WO2024112143A1 - Utilisation de stat1-tmd dans des maladies immunitaires - Google Patents

Utilisation de stat1-tmd dans des maladies immunitaires Download PDF

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WO2024112143A1
WO2024112143A1 PCT/KR2023/019074 KR2023019074W WO2024112143A1 WO 2024112143 A1 WO2024112143 A1 WO 2024112143A1 KR 2023019074 W KR2023019074 W KR 2023019074W WO 2024112143 A1 WO2024112143 A1 WO 2024112143A1
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disease
cells
cancer
tmd
ndstat1
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Korean (ko)
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김정호
김범석
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주식회사 굳티셀
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • 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.
  • STAT1 Signal transducer and activator of transcription 1, STAT1
  • PTD protein transduction domain
  • 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- ⁇ (IFN- ⁇ ), 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.
  • T helper 1 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).
  • STAT1 Signal transducer and activator of transcription 1
  • 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.
  • the protein transduction domain 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.
  • TMD transcription modulation domain
  • PTD protein transduction domain
  • 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.
  • 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.
  • 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).
  • PTD protein transduction domain
  • 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).
  • 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.
  • STAT1 Signal transducer and activator of transcription 1 regulates Th1 and Th17 cell lineage commitment at early stages and maintains immune function in vitro and in vivo.
  • ndSTAT1-TMD nuclear delivery form 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 na ⁇ ve 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.
  • 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- ⁇ to activate both macrophages and dendritic cells (DC). Secreted IFN- ⁇ 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- ⁇ 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).
  • 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- ⁇ t 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.
  • transcription modulation domain refers to a domain that constitutes a transcription factor and consists of only a DNA binding site without a transactivation domain.
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ORF open reading frame
  • an expression vector is provided into which the isolated nucleic acid molecule provided by the present invention is inserted.
  • the “vector” is a nucleic acid molecule capable of transporting another nucleic acid to which a nucleic acid molecule is linked.
  • a vector which refers to a circular double-stranded DNA into which additional DNA segments can be ligated.
  • 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).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors can enter a host cell and become integrated into the host cell's genome, thereby replicating along with the host genome.
  • 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”.
  • expression vectors useful in recombinant DNA techniques often exist in the form of plasmids.
  • plasmid and “vector” may be used interchangeably since plasmid is the most commonly used form of vector.
  • 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, Q ⁇ , 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.
  • 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.
  • 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.
  • a host cell line transformed with the expression vector provided by the present invention is provided.
  • 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.
  • 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.
  • bacterial cells such as Escherichia
  • lipid nanoparticles containing the nucleic acid molecules are provided.
  • 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).
  • auxiliary lipids selected from positively charged lipids, neutral lipids, and negatively charged lipids can be additionally used.
  • positively charged lipids and neutral lipids may be used
  • neutral liposomes neutral lipids
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • LNPs lipid nanoparticles
  • 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
  • broncho-pulmonary dysplasia it is not limited to this.
  • psoriasis is a recurrent inflammatory skin disease characterized by thick, non-pruritic plaques covered with silvery scales of the skin.
  • IBD Inflammatory bowel disease
  • CD Crohn's disease
  • UC ulcerative disease
  • 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.
  • IFN- ⁇ and IL-17A secreted by Th1 and Th17 cells, respectively, are important mediators that induce and accelerate the pathological progression of psoriasis and IBD.
  • therapeutic strategies targeting IFN- ⁇ or IL-17A for psoriasis and IBD still face several limitations that need to be overcome.
  • ndSTAT1-TMD nuclear delivery of the transcription modulation domain of STAT1
  • TMD transcription modulation domain
  • PTD human protein transduction domain
  • ndSTAT1-TMD nuclear delivery of the transcription modulation domain of STAT1
  • 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.
  • '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.
  • 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.
  • 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.
  • 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.
