordance with the guidelines of YLARC-IACUC for ethical use. (IACUC-A-202109-1327-01, IACUC-A-202201-1404-01).

13. 건선 유도 및 건선 부위 및 중증도 지수 (PASI) 점수 측정 (Psoriasis Induction and scoring of psoriasis area and severity index (PASI))13. Psoriasis Induction and scoring of psoriasis area and severity index (PASI)

After one week of acclimatization, 8-week-old female C57BL/6J mice were shaved and hair removal cream was applied to a 2*4cm area on the back on day 1 to completely remove hair. The normal group applied petroleum jelly to each exposed back skin (40 μg) and both ear skin (20 μg/ear) continuously for 7 days. In the disease-inducing group, 5% IMQ cream (Aldara Cream, Donga S&T, Seoul, Korea) was applied to the exposed back skin (40㎍) and both ear skin (20㎍/ear) from day 1. ndSTAT1-TMD (20 or 60 μg/mouse), ndSTAT1-TMD (V426D, T427D) (60 μg/mouse), or anti-IL-17A antibody (BioXCell, Lebanon, NH, USA) (60 μg/mouse). Intraperitoneal injections were administered every other day from days 1 to 5, and body weight and psoriasis area and severity index (PASI) scores were measured daily from days 1 to 7. The exposed back skin of the mice was photographed on

days

5 and 7, and both ears of the mice were photographed on day 7. The thickness of the back or right ear skin was measured with a Vernier caliper for 7 consecutive days. The Psoriasis Area and Severity Index (PASI) scores of all groups were assessed daily and measured in a blinded manner according to the following indices:

Erythema (redness): 0, flesh-colored pink; 1, slight redness over the entire surface; 2, medium red with dark red patches throughout the surface; 3, If there are dark red patches all over the surface.

Curing (thickness): 0, 1-5%; 1, 5-50%; 2, 50-100%; 3, >100%. Detachment (scaling): 0, no peeling of skin; 1, minor dry spots without peeling; 2, dry patches over most of the skin with peeling; 3. Dry patches over most of the skin, with moderate to severe peeling over a large surface area.

The experimental protocol was approved by the Yonsei University Institutional Animal Care and Use Committee (IACUC), and experiments on the psoriasis mouse model were performed in accordance with the IACUC Guidelines for the Ethical Use of Animals (IACUC-A-202201-1404-01).

13. 대장염 유발 및 질병 활동 지수(DAI) 점수 매기기 (Colitis Induction and Scoring of disease activity index)13. Colitis Induction and Scoring of disease activity index (DAI)

After one week of acclimatization, 8-week-old male C57BL/6N mice were provided with sterile water or water containing 2.5% DSS from days 1 to 6. Water containing dextran sulfate sodium (DSS) was changed every other day. After 6 days, all mice were provided with sterile water. Mice were administered ndSTAT1-TMD (20 or 100 μg/mouse), ndSTAT1-TMD (V426D, T427D) (100 μg/mouse), or anti-IL-17A antibody (BioXCell, Lebanon, NH, USA) (100 μg/mouse). was injected intraperitoneally. Body weight and disease activity index (DAI) scores were measured once a day from days 6 to 11, and daily from days 1 to 11. All mice were sacrificed on day 12 for analysis. The colon (caecum to rectum) was separated to measure length. The following indices were used to provide a blinded measure of DAI score:

Weight change: 0, 0-1%; 1, 1-5%; 2, 5-10%; 3, 10-20%; 4, >20%.

Stool: 0, normal; 1, some are soft but still formed; 2, very soft; 3, Diarrhea. Fecal occult blood: 0, normal; 1, benign hematoma; 2, traces of blood in stool; 3, rectal bleeding.

The experimental protocol was approved by the Yonsei University Institutional Animal Care and Use Committee (IACUC), and the colitis experiments followed the IACUC Guidelines for the Ethical Use of Animals (IACUC-A-202109-1327-01).

14. 조직학적 검사 (Histological Examination)14. Histological Examination

In the IMQ-induced psoriasis mouse model, 1 cm ² of back skin and right ear was collected from each mouse, fixed in 4% phosphate-buffered paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E) and Masson's trichrome. dyed with Histological samples were imaged through a light microscope (Olympus CX40) (Olympus Corporation, Shinjuku, Tokyo, Japan). The epidermal thickness of the back skin or right ear skin was measured using the Image J program.

In the dextran sulfate sodium (DSS)-induced colitis mouse model, distal sections of the colon were obtained from each mouse and fixed in 4% phosphate-buffered paraformaldehyde. Each sample was then embedded in paraffin, sectioned horizontally or vertically, and stained with H&E and PAS (periodic acid-Schiff). Histological samples were imaged via light microscopy (Olympus BX51) (Olympus Corporation). The extent of inflammatory cell infiltration, crypt damage, and goblet cell depletion were blindly measured using the following indicators:

Inflammatory cell infiltration: 0, no infiltration; 1, light penetration <25%; 2, moderate penetration <50%; 3, significant penetration >50%.

