WO2017119521A1 - Nouvelle protéine de fusion comprenant le domaine de modulation de transcription de p65 et domaine de transport de protéine et son utilisation - Google Patents

Nouvelle protéine de fusion comprenant le domaine de modulation de transcription de p65 et domaine de transport de protéine et son utilisation Download PDF

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WO2017119521A1
WO2017119521A1 PCT/KR2016/000104 KR2016000104W WO2017119521A1 WO 2017119521 A1 WO2017119521 A1 WO 2017119521A1 KR 2016000104 W KR2016000104 W KR 2016000104W WO 2017119521 A1 WO2017119521 A1 WO 2017119521A1
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fusion protein
protein
domain
disease
present
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PCT/KR2016/000104
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Korean (ko)
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이상규
박성동
양정진
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이상규
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Priority to CA3010811A priority Critical patent/CA3010811A1/fr
Priority to JP2018536197A priority patent/JP7051687B2/ja
Priority to AU2016385177A priority patent/AU2016385177B2/en
Priority to PCT/KR2016/000104 priority patent/WO2017119521A1/fr
Publication of WO2017119521A1 publication Critical patent/WO2017119521A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present invention is made by the task number NRF-2014R1A2A1A10052466, 2009-0083522 (ERC), 10044494 and 2015-22-0065 under the support of Yonsei University, under the support of the Ministry of Science, ICT and Future Planning, “Korea Research Foundation”, research project name is “medium researcher support project”, research project title “Treg customized immune disease new material, which is an immune activation control cell”, host organization is “Yonsei University Industry-Academic Cooperation Group”, research period is 2014.11.01 ⁇ 2017.10.31, and the research management specialized organization of the project number 2009-0083522 (ERC) is "Korea Research Foundation", the research project name is "Korea Research Foundation-Field of Science and Engineering (SRC / ERC)", and the research project name is "ERC / Research Center for Protein Function Control (3/3, Phase 2) ”, the lead institution is“ Yonsei University Industry-Academic Cooperation Group ”, and the research period is 2009.09.01 ⁇ 2016.02.
  • the present invention relates to a novel fusion protein comprising the transcriptional modulation domain (TMD) and protein transduction domain (PTD) of p65, a subunit of the transcription factor NF- ⁇ B and its use.
  • TMD transcriptional modulation domain
  • PTD protein transduction domain
  • Cytokines regulated by the transcription factor NF- ⁇ B include tumor necrosis factor- ⁇ (TNF- ⁇ ), interleukin-1 (IL-1), interleukin-6 and granulocytes. Macrophage colony stimulating factor (GM-CSF), and chemokines (chemokine) are interleukin-8, macrophage-inflammatory protein-1 ⁇ (MIP-1 ⁇ ), main Methyl accepting chemotaxis protein-1 (MCP-1) and eotaxin.
  • adhesion molecules regulated by NF- ⁇ B include E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1.
  • IL-1 endothelial leukocyte adhesion molecule-1, ELAM-1) and intercellular cell adhesion molecule-1 (ICAM-1)
  • the inducible enzyme is cyclooxygenase-2 (cyclooxygenase-2). 2 COX-2), NF- ⁇ B is involved in almost every physiological reaction in the body.
  • NF- ⁇ B The transcription factor NF- ⁇ B consists of different subunits composed of homologs or heterodimers.
  • NF- ⁇ B proteins include RelA (p65), c-Rel, Rel-B, NF- ⁇ B1 (p50) and NF- ⁇ B2 (p52), wherein p50 and p52 are their precursors NF- ⁇ B1 (p105) and Produced in NF- ⁇ B 2 (p100), respectively.
  • NF- ⁇ B proteins all have 300 amino acids in common called the N-terminal R-homology domain (RHD), which are involved in dimer formation, binding to specific DNA, and reaction with I ⁇ B proteins. It also contains a nuclear localization signal (NLS) to move into the nucleus and act as a transcription factor.
  • RHD N-terminal R-homology domain
  • the constitutive proteins of NF- ⁇ B are class I (NF- ⁇ B1, NF- ⁇ B2) and class II (RelA / p65, RelB, c-Rel) depending on the presence of the C-terminal transactivation domain (TAD).