  • the “neurodegenerative disease” or “degenerative brain or vascular disease” may refer to a disease caused by decreased or lost function of nerve cells
  • 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.
  • 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.
  • the “object of interest” refers to an individual who has developed, or is likely to develop, an immune-related disease.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a pharmaceutically acceptable carrier as described above.
  • 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.
  • it can be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • PTD protein transduction domain
  • STAT1 provides a fusion protein represented by the amino acid sequence of SEQ ID NO: 13.
  • a fusion protein in which the transcriptional regulatory domain of STAT1 is represented by the amino acid sequence of SEQ ID NO: 14.
  • 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.
  • LNPs lipid nanoparticles
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • IBD inflammatory bowel disease
  • COPD chronic obstructive pulmonary disease
  • rheumatoid arthritis acute lung injury
  • broncho-pulmonary dysplasia broncho-pulmonary dysplasia.
  • 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.
  • 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
  • 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 ⁇ M) or STAT1-TMD (2 ⁇ M) 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 ⁇ m.
  • Figure 1G shows cell cytotoxicity evaluation of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) in mouse splenocytes analyzed by CCK-8 assay.
  • Figure 2 shows the functional impact of ndSTAT1-TMD on the differentiation ability of CD4 + naive T cells into various T cell subsets.
  • Figure 2A shows flow cytometric analysis of intracellular T-bet and IFN- ⁇ in Th1 cells treated with various concentrations (0.5-2 ⁇ M) of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) under Th1-skewing conditions. It represents.
  • Figure 2B shows the level of IFN- ⁇ 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- ⁇ t and intracellular IL- levels in Th17 cells treated with various concentrations (0.5-2 ⁇ M) 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- ⁇ 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.
  • 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 .
  • FIGS 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.
  • Figure 4A shows PBS (IMQ group), anti-IL-17A antibody (60 ⁇ g/mouse), and STAT1-TMD (20 ⁇ g/mouse or 60 ⁇ g/mouse), or ndSTAT1-TMD (V426D, T427D).
  • This shows a treatment method for 5% IMQ-induced psoriasis mice using (60 ⁇ g/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.
  • 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- ⁇ , IL-1 ⁇ , or IL-6) in the serum of mice from each group measured by ELISA.
  • TNF- ⁇ proinflammatory cytokines
  • IL-1 ⁇ IL-1 ⁇
  • IL-6 proinflammatory cytokines
  • Figure 5 shows that ndSTAT1-TMD treatment restores the destroyed functional state of CD4 + T cells in a psoriasis animal model.
  • 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- ⁇ t + 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- ⁇ + (left panels of (C) and (D)) or CD4 + IL-17A + ( (C) and (D) right panel) levels were analyzed on day 7.
  • Figure 6 shows that ndSTAT1-TMD treatment of a psoriasis animal model prevents leukocytes from infiltrating the back skin.
  • 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 ⁇ m for 100X and 100 ⁇ m 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.
  • 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.
  • FIGS 6F and 6G show infiltration of T-bet + (Figure 6F) or ROR- ⁇ t + ( 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- ⁇ + (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.
  • Figure 7 shows that ndSTAT1-TMD alleviates the disease progression of DSS-induced colitis.
  • Figure 7A shows 100 ⁇ g of PBS (DSS group), anti-IL-17A antibody (100 ⁇ g/mouse), ndSTAT1-TMD (20 ⁇ g/mouse or 100 ⁇ g/mouse), or ndSTAT1-TMD (V426D, T427D) (100 ⁇ g). /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 ⁇ m for 40X and 100 ⁇ m for 200X.
  • Figure 7F shows the thickness of the colonic muscle layer of each treatment group measured from the image in Figure 7E.
  • 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).
  • 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.
  • 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).