Crypt damage: 0, none; 1, Some crypt damage; 2, larger space between crypts; 3, Large area without crypts.

Goblet cell depletion: 0, none; 1, mild depletion <25%; 2, moderate depletion <50%; 3, significant depletion >50%.

The thickness of the muscle layer of the large intestine was measured using the Image J program. All tissue staining was performed at Korea CFC (Seoul, South Korea).

15. 유세포 분석(Flow Cytometry)15. Flow Cytometry

The transduction efficiency of ndSTAT1-TMD in mouse splenocytes was analyzed by intracellular staining using anti-FLAG-PE (BioLegend) after protein treatment for the indicated times.

For analysis, activated CD4 ⁺ T cells were stained with anti-CD69-FITC (BD Bioscience) or anti-CD25-PE (BD Bioscience). After 72 hours of incubation with recombinant proteins, differentiated CD4 ⁺ effector T cell subsets were resuspended with Cell-Stimulation Cocktail (500X) (eBioscience, San Diego, CA, USA) for 4 hours ^. provoked. Each subset was then fixed and permeabilized with Fixation/Permeabilization Buffer (eBioscience) and intracellularly stained for appropriate lineage-defining transcription factors and cytokines. All antibodies were purchased from eBioscience.

Immune cells isolated from the spleen or draining lymph nodes (dLN) of IMQ-induced psoriasis mice and the spleen or mesenteric lymph nodes (mLN) of DSS-induced colitis mice were restimulated with Cell Stimulation Cocktail (500X) (eBioscience) for 4 hours. After restimulation, cells were first stained with anti-CD4-AF700 (eBioscience) before fixation and permeabilization. Intracellular cytokines and transcription factors were added with anti-IFN-γ-APC, anti-IL-17A-APC, anti-T-bet-PE, anti-ROR-γt-PE, or anti-Foxp3-PE (eBioscience). was dyed with

Skin-infiltrating leukocytes were isolated from the back and ear skin of mice with IMQ-induced psoriasis. Their properties were analyzed by staining for appropriate surface markers or staining for intracellular transcription factors or cytokines. For intracellular staining, cells were restimulated for 4 h using Cell Stimulation Cocktail (500X) (eBioscience), fixed, and permeabilized. Cells were incubated with anti-CD45-AF700, anti-CD11b-APC, anti-IFN-γ-PE, anti-IL-17A-PE, anti-T-bet-PE, anti-ROR-γt-PE (eBioscience), and anti- Stained with -F4/80-PE (BD Bioscience).

All cells restimulated using the cell stimulation cocktail (500x) were cultured at 37°C in humidified conditions with 5% CO ₂ . Flow cytometry was performed using SA3800 (Sony, Tokyo, Japan), and data analysis was performed using the Flowjo V10 program.

16. 산화질소 생산 분석 (Nitro Oxide production assay)16. Nitro Oxide production assay

On the 7th day of disease induction, blood was collected from IMQ-induced psoriasis mice in each group, and serum was separated by centrifugation. Nitro oxide (NO) production levels were measured using the Griess Reagent System according to the manufacturer's protocol (Peprotech). Nitric oxide levels in blood supernatant samples were measured by analyzing absorbance at 540 nm using a microplate reader (Bio-Rad).

17. 피부 침윤 백혈구 분리 (Isolation of Skin-infiltrating Leukocytes)17. Isolation of Skin-infiltrating Leukocytes

Skin-infiltrating leukocytes were isolated from the back skin or left ear skin of IMQ-induced psoriasis mice. Seven days after IMQ application, a 1 cm ² area was cut from the mouse back skin and left ear, the remaining IMQ cream was removed by washing with HBSS (Gibco), and the epidermis and dermis were separated using curved forceps. The separated epidermis and dermis were digested at 37°C using Dispase II solution (5 mg/ml, Sigma Aldrich). After the first digestion, the dermis was dissected into small pieces (<0.5%) using curved scissors in dermal dissociation buffer (DMEM (Hyclone, Roche) containing 1 mg/ml collagenase P and 100 μg/ml DNaseI (Hyclone, Roche)). mm) and incubated at 37°C for 1 hour. The fully digested dermal suspension was filtered through a 40-μm cell strainer (Falcon), rinsed with DMEM medium containing 10% FBS (Hyclone), and separated dermal-infiltrating leukocytes were collected by centrifugation. After the first digestion, the front and back were quickly cut into small pieces (< 2 mm) with curved scissors, transferred to 0.05% TE (Trypsin-EDTA, Hyclone) buffer, and incubated at 37°C for 5 minutes. After incubation, trypsin neutralizing solution (5% FBS diluted in HBSS) was added to the completely digested epidermis, and the epidermal suspension was filtered through a 40-μm cell strainer (Falcon). Isolated epidermal-infiltrating leukocytes were collected by centrifugation.