  • Class II has a transactivation domain that can act as a transcription factor without other NF- ⁇ B constitutive proteins, and class I lacks a transactivation domain. Dimers form between these two classes of NF- ⁇ B to act as DNA transcription factors, the most common of which is the form of p50 / p65.
  • RelA (p65), RelB and c-Rel have a transactivation domain at the C terminus and can activate the expression of a target gene.
  • p50 and p52, NF- ⁇ B1 and NF- ⁇ B2 do not have a transactivation domain at the C terminus, so the homodimers of p50 and p52 are transcribed without binding of proteins such as co-activators with transactivation domains. Can't.
  • Protein transduction domain is a short hydrophobic short peptide, and is known to effectively transfer physiologically active molecules such as proteins or DNA and RNA fused together into cells.
  • the present inventors have developed two PTDs to date and are described in detail in WO 2003059940 and WO 2003059941. Since the protein transport domain can carry bioactive molecules not only into the cytoplasm but also into the nucleus, the protein transport domain has properties suitable for delivering a modified transcription factor, which is a key substance of the present invention, into the nucleus.
  • the present inventors inhibited the transcription and activity of NF- ⁇ B by competitive inhibition using a fusion protein comprising a transcriptional regulatory domain and a protein transport domain of p65, a subunit of NF- ⁇ B. It was intended to effectively treat the resulting disease.
  • the present inventors have made diligent research efforts to develop substances that can effectively prevent, ameliorate or treat diseases caused by excessive activity of NF- ⁇ B.
  • the present invention was completed by confirming that, when using a fusion protein comprising a transcriptional regulatory domain and a protein transport domain of p65, which is a subunit of NF- ⁇ B, the transcription and activity of NF- ⁇ B can be inhibited by competitive inhibition. It was.
  • Another object of the present invention is to provide an NF- ⁇ B transcription or activity inhibitor comprising the fusion protein of the present invention.
  • Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating NF- ⁇ B overactivity-related diseases comprising the fusion protein of the present invention as an active ingredient.
  • Another object of the present invention is to provide a method for inhibiting NF- ⁇ B transcription or activity and a method for preventing, ameliorating or treating NF- ⁇ B overactivity-related diseases.
  • the invention provides a fusion protein comprising a transcriptional regulatory domain and a protein transport domain of p65, which is a subunit of NF- ⁇ B. Fusion proteins of the invention inhibit NF- ⁇ B transcription by competitive inhibition.
  • the present inventors have made diligent research efforts to develop substances that can effectively prevent, ameliorate or treat diseases caused by excessive activity of NF- ⁇ B. As a result, it was confirmed that in the case of using a fusion protein comprising a transcriptional regulatory domain and a protein transport domain of p65, which is a subunit of NF- ⁇ B, the transcription and activity of NF- ⁇ B can be inhibited by competitive inhibition.
  • transcriptional regulatory domain refers to a domain constituting a transcription factor and refers to a domain consisting of only DNA binding sites without a transactivation domain.
  • the fusion protein of the present invention can bind to the desired promoter but cannot promote transcription because it has no transactivation domain but has a DNA binding site. Therefore, since the fusion protein of the present invention is a dominant negative mutant for the NF- ⁇ B gene, it can act as a competitive inhibitor against wild-type NF- ⁇ B in cells and inhibit the transcription and activity of NF- ⁇ B.
  • the NF- ⁇ B of the present invention is NF- ⁇ B selected from the group consisting of RelA (p65), c-Rel, Rel-B, NF- ⁇ B1 (p50) and NF- ⁇ B2 (p52) to be.
  • the NF- ⁇ B of the invention is RelA (p65).
  • the transcriptional regulatory domain of NF- ⁇ B of the present invention consists of the amino acid sequence of SEQ ID NO: 1.
  • the transcriptional regulatory domain of NF- ⁇ B consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention is encoded by the nucleotide sequence of SEQ ID NO: 3.
  • protein transport domain refers to a short hydrophobic short peptide composed of 7-50 amino acids, and refers to a domain capable of delivering DNA or RNA into cells as well as proteins with a molecular weight of 120 kDa or more.
  • proteins without the protein transport domain of the present invention i.e., p65-TMD consisting only of the transcriptional regulatory domain
  • NF- ⁇ B and IL-2 Inhibition of transcription, inhibition of inflammatory cytokine secretion by LPS, and inhibition of production of IL-2, IFN- ⁇ , IL-4, IL-17A and IL-10 in splenocytes were confirmed.