  • HEK293T cells were incubated with 10% heat-inactivated fetal bovine serum (FBS, Hyclone), 2mM L-glutamate (Lonza), 100 ⁇ g/ml penicillin-streptomycin (Lonza), 1mM sodium pyruvate (Lonza), and NEAA (Gibco, Waltham). , MA, USA) was cultured in DMEM medium (Lonza, Basel, Switzerland) supplemented with .
  • 2x105 mouse splenocytes were cultured with recombinant proteins in 96-well cell culture plates (Eppendorf) in a dose-dependent manner (0.5-2 ⁇ M, 1 h) or time-dependent manner (2 ⁇ M, 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 ⁇ M 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 na ⁇ ve 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 ⁇ M).
  • 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 ⁇ M STAT1-TMD, or 1 ⁇ M ndSTAT1-TMD for 1 hour.
  • SPL 12-well cell culture plates
  • 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).
  • ndSTAT1-TMD and ndSTAT1-TMD were 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.
  • 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).
  • 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 ⁇ g/ml anti-CD3 ⁇ (BD Bioscience, San Diego, CA, USA) and anti-CD28 (BD Bioscience) for 72 h. activated.
  • Th1 25 ng/ml of recombinant mouse IL-12 and 2 ⁇ g/ml of anti- IL-4 antibody
  • Th2 200 ng/ml recombinant mouse IL-4 and 2 ⁇ g/ml anti-IFN- ⁇ antibody
  • Th17 1 ng/ml recombinant mouse TGF- ⁇ 1, 25 ng/ml recombinant mouse IL) -6, 2 ⁇ g/ml anti-IFN- ⁇ antibody and 2 ⁇ g/ml anti-IL-4 antibody
  • iTreg 5 ng/ml recombinant mouse TGF- ⁇ 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 .
  • 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- ⁇ , IL-1 ⁇ , 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).
  • ndSTAT1-TMD and ndSTAT1-TMD 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 ⁇ g of pCMV6 vector containing mouse wild-type STAT1 and 1 ⁇ g of IL using Lipofectamine Reagents (Invitrogen) diluted in Opti-MEM (Gibco). -17A promoter-luciferase vector was co-transfected.
  • ndSTAT1-TMD or ndSTAT1-TMD V426D, T427D
  • V426D, T427D a dose-dependent manner (0.5-2 ⁇ M).
  • 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).
  • 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.
  • GSEA Gene set enrichment analysis
  • mice 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).
  • SPPF semi-specific-pathogen-free
  • YLARC Yonsei Animal Research Center
  • Psoriasis Induction and scoring of psoriasis area and severity index (PASI)
  • ndSTAT1-TMD (20 or 60 ⁇ g/mouse), ndSTAT1-TMD (V426D, T427D) (60 ⁇ g/mouse), or anti-IL-17A antibody (BioXCell, Lebanon, NH, USA) (60 ⁇ g/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 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 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.
  • mice 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 ⁇ g/mouse), ndSTAT1-TMD (V426D, T427D) (100 ⁇ g/mouse), or anti-IL-17A antibody (BioXCell, Lebanon, NH, USA) (100 ⁇ g/mouse). was injected intraperitoneally.
  • DAI Body weight and disease activity index
  • Weight change 0, 0-1%; 1, 1-5%; 2, 5-10%; 3, 10-20%; 4, >20%.
  • DSS dextran sulfate sodium
  • 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).
  • ndSTAT1-TMD 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.
  • 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- ⁇ -APC, anti-IL-17A-APC, anti-T-bet-PE, anti-ROR- ⁇ t-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- ⁇ -PE, anti-IL-17A-PE, anti-T-bet-PE, anti-ROR- ⁇ t-PE (eBioscience), and anti- Stained with -F4/80-PE (BD Bioscience).
  • Cell Stimulation Cocktail 500X
  • eBioscience Cell Stimulation Cocktail
  • 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).
  • 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).
  • 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 ⁇ g/ml DNaseI (Hyclone, Roche)). mm) and incubated at 37°C for 1 hour.