18. 통계 분석18. Statistical analysis

Results are expressed as mean ± SEM (n ≥3). Statistical analysis of group differences used unpaired Student's t-test or ANOVA analysis followed by Dunnett's multiple comparison test. The number of asterisks indicates the results of statistical analysis as follows: ns; Not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. GraphPad Prism 9 was used for analysis of all data.

[실시예][Example]

1. 일차 T 세포의 핵으로의 ndSTAT1-TMD의 세포내 전달 동역학 (Intracellular delivery kinetics of ndSTAT1-TMD into the nucleus of primary T cells)1. Intracellular delivery kinetics of ndSTAT1-TMD into the nucleus of primary T cells

CD4 ⁺ naive T cells can differentiate into Th1 cells by adopting the STAT1 signaling pathway, which includes phosphorylation (p-STAT1), homodimerization, and subsequent processes of STAT1 translocation. Various genetic and therapeutic approaches have been used to analyze STAT1 function in vitro and in vivo . However, the regulatory role of STAT1 in the functional context of T cell networks in vitro and in vivo remains fragmented. To investigate the role of STAT1 in the functional network of T cell subsets in vitro and in vivo under normal physiological conditions, ndSTAT1-TMD, a fusion protein between Hph-1-PTD and STAT1-TMD, was used as shown in Figure 1A. created. As a negative control, STAT1-TMD without Hph1-PTD was produced, and two essential amino acids (V426, T427) important for DNA binding were changed to D426 and D427. All these fusion proteins were expressed in the Escherichia coli system and purified under basic conditions using Ni-NTA agarose. These purified proteins were confirmed by Western blot using anti-6xHis and SDS-PAGE (Figure 1B).

Next, we investigated the intranuclear transduction kinetics of ndSTAT1-TMD in primary T cells. Unlike STAT1-TMD without Hph-1-PTD, ndSTAT1-TMD was efficiently delivered into cells in a dose- and time-dependent manner when treated at a concentration of 2 μM (Figure 1C-1D). The delivered ndSTAT1-TMD remained stably inside the cells up to 72 hours after delivery (Figure 1E). Additionally, when the subcellular localization of delivered ndSTAT1-TMD within primary T cells was visualized using confocal microscopy, ndSTAT1-TMD was localized to the nucleus of treated cells but not found in cells treated with STAT1-TMD. did (Figure 1F).

To test whether these fusion proteins have cytotoxicity, primary T cells were treated with ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) at various concentrations for 1 hour, and then the cytotoxic effect was analyzed through CCK-8 analysis. Neither ndSTAT1-TMD nor ndSTAT1-TMD(V426D, T427D) showed significant cytotoxicity (Figure 1G). These data show that ndSTAT1-TMD can be effectively and stably delivered into the nucleus of primary T cells without affecting cell viability.

2. ndSTAT1-TMD는 나이브 CD4+ T 세포가 Th1 및 Th17 T 세포 하위 집합으로 분화되는 것을 특이하게, 그리고 동시에 억제함.2. ndSTAT1-TMD specifically and simultaneously inhibits the differentiation of naïve CD4+ T cells into Th1 and Th17 T cell subsets.

T cell receptor (TcR) stimulation is an essential factor required for the successful differentiation of naïve CD4 ⁺ T cells into various T cell subsets. To investigate whether ndSTAT1-TMD or ndSTAT1-TMD(V426D, T427D) treatment of T cells affects T cell receptor-mediated early activation signaling events, T The induced surface expression levels of CD69, CD25, and IL-2 secretion were measured after cell receptor stimulation. As shown in Figures 1H and 1I, the expression levels of CD25, CD69, and interleukin-2 (IL-2) were not affected by treatment with ndSTAT1-TMD or ndSTAT1-TMD (V426D, V427D).

Next, to investigate the function of ndSTAT1-TMD during the differentiation of multiple CD4 ⁺ T cell subsets, including Th1, Th2, Th17, and regulatory T cells (Treg), naïve CD4+ T cells were transfected with ndSTAT1-TMD or ndSTAT1-TMD ( Different concentrations of V426D, T427D) were induced to differentiate into each T cell subset under the appropriate T cell subset-skewing conditions. T-bet expression, levels of intracellular and secreted IFN-γ in Th1 cells were significantly reduced by ndSTAT1-TMD treatment in a concentration-dependent manner, and the functional impact of ndSTAT1-TMD (V426D, T427D) on Th1 cell differentiation was significantly reduced by ndSTAT1 It was significantly less than -TMD (Figures 2A-2B).