  • the protein transport domain of the present invention is Hph-1, Sim-2, Tat, VP22, Antp (antennapedia), Pep-1 (peptide-1), PTD-5 (protein transduction domain- 5), 7R, 9R, 11R and CTP (cytoplamic transduction peptide).
  • the terms “7R”, “9R” and “11R” refer to peptides consisting of seven, nine and eleven arginine, respectively.
  • the protein transport domain of the invention is Hph-1.
  • the protein transport domain of the invention consists of the amino acid sequence of SEQ ID NO: 2 sequence.
  • the protein transport domain consisting of the amino acid sequence of SEQ ID NO: 2 of the present invention is encoded by the nucleotide sequence of SEQ ID NO: 4.
  • the fusion protein of the invention comprises the amino acid sequence of SEQ ID NO: 5 sequence.
  • the fusion protein comprising the amino acid sequence of SEQ ID NO: 5 of the present invention is encoded by the nucleotide sequence of SEQ ID NO: 6.
  • the invention provides an NF- ⁇ B transcriptional or activity inhibitor comprising the fusion protein of the invention.
  • the fusion proteins of the present invention inhibit the transcription and activity of NF- ⁇ B and IL-2 (FIG. 6), inhibit the secretion of inflammatory cytokines by LPS (FIG. 7), in splenocytes.
  • T cells by NF- ⁇ B inhibiting the production of IL-2, IFN- ⁇ , IL-4, IL-17A and IL-10 expressed by anti-CD3 / CD28 stimulated T cell activation (FIG. 9). It was confirmed that it is possible to inhibit the proliferation, differentiation and secretion-inducing activity of inflammatory cytokines, and thus it was confirmed that the disease caused by excessive activity of NF- ⁇ B can be effectively prevented, improved or treated.
  • the present invention provides a pharmaceutical composition for preventing or treating NF- ⁇ B overactivity-related disease comprising the fusion protein of the present invention as an active ingredient.
  • the term “treatment” means (a) inhibiting the development of a disease, disorder or condition; (b) alleviation of a disease, illness or condition; Or (c) eliminating a disease, condition or symptom.
  • the composition of the present invention serves to inhibit, eliminate or alleviate the development of the symptoms of metabolic disease.
  • the composition of the present invention may itself be a composition for the treatment of NF- ⁇ B overactive-related diseases, or is administered together with a composition for the treatment of other NF- ⁇ B overactive-related diseases to aid in the treatment of these diseases. It may be applied as.
  • the term “treatment” or “therapeutic agent” in this specification includes the meaning of "treatment aid” or "treatment aid”.
  • the present invention is the fusion protein of the present invention inhibits the transcription of NF- ⁇ B and IL-2, inhibits the secretion of inflammatory cytokines by LPS, IL-2 in splenocytes Inhibits the production of IFN- ⁇ , IL-4, IL-17A and IL-10. Therefore, the fusion protein of the present invention can be usefully used as an effective prophylactic or therapeutic composition for various NF- ⁇ B overactivity-related diseases.
  • the NF- ⁇ B overactive-related diseases of the invention are inflammatory diseases or autoimmune diseases.
  • the NF- ⁇ B overactive-related disease of the present invention is sepsis shock, allergic asthma, allergic rhinitis, atopic dermatitis, systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, lacrimitis , Alzheimer's disease, stroke, arteriosclerosis, vascular restenosis, type I diabetes, type II diabetes, gallbladder, conjunctivitis, psoriasis, systemic inflammatory syndrome, multiple myositis, dermatitis, nodular polyarthritis, mixed connective tissue syndrome, Scheren's syndrome , Gout, Parkinson's disease, Amyotrophic lateral sclerosis, diabetic retinopathy, multiple sclerosis, Crohn's disease, chronic thyroiditis, ceriac disease, myasthenia gravis,
  • the NF- ⁇ B overactive-related disease of the invention is sepsis shock.
  • Septic shock which is caused by microorganisms and causes severe inflammatory reactions throughout the body, causes cardiovascular and vascular dysfunction caused by endotoxins and inflammatory mediators that circulate in blood vessels. This results in hypovolemia due to dehydration and the leakage of intravascular fluid, constriction and relaxation of blood vessels on capillaries, resulting in abnormal blood supply.