  • the fully digested dermal suspension was filtered through a 40- ⁇ m cell strainer (Falcon), rinsed with DMEM medium containing 10% FBS (Hyclone), and separated dermal-infiltrating leukocytes were collected by centrifugation.
  • 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- ⁇ m cell strainer (Falcon). Isolated epidermal-infiltrating leukocytes were collected by centrifugation.
  • 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.
  • 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.
  • STAT1 signaling pathway which includes phosphorylation (p-STAT1)
  • p-STAT1 phosphorylation
  • homodimerization homodimerization
  • subsequent processes of STAT1 translocation Various genetic and therapeutic approaches have been used to analyze STAT1 function in vitro and in vivo .
  • STAT1 signaling pathway which includes phosphorylation (p-STAT1), homodimerization, and subsequent processes of STAT1 translocation.
  • ndSTAT1-TMD specifically and simultaneously inhibits the differentiation of na ⁇ ve 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 na ⁇ ve CD4 + T cells into various T cell subsets.
  • TcR T cell receptor
  • 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.
  • 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).
  • na ⁇ ve 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.
  • interferon regulatory factor 1 known as a primary responder to IFN- ⁇ -STAT1 signaling in Th1 cells
  • IRF1 interferon regulatory factor 1
  • the level of interferon regulatory factor 1 (IRF1) decreased after ndSTAT1-TMD treatment, which is consistent with ndSTAT1 Unlike the Th1 group treated with -TMD (V426D, T427D), it decreased ( Figure 2c).
  • the expression levels of ROR- ⁇ t and intracellular IL-17A in Th17 cells were also significantly decreased by ndSTAT1-TMD in a concentration-dependent manner (Figure 2D).
  • ndSTAT1-TMD treatment reduced the secretion of IL-17A and IFN- ⁇ from Th17 cells ( Figures 2E-2F). Meanwhile, it has been suggested that pathogenic Th17 cells can secrete IL-17A and IFN- ⁇ .
  • 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.
  • the level of reduction was substantially lower compared to cells treated with the same concentration of ndSTAT1-TMD.
  • 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).
  • ndSTAT1-TMD during Th1 and Th17 cell differentiation is due to disruption of endogenous STAT1 binding to target gene promoters at the transcriptional level.
  • 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).
  • Activated STAT1 is involved in the generation of Th1 cells secreting IFN- ⁇ 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.
  • GSEA Gene set enrichment analysis
  • 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- ⁇ response, such as Irf5 , were relatively downregulated. was regulated ( Figure 3D).
  • 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).
  • ndSTAT1-TMD alleviates psoriasis symptoms induced by IMQ (The ndSTAT1-TMD treatment alleviates the symptoms of psoriasis induced by IMQ)
  • 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).
  • ndSTAT1-TMD treatment showed significant improvement in pathological changes compared to anti-IL-17A antibody treatment ( Figure 4C).
  • the thickness of the back skin doubled in the psoriasis-induced group.
  • ndSTAT1-TMD treatment showed the most effective treatment function in the ear.
  • major pro-inflammatory cytokines secreted in the serum of each group of mice 7 days after IMQ induction such as TNF- ⁇ , IL-1 ⁇ , and IL-6
  • 60 ⁇ g of ndSTAT1-TMD or anti-IL-17A antibody A significant decrease in serum concentration was observed in the treated group ( Figure 4G).
  • 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- ⁇ + CD4 + T cells and ROR- ⁇ t + in psoriasis-induced mice.
  • 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.
  • ndSTAT1-TMD V426D, T427D
  • ndSTAT1-TMD improves the inflammatory state of the dermal region of psoriatic skin by limiting activated Th1 and Th17 cells as well as macrophages.
  • Colitis is another inflammatory disease in which Th1 or/and Th17 cells play a pathogenic role.
  • DSS dextran sulfate sodium
  • ndSTAT1-TMD nti-IL-17A antibody treatment served as a positive control.