In addition, the level of interferon regulatory factor 1 (IRF1), known as a primary responder to IFN-γ-STAT1 signaling in Th1 cells, decreased after ndSTAT1-TMD treatment, which is consistent with ndSTAT1 Unlike the Th1 group treated with -TMD (V426D, T427D), it decreased (Figure 2c). In addition, the expression levels of ROR-γt and intracellular IL-17A in Th17 cells were also significantly decreased by ndSTAT1-TMD in a concentration-dependent manner (Figure 2D). Additionally, we found that ndSTAT1-TMD treatment reduced the secretion of IL-17A and IFN-γ from Th17 cells (Figures 2E-2F). Meanwhile, it has been suggested that pathogenic Th17 cells can secrete IL-17A and IFN-γ.

Meanwhile, ndSTAT1-TMD appeared to have no effect on regulating the expression of GATA3 or interleukin-4 (IL-4) in Th2 cells and Foxp3 expression in Treg cells. ndSTAT1-TMD (V426D, T427D) treated cells showed a perceptible decrease in the expression of genes associated with immunological functions of Th1 or Th17 cells. However, the level of reduction was substantially lower compared to cells treated with the same concentration of ndSTAT1-TMD. These results suggest that, in addition to STAT1 binding to the promoter region of target genes, another functional domain of STAT1 is partially involved in regulating Th1 or Th17 cell differentiation. Taken together, these results show that ndSTAT1-TMD specifically inhibits the differentiation and function of both Th1 and Th17 cells without affecting T cell receptor-mediated early T cell activation signaling events.

3. ndSTAT1-TMD에 의한 Th1/17 세포 분화의 억제는 Th1/17 세포의 기능과 관련된 STAT1 표적 유전자의 전사 억제에 기인함 (Inhibition of Th1/17 cell differentiation by ndSTAT1-TMD is attributed to the transcriptional suppression of STAT1 target genes associated with the functions of Th1/17 cells).3. Inhibition of Th1/17 cell differentiation by ndSTAT1-TMD is attributed to the transcriptional suppression of STAT1 target genes related to the function of Th1/17 cells of STAT1 target genes associated with the functions of Th1/17 cells).

Next, we sought to determine whether the suppressive function of ndSTAT1-TMD during Th1 and Th17 cell differentiation is due to disruption of endogenous STAT1 binding to target gene promoters at the transcriptional level. Given that STAT1 binds to the IL-17A promoter, a plasmid in which induction of the IL-17A promoter induces expression of a luciferase reporter gene was co-transfected into HEK293T cells together with a plasmid containing the gene encoding wild-type STAT1. . The transfected cells were then incubated with various concentrations of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D). In the ndSTAT1-TMD treatment group, luciferase activity was significantly decreased in a concentration-dependent manner. On the other hand, the ndSTAT1-TMD treated group (V426D, T427D) did not show an inhibitory function (Figure 3A). Therefore, these results show that ndSTAT1-TMD specifically affects STAT1-mediated transcription within target cells.

Activated STAT1 is involved in the generation of Th1 cells secreting IFN-γ and Th17 cells secreting IL-17A in the early stage of lineage commitment. Disruption of such functional commitment of Th1 and/or Th17 cells during differentiation often leads to autoimmune diseases and immune dysfunction. The mRNA expression profile of Th1 and Th17 cells treated with ndSTAT1-TMD was analyzed to evaluate the intrinsic role of ndSTAT1-TMD on the expression of genes related to the function of Th1 and Th17 cells. Gene set enrichment analysis (GSEA) showed that ndSTAT1-TMD-treated Th1 cells showed significant downregulation in signature genes associated with the IFN-γ response (Figure 3B), while ndSTAT1-TMD-treated Th17 cells showed significant downregulation in STAT3-knockout CD4+ T cells. follows the gene enrichment pattern of (Figure 3C).

Consistent with this GSEA, mRNA sequencing data from ndSTAT1-TMD treated Th1 cells showed that genes involved in Th1 cell differentiation and function, such as Stat1 and Tbx21 , and genes required for the IFN-γ response, such as Irf5 , were relatively downregulated. was regulated (Figure 3D). Likewise, mRNA sequencing data generated from Th17 cells treated with ndSTAT1-TMD showed that the expression of genes involved in the function of Th17 cells, such as Rorc , Il17ra , and Ccl20, was significantly reduced, while the activity of Th17 cells, such as Osm and Eomes, was significantly reduced. Expression of genes known to be restrictive showed increased (Figure 3E). Additionally, Stat1 expression in Th17 cells was reduced, as observed in Th1 cells (Figure 3E). Additionally, we found that genes associated with naïve T cells were more abundant in the Th1 and Th17 populations treated with ndSTAT1-TMD compared to the untreated group (Figure 3D-3E). Taken together, these results suggest that the inhibition of Th1 and Th17 cell differentiation by ndSTAT1-TMD is due to competitive binding to the promoter region of STAT1 target genes, resulting in downregulation of the expression of genes related to the function of Th1 and Th17 cells. It has been done.