  • Vasculitis and thrombus make reflux into tissue more difficult, resulting in hypoxia in tissues. Induces metabolic acidosis.
  • endotoxins activate NF- ⁇ B in immune cells (macrophages, granules, dendritic cells, lymphocytes) to secrete cytokines such as tumor necrosis factor, interleukin 1 and 6, and these cytokines may be neutrophils, endothelial cells Cells that release platelets, or inflammatory mediators, stimulate the systemic response.
  • cytokines such as tumor necrosis factor, interleukin 1 and 6
  • these cytokines may be neutrophils, endothelial cells Cells that release platelets, or inflammatory mediators, stimulate the systemic response.
  • the fusion protein of the present invention inhibits inflammatory cytokine secretion and increases survival in the pulmonary pulmonary shock animal model induced by LPS (FIG. 10).
  • the fusion protein of the present invention has the effect of preventing or treating NF- ⁇ B overactivity-related diseases.
  • the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, and the like. It doesn't happen.
  • the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.
  • a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, a kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mann
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like. According to one embodiment of the invention, the pharmaceutical composition of the invention may be administered by intraperitoneal infusion.
  • Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, pathological condition, food, time of administration, route of administration, rate of excretion, and response to response of the patient. Can be.
  • the daily dose of the pharmaceutical composition of the present invention is, for example, 0.0001-1000 mg / kg.
  • compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or may be in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
  • the NF- ⁇ B transcriptional or activity inhibitor of the present invention and the pharmaceutical composition for preventing or treating NF- ⁇ B overactivity-related diseases have the above-described fusion proteins in common, the contents common to the relationship with the fusion proteins are the present invention. The description is omitted to avoid excessive complexity.
  • the invention provides a method for inhibiting NF- ⁇ B transcription or activity comprising the step of administering a composition comprising the fusion protein of the invention as an active ingredient.
  • the present invention provides a method for preventing, ameliorating, or preventing NF- ⁇ B overactivity-related diseases comprising administering to a subject in need thereof a composition comprising the fusion protein of the invention as an active ingredient. Provide treatment.
  • the term “administration” or “administer” refers to the formation of the same amount in a subject's body by directly administering a therapeutically effective amount of a composition of the invention to an individual in need thereof (ie, a subject). Say what you can. Therefore, the term “administration” includes the injection of the active ingredient of the present invention (fusion protein of the present invention) to the lesion site, so the term “administer” is used in the same meaning as "inject”.
  • a “therapeutically effective amount” of a composition means a content of an extract sufficient to provide a therapeutic or prophylactic effect to the individual to whom the composition is to be administered, and includes a “prophylactically effective amount”.
  • the term “individual” includes, without limitation, human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. Specifically, the subject of the present invention is a human.
  • the method for inhibiting NF- ⁇ B transcription or activity of the present invention and the method for preventing, ameliorating or treating NF- ⁇ B overactivity-related diseases have the same fusion protein and its use as described above, the fusion protein, NF- ⁇ B transcription Or common in relation to inhibition of activity and NF- ⁇ B overactivity-related diseases, the description of which is omitted to avoid excessive complexity of the present invention.
  • the present invention provides a fusion protein comprising the transcriptional regulatory domain and protein transport domain of p65, which is a subunit of NF- ⁇ B, and its use.
  • the fusion protein of the present invention inhibits the transcription of NF- ⁇ B and IL-2 by competitive inhibition, inhibits the secretion of inflammatory cytokines by LPS, IL-2, IFN- ⁇ , Since there is an effect of inhibiting the production of IL-4, IL-17A and IL-10, it can be usefully used as a prophylactic or therapeutic composition for NF- ⁇ B overactivity-related diseases.
  • nt-p65-TMD p65 transcriptional regulatory domain
  • PTD Hph-1
  • Figure 2 shows Coomassie blue staining results of the fusion protein (nt-p65-TMD) of the present invention.
  • Figure 3 shows the results of intracellular delivery of BV2 and Jurkat T cells of the fusion protein (nt-p65-TMD) of the present invention.
  • Figure 4 shows the results of intranuclear delivery of BV2 and HeLa cells of the fusion protein (nt-p65-TMD) of the present invention.