  • 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.
  • 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).
  • ndSTAT1-TMD could improve colitis symptoms by suppressing the pathogenic functions of inflammatory Th1 and Th17 cells.
  • the levels of T-bet + or IFN- ⁇ + CD4 + T cells and ROR- ⁇ t + 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- ⁇ + CD4 + T cells and ROR- ⁇ t + 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.

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Abstract

Une protéine de fusion (ndSTAT1-TMD) selon la présente invention peut être efficacement administrée au noyau de cellules sans affecter la cytotoxicité et un événement de signalisation à médiation par TcR. De plus, la protéine de fusion selon la présente invention inhibe spécifiquement l'activité transcriptionnelle de STAT1 endogène dans des cellules Th1 et Th17, et présente un potentiel thérapeutique similaire à un anticorps anti-IL -17A lorsqu'elle est administrée au modèles de souris atteintes de psoriasis et de souris IBD. Par conséquent, la protéine de fusion selon la présente invention régule les fonctions de cellules Th1 et Th17 pathogènes ensemble et peut ainsi être un nouveau médicament pour des maladies auto-immunes médiées par les cellules Th1/17.
PCT/KR2023/019074 2022-11-24 2023-11-24 Utilisation de stat1-tmd dans des maladies immunitaires WO2024112143A1 (fr)

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Publication number Priority date Publication date Assignee Title
US5716622A (en) * 1995-01-06 1998-02-10 The Rockefeller University Functionally active regions of signal transducer and activators of transcription
US6160092A (en) * 1998-05-29 2000-12-12 The Rockefeller University Crystal of the core portion of a signal transducer and activator of transcription (STAT)
US20130231274A1 (en) * 2010-08-20 2013-09-05 Sang-Kyou Lee Fusion protein having transcription factor transactivation-regulating domain and protein transduction domain, and transcription factor function inhibitor comprising the same
US20190315820A1 (en) * 2016-01-25 2019-10-17 Good T Cells, Inc. A Composition Containing SMAD Protein for Treatment of Autoimmune Diseases, a Fusion Protein Comprising SMAD Protein, an Expression Vector and a Method for Preparing the Same
CN112807425A (zh) * 2021-01-14 2021-05-18 南方医科大学深圳医院 一种tTIM融合蛋白疫苗、制备方法及应用

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US5716622A (en) * 1995-01-06 1998-02-10 The Rockefeller University Functionally active regions of signal transducer and activators of transcription
US6160092A (en) * 1998-05-29 2000-12-12 The Rockefeller University Crystal of the core portion of a signal transducer and activator of transcription (STAT)
US20130231274A1 (en) * 2010-08-20 2013-09-05 Sang-Kyou Lee Fusion protein having transcription factor transactivation-regulating domain and protein transduction domain, and transcription factor function inhibitor comprising the same
US20190315820A1 (en) * 2016-01-25 2019-10-17 Good T Cells, Inc. A Composition Containing SMAD Protein for Treatment of Autoimmune Diseases, a Fusion Protein Comprising SMAD Protein, an Expression Vector and a Method for Preparing the Same
CN112807425A (zh) * 2021-01-14 2021-05-18 南方医科大学深圳医院 一种tTIM融合蛋白疫苗、制备方法及应用

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PARK JIYOON, SON MIN-JI; HO CHUN-CHANG; KIM YUNA; AN JAEKYEUNG; LEE SANG-KYOU: "Transcriptional inhibition of STAT1 functions in the nucleus alleviates Th1 and Th17 cell-mediated inflammatory diseases", FRONTIERS IN IMMUNOLOGY, FRONTIERS MEDIA, LAUSANNE, CH, vol. 13, 15 December 2022 (2022-12-15), Lausanne, CH , pages 1 - 19, XP093174175, ISSN: 1664-3224, DOI: 10.3389/fimmu.2022.1054472 *

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