4. ndSTAT1-TMD는 IMQ에 의해 유발된 건선 증상을 완화함 (The ndSTAT1-TMD treatment alleviates the symptoms of psoriasis induced by IMQ)4. ndSTAT1-TMD alleviates psoriasis symptoms induced by IMQ (The ndSTAT1-TMD treatment alleviates the symptoms of psoriasis induced by IMQ)

Since psoriasis is known to be a disease caused by overactivated Th1 and Th17 cells, we next evaluated whether ndSTAT1-TMD had therapeutic potential for psoriasis. Psoriasis was induced by applying IMQ (imiquimod) cream to the back and ear skin of mice for 7 consecutive days, and as a positive control, ndSTAT1-TMD, ndSTAT1-TMD (V426D, T427D) or anti-IL-17A antibody was applied 2 days from the initial disease induction date. It was administered via intraperitoneal injection at daily intervals (Figure 4A). The psoriasis area and severity index (PASI) score, measured by analyzing the back skin of psoriasis-induced mice, peaked on the 5th day. It then gradually decreased on

day

6 or 7 throughout modeling (Figure 4B). The group treated with 60 μg of ndSTAT1-TMD had the lowest PASI score on day 5 among the treatment groups (Figure 4B). Likewise, with regard to the dorsal skin appearance status on

days

5 and 7, ndSTAT1-TMD treatment showed significant improvement in pathological changes compared to anti-IL-17A antibody treatment (Figure 4C). On the 5th day of onset, the thickness of the back skin doubled in the psoriasis-induced group. At the same time, treatment with ndSTAT1-TMD or anti-IL-17A antibody substantially reduced dorsal skin thickness closer to the normal group at day 5 and throughout the modeling period (Figures 4D, 4E). Weight loss in mice is also considered one of the criteria for measuring disease severity. Administration of ndSTAT1-TMD or anti-IL-17A antibody partially prevented weight loss, consistent with the treatment effect (Figure 4F). However, the therapeutic activity of ndSTAT1-TMD (V426D, T427) was partial or minimal (Figures 4B-4F).

Despite the fact that the pathological patterns, such as the PASI score plot and skin thickness changes in the ear skin, were different from those in the back skin, ndSTAT1-TMD treatment showed the most effective treatment function in the ear. When evaluating the concentrations of major pro-inflammatory cytokines secreted in the serum of each group of mice 7 days after IMQ induction, such as TNF-α, IL-1β, and IL-6, 60 μg of ndSTAT1-TMD or anti-IL-17A antibody A significant decrease in serum concentration was observed in the treated group (Figure 4G). These results show that ndSTAT1-TMD prevents psoriasis progression as effectively as anti-IL-17A antibody.

5. 건선 동물 모델의 2차 림프 기관에서 CD4+ T 세포 서브세트의 파괴된 상태는 ndSTAT1-TMD 처리에 의해 복원된다 (The disrupted status of CD4+ T cell subsets in secondary lymphoid organs of psoriasis animal model is restored by ndSTAT1-TMD treatment)5. The disrupted status of CD4+ T cell subsets in secondary lymphoid organs of psoriasis animal model is restored by ndSTAT1-TMD treatment -TMD treatment)

Because Th1 cells and Th17 cells are important factors in the development of psoriasis and ndSTAT1-TMD inhibited the function and differentiation program of both Th1 and Th17 cells in vitro and in vivo, ndSTAT1-TMD treatment on animal models of psoriasis may be effective in vivo. It was hypothesized to alter the functional status of CD4 ⁺ T cell subsets. Profiles of CD4 ⁺ T cell subsets in the spleen and draining lymph nodes (dLNs) of animal models of psoriasis revealed T-bet ⁺ or IFN-γ ⁺ CD4 ⁺ T cells and ROR-γt ⁺ in psoriasis-induced mice. Alternatively, there were higher levels of IL-17A ⁺ CD4 ⁺ T cells (Figures 5A-5D). ndSTAT1-TMD treatment was observed to substantially reduce the levels of both T-bet ⁺ and ROR-γt ⁺ CD4 ⁺ T cells in the spleen and draining lymph nodes (dLN). Anti-IL-17A antibody treatment showed relatively specific therapeutic function on ROR-γt CD4 ⁺ T cells (Figures 5A-5B). Consistent results were also obtained when we examined levels of IFN-γ and IL-17A secretion in secondary lymphoid organs after treatment with ndSTAT1-TMD or anti-IL-17A antibody (Figures 5C-5D). In contrast to the increase in Th1 and Th17 cells, the levels of CD4 ⁺ Foxp3 ⁺ T cells were decreased in psoriasis-induced mice. Functional inhibition of Th1 and Th17 cells by ndSTAT1-TMD in psoriasis-induced mice substantially restored the levels of CD4 ⁺ Foxp3 ⁺ T cells (Figure 5E). Overall, ndSTAT1-TMD treatment of psoriasis-induced animals showed therapeutic efficacy and restored the functional status of various T cell subsets.