  • FIG. 5 shows cytotoxicity results in BV2 and spleen cells of the fusion protein (nt-p65-TMD) of the present invention.
  • Figure 6 shows the competitive transcriptional inhibitory effect of the fusion protein (nt-p65-TMD) of the present invention.
  • Figure 7 shows the expression inhibitory effect of the fusion protein (nt-p65-TMD) of the present invention on TNF- ⁇ , IL-1 ⁇ and IL-6 expressed by LPS.
  • 8A shows that the fusion protein (nt-p65-TMD) of the present invention specifically inhibits the transcription of NF- ⁇ B by T cell activation stimulated with anti-CD3 / CD28.
  • 8B shows that the fusion protein (nt-p65-TMD) of the present invention does not affect the signaling pathway by T cell activation stimulated with anti-CD3 / CD28.
  • FIG. 9 shows IL-2, IFN- ⁇ , IL-4, IL-17A and IL-10 in which the fusion proteins of the present invention (nt-p65-TMD) are expressed by T cell activation stimulated with anti-CD3 / CD28. The expression inhibition result of this is shown.
  • fusion protein (nt-p65-TMD) of the present invention increases the survival rate by inhibiting the secretion of inflammatory cytokines TNF- ⁇ , IL-1 ⁇ and IL-6 in septic shock animal model .
  • Example 1 Preparation of recombinant fusion protein comprising a transcriptional regulatory domain and a protein transport domain of p65
  • the recombinant fusion DNA was transformed into BL21 CodonPlus (DE3) -RIPL E. coli strain (Invitogen). After incubating the transformed strain, 1 mM IPTG (isopropyl- ⁇ -D-thiogalactopyranoside, Duchefa) was added thereto, and induced to express the protein at 37 ° C. for 5 hours.
  • digestion buffer (10 mM imidazole, 50 mM NaH 2 PO 4 , 300 mM NaCl and pH 8.0), and the cells were digested with a grinder.
  • the fusion protein was bound to Ni-NTA beads (Qiagen) using six histidines artificially bound to the front of the protein.
  • the protein was added to a column (HisTrap chromatography columns, Bio-Rad), washed well with washing buffer (30 mM imidazole, 50 mM NaH 2 PO 4 , 300 mM NaCl and pH 8.0), and elution buffer (250 mM imidazole).
  • nt-p65-TMD of Example 1 was concentrated (0, 0.1, 0.5, 1, 2 and 5 ⁇ M) or hourly (0, 1, 2, 4) using BV2 microglia and Jurkat T cells. , 6, 12, 24 and 48 h) was incubated with the recombinant fusion protein and confirmed by protein delivery by Western blot.
  • nt-p65-TMD recombinant fusion protein of Example 1 was incubated with BV2 microglia and HeLa cells for 1 hour, washed with PBS, and then washed with 0.2% Triton X-100 (Sigma-Aldrich). Fluorescently labeled antibody was bound to the recombinant fusion protein in between. Next, after staining the nucleus of the cell using a DAPI stain (Invitrogen), the position of the fluorescence was confirmed by fluorescence microscopy to confirm the position where the recombinant fusion protein was delivered.
  • DAPI stain Invitrogen
  • Cytotoxicity tests were performed to confirm that LPS was completely removed from the protein obtained from the E. coli strains, thereby showing no toxicity in cells or animals. After delivery of various concentrations of protein to BV2 microglia and splenocytes, the cells were incubated with WST-8, a colored substrate by dehydrogenase in living cells.
  • nt-p65-TMD recombinant fusion protein of Example 1 binds to the promoters of the NF- ⁇ B and IL-2 cytokine genes instead of wild-type p65 to inhibit expression
  • a luciferase reporter gene was identified.
  • nuclear transfection of NF- ⁇ B and IL-2 promoters with wild luciferase and wild-type p65 genes into HEK293T cells was performed, followed by treatment with the nt-p65-TMD recombinant fusion protein.
  • nt-p65-TMD acted as a competitive inhibitor of wild type p65, blocking the binding site of p65 of the NF- ⁇ B and IL-2 promoters (see FIG. 6).
  • NF nt-p65-TMD recombinant fusion protein of Example 1 activated by picket T cell activation stimulated with anti-CD3 (1 ⁇ g / ml, BD Pharmingen) and anti-CD28 (1 ⁇ g / ml, BD Pharmingen) It was confirmed whether or not specifically inhibit the transcription of - ⁇ B.