6. ndSTAT1-TMD는 건선 유발 쥐의 피부에 백혈구가 침투하는 것을 제한 (ndSTAT1-TMD restricts the infiltration of leukocytes into the skin of psoriasis-induced mice)6. ndSTAT1-TMD restricts the infiltration of leukocytes into the skin of psoriasis-induced mice.

The histopathological features of the IMQ-induced psoriasis mouse model are human plaque-type psoriasis, such as acanthosis, parakeratosis, and neovascularization, along with infiltration of various leukocytes consisting of dendritic cells, neutrophils, macrophages, and T cells. It has been proven to be very similar to the multifactorial characteristics of . Additionally, several studies have reported that depletion of CD4 ⁺ T cells significantly reduces IMQ-induced skin inflammation. H&E-stained sections of the back and ear skin of psoriasis-induced mice showed epidermal thickening, elongated epidermal network ridges, keratosis, and dyskeratosis (Figure 6A). As shown in Figures 6A-6B, psoriasis-related inflammatory parameters of back and ear skin were significantly reduced after administration of 60 μg of ndSTAT1-TMD or anti-IL-17A antibody, but the effect of ndSTAT-TMD (V426D, T427D) was minimal. . In addition, histological observation of Masson's trichrome staining areas in the group treated with 60 μg of ndSTAT1-TMD or anti-IL-17A antibody showed that the infiltration of keratinocytes in the dermal area was significantly reduced and the occupancy of collagen fibers was reduced. It was found that (Figure 6A).

Next, we isolated leukocytes from the dermis and epidermis of the ear and back skin of treated mice to elucidate changes in the population of infiltrating leukocytes. In groups treated with 60 μg of ndSTAT1-TMD or anti-IL-17A antibody, the level of CD45 ⁺ leukocytes was reduced in the dermis of the ears and back skin, which was not observed in the epidermal area (Figure 6C). Considering that macrophages located in the dermal region of psoriatic skin are activated by IFN-γ or/and IL-17A, we investigated whether ndSTAT1-TMD could limit the activation and recruitment of macrophages. Administration of anti-IL-17A antibody or ndSTAT1-TMD resulted in a significant reduction of the CD11b ⁺ F4/80 ⁺ macrophage population in the dermal regions of the back and ear skin (Figure 6D). Consistent with these results, the nitric oxide (NO) level in serum at day 7 was substantially reduced in groups treated with ndSTAT1-TMD or anti-IL17A antibody, suggesting that ndSTAT1-TMD activates and recruits macrophages. (Figure 6E).

To investigate whether the reduction in macrophage activation and recruitment to the skin is due to an overall reduction in pathogenic T cell subsets, we measured the expression of T cell subset-specific transcription factors and cytokines in leukocytes in the dermal and epidermal regions of ear and back skin. analyzed. Groups treated with 60 μg or 20 μg ndSTAT1-TMD showed a significant decrease in CD45 ⁺ T-bet ⁺ T cells in dermal leukocytes (Figure 6F). In contrast, the levels of the CD45 ⁺ ROR-γt ⁺ T cell population in dermal leukocytes were not significantly affected by administration of ndSTAT1-TMD or anti-IL-17A antibodies (Figure 6G). Consistently, the group treated with 60 μg of ndSTAT1-TMD showed a substantial reduction in IFN-γ secretion from dermal leukocytes (Figure 6H). IL-17A secretion from dermal leukocytes was also reduced by treatment with ndSTAT1-TMD or anti-IL-17A antibody (Figure 6I). Meanwhile, ndSTAT1-TMD and anti-IL-17A antibody treatment did not exert significant inhibitory effects on macrophages or T cells within the epidermal leukocytes of the ear and back skin (Figures 6D and 6F-6I). Additionally, the ndSTAT1-TMD (V426D, T427D) treatment group showed no effect on the functional regulation of macrophages and T cells (Figures 6D-6I). These results demonstrate that ndSTAT1-TMD improves the inflammatory state of the dermal region of psoriatic skin by limiting activated Th1 and Th17 cells as well as macrophages.

7. DSS 유발 대장염의 중증도는 ndSTAT1-TMD 치료에 의해 약화 (The severity of DSS-induced colitis is attenuated by ndSTAT1-TMD treatment)7. The severity of DSS-induced colitis is attenuated by ndSTAT1-TMD treatment

Colitis is another inflammatory disease in which Th1 or/and Th17 cells play a pathogenic role. To evaluate the therapeutic potential of ndSTAT1-TMD in colitis animal models, we generated a dextran sulfate sodium (DSS)-induced colitis mouse model and animals were treated with ndSTAT1-TMD, and nti-IL-17A antibody treatment served as a positive control. was used (Figure 7A). The severity of IBD was assessed by starting treatment with ndSTAT1-TMD, ndSTAT1-TMD (V426D, T427D), or anti-IL-17A antibody on day 6 after colitis induction via intraperitoneal injection and monitoring disease progression daily for another 6 days. monitored.

The weight loss rate was significantly improved in the group treated with ndSTAT1-TMD or anti-IL-17A antibody, whereas the group treated with ndSTAT1-TMD (V426D, T427D) continued to lose weight (Figure 7B). The overall disease activity index (DAI) score of colitis was substantially reduced in the ndSTAT1-TMD treatment group, similar to the anti-IL-17A antibody treatment group (Figure 7C). Consistent with these data, colon length was well maintained in groups treated with every 100 μg of ndSTAT1-TMD or anti-IL-17A antibody. However, no improvement in disease progression or colon length was seen in the ndSTAT1-TMD (V426D, T427D) treatment group (Figures 7C-7D). To determine the histopathological status of the colon, colon sections were stained with H&E or PAS 6 days after intraperitoneal administration. Signs of inflammatory colon, such as increased infiltrating inflammatory cells, goblet cell depletion, crypt damage, and changes in muscle layer thickness, were improved in the anti-IL-17A antibody or ndSTAT1-TMD treated groups (Figures 7E-7G). On the other hand, in the ndSTAT1-TMD treatment group (V426D, T427D), the severity of colitis remained at a severe level without any improvement (Figures 7E-7G).

On day 12, the serum of each treatment group was tested for the levels of secreted pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. The secretion of these pro-inflammatory cytokines was significantly reduced in the ndSTAT1-TMD-treated group in a dose-dependent manner (Figure 7H).

Based on the results of psoriasis animal models, we hypothesized that ndSTAT1-TMD could improve colitis symptoms by suppressing the pathogenic functions of inflammatory Th1 and Th17 cells. The levels of T-bet ⁺ or IFN-γ ⁺ CD4 ⁺ T cells and ROR-γt ⁺ or IL-17A ⁺ CD4 ⁺ T cells in the spleen and mesenteric lymph nodes (mLN) in IBD-induced mice treated with PBS It was significantly higher than that of healthy controls. ndSTAT1-TMD or anti-IL-17A antibody treatment resulted in decreased T-bet ⁺ or IFN-γ ⁺ CD4 ⁺ T cells and ROR-γt ⁺ or IL-17A ⁺ CD4 ⁺ T cells in both the spleen and mesenteric lymph nodes (mLNs). The number of cells was greatly reduced. Consistent with the therapeutic potential of ndSTAT1-TMD in psoriasis animal models, ndSTAT1-TMD showed dual inhibitory functions against Th1 cells and Th17 cells. The decrease in Th1 and Th17 cells by ndSTAT1-TMD treatment was accompanied by an increase in Foxp3 expressing CD4 ⁺ Treg cells in the spleen and mesenteric lymph nodes (mLN). Taking these results together, ndSTAT1-TMD treatment showed a therapeutic effect in DSS-induced colitis comparable to anti-IL-17A antibody treatment.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims

Fusion protein containing the transcriptional regulatory domain and protein transduction domain (PTD) of STAT1 (Signal transducer and activator of transcription 1).
According to paragraph 1,

The STAT1 is a fusion protein represented by the amino acid sequence of SEQ ID NO: 13.
According to paragraph 1,

The transcriptional regulatory domain of STAT1 is a fusion protein represented by the amino acid sequence of SEQ ID NO: 14.
According to paragraph 3,

The protein transport domains include Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (antennapedia), Pep-1 (peptide-1), PTD-5 (protein transduction domain-5), 11R, 7R and A fusion protein selected from the group consisting of CTP (cytoplamic transduction peptide).
According to paragraph 4,

A fusion protein wherein the protein transport domain is represented by any one of the amino acid sequences of SEQ ID NOs: 1 to 12.
According to clause 5,

The fusion protein competitively inhibits STAT1-mediated transcription.
According to clause 6,

The fusion protein inhibits Th1 or Th17 differentiation of naïve CD4+ T cells.
A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 7.
Lipid nanoparticles (LNPs) comprising the nucleic acid molecule of claim 8.
An expression vector into which the nucleic acid molecule of claim 9 is inserted.
A host cell transfected with the expression vector of claim 10.
Containing as an active ingredient any one of the fusion protein of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the lipid nanoparticle (LNP) of claim 9, the expression vector of claim 10, and the host cell of claim 11. Pharmaceutical composition for preventing or treating immune-related diseases.
According to clause 12,

A pharmaceutical composition for preventing or treating an immune-related disease, wherein the immune-related disease occurs due to activation or dysfunction of Th1 cells or Th17 cells.
According to clause 12,