  • T cells are activated, not only NF- ⁇ B but also NFAT transcription is activated, if nt-p65-TMD fusion protein inhibits NF- ⁇ B without affecting NFAT transcription, nt-p65-TMD recombinant fusion protein It can be seen that it acts as a competitive inhibitor specific to NF- ⁇ B.
  • Luciferase reporter gene was used to confirm this.
  • NF- ⁇ B and NFAT reporter genes having luciferases in the nucleus were first injected into the nucleus by electroporation, followed by nt- to the anti-CD3 and anti-CD28 stimulated nt- cells.
  • p65-TMD recombinant fusion protein was treated.
  • nt-p65-TMD recombinant fusion protein of Example 1 is involved in tyrosine phosphorylation of proteins involved in various signal transduction systems in cells.
  • Splenocytes were isolated from the spleens of 6-8 week old female C57BL / 6 mice and treated with nt-p65-TMD of Example 1 for 1 hour to deliver the recombinant fusion protein into the cells.
  • the cells were stimulated with anti-CD3 (1 ⁇ g / ml) and anti-CD28 (1 ⁇ g / ml) and incubated for 72 hours. Thereafter, the amount of cytokines present in the culture was measured by ELISA.
  • Sepsis shock animal models were made by intraperitoneally injecting LPS (20 mg / kg) into male BALB / c mice aged 6-8 weeks. After 2 hours and 14 hours, the nt-p65-TMD recombinant fusion protein of Example 1 was intraperitoneally injected and observed for 6 days, respectively.
  • Example 1 of the present invention in 2 animals per group After oral administration of the recombinant fusion protein at a dose of 1 g / kg, the animals were examined for mortality, clinical symptoms, and weight changes. Hematological and hematological and biochemical tests were performed. Abnormalities of thoracic organs were observed.
  • the recombinant fusion protein of Example 1 of the present invention did not show toxicity even in rats up to 1 g / kg, and it was confirmed that the oral administration minimum dose (LD 50 ) was determined to be a safe substance of 1 g / kg or more.

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Abstract

La présente invention concerne une nouvelle protéine de fusion comprenant un domaine de modulation de transcription de p65 qui est une sous unité du facteur de transcription NF-κB, et un domaine de transport de protéine et son utilisation. La protéine de fusion selon la présente invention présente les effets d'inhibition de la transcription de NF-κB et IL-2 par inhibition compétitive, inhibition de la sécrétion de cytokines inflammatoires par LPS, et inhibition de la production d'IL-2, IFN-γ, IL-4, IL-17A et IL-10 dans des splénocytes, et peut ainsi être utile en tant que composition pour la prévention ou le traitement de maladies liées à l'hyperactivité de NF-κB.
PCT/KR2016/000104 2016-01-06 2016-01-06 Nouvelle protéine de fusion comprenant le domaine de modulation de transcription de p65 et domaine de transport de protéine et son utilisation WO2017119521A1 (fr)

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CA3010811A CA3010811A1 (fr) 2016-01-06 2016-01-06 Nouvelle proteine de fusion comprenant le domaine de modulation de transcription de p65 et domaine de transport de proteine et son utilisation
JP2018536197A JP7051687B2 (ja) 2016-01-06 2016-01-06 Ｐ６５の転写調節ドメインと蛋白質運搬ドメインとを含む新規融合蛋白質およびその用途
AU2016385177A AU2016385177B2 (en) 2016-01-06 2016-01-06 Novel fusion protein comprising transcription modulation domain of P65 and protein transport domain and use thereof
PCT/KR2016/000104 WO2017119521A1 (fr) 2016-01-06 2016-01-06 Nouvelle protéine de fusion comprenant le domaine de modulation de transcription de p65 et domaine de transport de protéine et son utilisation

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PCT/KR2016/000104 WO2017119521A1 (fr) 2016-01-06 2016-01-06 Nouvelle protéine de fusion comprenant le domaine de modulation de transcription de p65 et domaine de transport de protéine et son utilisation

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CA3010811A1 (fr) 2017-07-13
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JP2019507117A (ja) 2019-03-14
AU2016385177A1 (en) 2018-08-09

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