The immune-related disease is at least one selected from the group consisting of autoimmune disease, degenerative brain or vascular disease, metabolic disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease. , Pharmaceutical composition for preventing or treating immune-related diseases.
According to clause 14,

The autoimmune diseases include psoriasis, acanthosis, parakeratosis, neo-angiogenesis, inflammatory bowel disease (IBD), rheumatoid arthritis, systemic scleroderma, and systemic lupus erythematosus. , atopic dermatitis, alopecia areata, asthma, Crohn's disease, Behce's disease, Sjögren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyositis, and polyarteritis nodosa. A pharmaceutical composition for preventing or treating immune-related diseases.
According to clause 14,

The acute or chronic inflammatory diseases include colitis, sepsis, septic shock, inflammatory bowel disease (IBD), psoriasis, peritonitis, nephritis, acute bronchitis, chronic bronchitis, osteoarthritis, enteropathy spondylitis, and chronic obstructive inflammatory disease. Characterized by at least one selected from the group consisting of chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, acute lung injury, and broncho-pulmonary dysplasia , Pharmaceutical composition for preventing or treating immune-related diseases.
According to clause 14,

The degenerative brain or blood vessel diseases include stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, and prion disease. ), Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, Amyotrophic lateral sclerosis, Paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear disease A pharmaceutical composition selected from the group consisting of paralysis, nervous system autoimmune diseases, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular diseases, spinal cord injury and tauopathy.
According to clause 14,

The metabolic diseases include insulin resistance disease, fatty liver, diabetes, type 2 diabetes, hyperlipidemia, hypertriglyceridemia, dyslipidemia, cardiovascular disease, stroke, myocardial infarction, hyperglycemia, hyperinsulinemia, arteriosclerosis, hypertension, heart disease, and obesity. , a pharmaceutical composition that is one or more selected from the group consisting of liver disease, kidney damage, and high blood pressure.
Containing as an active ingredient any one of the fusion protein of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the lipid nanoparticle (LNP) of claim 9, the expression vector of claim 10, and the host cell of claim 11. Pharmaceutical composition for preventing or treating cancer.
According to clause 19,

A pharmaceutical composition for preventing or treating cancer, wherein the cancer occurs due to activation or dysfunction of Th1 cells or Th17 cells.
According to clause 20,

The pharmaceutical composition, wherein the cancer is stomach cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer.
Administering to a subject any one of the fusion protein of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the lipid nanoparticle (LNP) of claim 9, the expression vector of claim 10, and the host cell of claim 11. , Treatment method for immune-related diseases.
According to clause 22,

A treatment method in which the immune-related disease occurs due to activation or dysfunction of Th1 cells or Th17 cells.
According to clause 23,

The immune-related disease is at least one selected from the group consisting of autoimmune disease, degenerative brain or vascular disease, metabolic disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease. , Treatment method for immune-related diseases.
According to clause 24,

The autoimmune diseases include psoriasis, acanthosis, parakeratosis, neo-angiogenesis, inflammatory bowel disease (IBD), rheumatoid arthritis, systemic scleroderma, and systemic lupus erythematosus. , atopic dermatitis, alopecia areata, asthma, Crohn's disease, Behce's disease, Sjögren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyositis, and polyarteritis nodosa. A method of treating immune-related diseases, characterized in that.
According to clause 24,

The acute or chronic inflammatory diseases include colitis, sepsis, septic shock, inflammatory bowel disease (IBD), psoriasis, peritonitis, nephritis, acute bronchitis, chronic bronchitis, osteoarthritis, enteropathy spondylitis, and chronic obstructive inflammatory disease. Characterized by at least one selected from the group consisting of chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, acute lung injury, and broncho-pulmonary dysplasia , Treatment method for immune-related diseases.
According to clause 24,

The degenerative brain or blood vessel diseases include stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, and prion disease. ), Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, Amyotrophic lateral sclerosis, Paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear disease A method of treatment selected from the group consisting of paralysis, nervous system autoimmune diseases, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular diseases, spinal cord injury, and tauopathy.
According to clause 14,

The metabolic diseases include insulin resistance disease, fatty liver, diabetes, type 2 diabetes, hyperlipidemia, hypertriglyceridemia, dyslipidemia, cardiovascular disease, stroke, myocardial infarction, hyperglycemia, hyperinsulinemia, arteriosclerosis, hypertension, heart disease, and obesity. , liver disease, kidney damage, and high blood pressure.
Cancer in which any one of the fusion protein of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the lipid nanoparticle (LNP) of claim 9, the expression vector of claim 10, or the host cell of claim 11 is administered to a subject. treatment method.
According to clause 29,

A method of treating cancer, wherein the cancer occurs due to activation or dysfunction of Th1 cells or Th17 cells.
According to clause 30,

The cancer is stomach cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer.