WO2017034244A1 - Peptide ayant pour effet de prévenir ou traiter des maladies du système nerveux central et composition pharmaceutique pour la prévention et le traitement de maladies du système nerveux central, contenant ce dernier en tant que principe actif - Google Patents

Peptide ayant pour effet de prévenir ou traiter des maladies du système nerveux central et composition pharmaceutique pour la prévention et le traitement de maladies du système nerveux central, contenant ce dernier en tant que principe actif Download PDF

Info

Publication number
WO2017034244A1
WO2017034244A1 PCT/KR2016/009201 KR2016009201W WO2017034244A1 WO 2017034244 A1 WO2017034244 A1 WO 2017034244A1 KR 2016009201 W KR2016009201 W KR 2016009201W WO 2017034244 A1 WO2017034244 A1 WO 2017034244A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
ctctla
peptide
dnp2
amino acid
Prior art date
Application number
PCT/KR2016/009201
Other languages
English (en)
Korean (ko)
Inventor
최제민
임상호
Original Assignee
한양대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 한양대학교 산학협력단 filed Critical 한양대학교 산학협력단
Priority to EP16839533.3A priority Critical patent/EP3336100B1/fr
Priority to CN201680056710.3A priority patent/CN108137659B/zh
Priority to US15/753,915 priority patent/US11053295B2/en
Priority claimed from KR1020160105642A external-priority patent/KR101969513B1/ko
Publication of WO2017034244A1 publication Critical patent/WO2017034244A1/fr
Priority to US16/544,671 priority patent/US11351224B2/en

Links

Images

Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • 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

Definitions

  • the present invention relates to a peptide having a preventive or therapeutic effect on diseases of the central nervous system and a pharmaceutical composition for preventing and treating central nervous system diseases using the same as an active ingredient.
  • MS Multiple sclerosis
  • BBB blood-brain barrier
  • IFN-y interleukin-17A
  • Th1 and Th17 cells induce inflammation and neuronal cell necrosis at the central nervous system, including the brain and spinal cord, and at high levels in these cells.
  • Non-Patent Documents 1 and 2 are examples of the effector cells.
  • the developed therapeutic biomolecules are derived from synthetic compounds or other organisms, have not only long-term toxicity or other side effects, but are still limited by BBB and BSCB, There was a problem that did not show the effect.
  • Patent Literature 1 A blood-brain barrier comprising peptides with NH2-arginine-proline-hydroxyproline-glycine-thienyl alanine-serine-proline-4-Me-tyrosine? (CH2NH) -arginine-COOH or structural analogues thereof with certain modifications (Patent Literature 1), and Korean Patent Laid-Open Publication No. 10-2014-0026372 discloses an amino acid sequence having a blood-brain barrier permeability function as an amino acid sequence represented by a predetermined sequence list Document 2).
  • the present invention has been made in view of the above problems, and it is an object of the present invention to provide a peptide having excellent transmission efficiency against blood-brain barrier or blood-spinal cord barrier and having excellent IL-2 inhibitory activity, And to provide a method for preventing or treating such diseases.
  • the present invention provides a peptide having an IL-2 inhibitory activity comprising the amino acid sequence represented by SEQ ID NO: 1.
  • the present invention provides a peptide having an IL-2 inhibitory activity comprising a fragment of the cytoplasmic region of the CTLA-4 protein represented by SEQ ID NO: 2 or SEQ ID NO: 3, or a fusion peptide thereof.
  • the fusion peptide is characterized by being composed of the amino acid sequence shown in SEQ ID NO: 4.
  • the present invention provides a peptide comprising an amino acid sequence represented by SEQ ID NO: 1, a fusion peptide thereof or a fragment thereof; And a cell permeable peptide.
  • the fragment is characterized by being composed of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3.
  • the fusion peptide is characterized by being composed of the amino acid sequence shown in SEQ ID NO: 4.
  • the cell permeable peptides include HIV-1 tat (47-57), D-amino acid substituted HIV-1 tat (48-60), arginine substituted HIV-1 tat (48-60), Drosophila Antennapaedia (43-58), viral RNA-binding peptides comprising 7 or more amino acids, DNA binding peptides comprising 7 or more arginines, polyarginine polypeptides with 6 to 8 arginines, poly Lysine polypeptide, dNP2 protein of the amino acid sequence of SEQ ID NO: 5, Hph-1 (SEQ ID NO: 6), Transportan (SEQ ID NO: 8), Pep-1 (SEQ ID NO: 9), pVEC (SEQ ID NO: 11), TP10 (SEQ ID NO: 12), VP22 (SEQ ID NO: 13), Buforin 2 (SEQ ID NO: 14), KALA (SEQ ID NO: 15), CL22 And may be any one selected.
  • cell permeable peptide is a dNP2 protein having an amino acid sequence represented by SEQ ID NO: 5.
  • the present invention provides a peptide comprising an amino acid sequence represented by SEQ ID NO: 1, a fusion peptide thereof or a fragment thereof; And a gene for coating a fusion substance comprising a cell permeable peptide.
  • the cell permeable peptide in the fusant is characterized by having blood-brain barrier or blood-spinal cord barrier permeability.
  • central nervous system disease is any one selected from the group consisting of spinal cord injury, stroke, cerebral infarction, cerebral ischemia, Alzheimer's disease, and multiple sclerosis.
  • Another object of the present invention is to provide a method for preventing or treating a central nervous system disease in an animal other than a human by administering a pharmaceutical composition for prevention or treatment of the central nervous system disease to an individual.
  • the present invention has the ability to penetrate the blood-brain barrier and blood-spinal cord barrier of the central nervous system which can not be passed through with good efficiency, and at the same time has the IL-2 inhibitory activity and is administered rapidly, An effective therapeutic effect can be obtained.
  • the present invention can deliver local administration compared to conventional therapeutic agents, so that side effects are reduced and local high dose administration of the therapeutic agent is possible, so that potential new treatments and prescriptions are possible.
  • FIG. 1A is a diagram showing the results of identification and mutation of a fragment of the cytoplasmic region of the CTLA-4 protein according to the present invention.
  • FIG. 1A is a diagram showing the results of identification and mutation of a fragment of the cytoplasmic region of the CTLA-4 protein according to the present invention.
  • 1B is a diagram showing the result of identification of dNP2 as a human-derived cell permeable peptide according to the present invention.
  • Figure 2 shows the result of analysis of the structure of dNP2-ctCTLA-4 fusion protein of the present invention and dNP2-ctCTLA-4 fusion protein by SDS-PAGE.
  • FIG. 3 is a graph showing the intracellular delivery efficiency of ctCTLA-4 peptide, dNP2-ctCTLA-4, and Hph-1-ctCTLA-4 fusions in primary mouse CD4-T cells.
  • FIG. 4 is a graph showing the results of measuring the inhibitory effect of IL-2 on 1 ⁇ M PBS, dNP2-ct CTLA-4 fusions and TAT-ct CTLA-4 fusions. Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • FIGS. 5A and 5B are graphs showing the results of measuring the inhibitory effect of IL-2 on dNP2-ctCTLA-4 fused dNP2-EGFP fusions. Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • FIG. 6A is a graph showing the results of measuring the inhibitory effect of dNP2-ctCTLA-4 fusion and dNP2-EGFP fusion on the expression of IFN-y.
  • FIG. 6B is a graph showing the inhibitory effect of dNP2-ctCTLA-4 fusion and dNP2- As shown in FIG. Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • FIG. 7 is a graph showing the experimental results of Experiment 3 (1), which graph shows the clinical scores for EAE animal models (in the y-axis) versus days after disease induction (in the x-axis) .
  • Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • FIG. 8 is a graph showing the experimental results of Experiment 3 (2), which shows the clinical score for the EAE animal model (in the y-axis) on days after disease induction (in the x-axis) .
  • Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • FIG. 9 is a graph showing the experimental results from Experiment 3 (3), which shows the clinical scores for EAE animal models (in the y-axis) versus days after disease induction (in the x-axis) .
  • Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • FIG. 10 is a graph showing experimental results from Experimental Example 3, 4). This graph shows the clinical scores for EAE animal models (in the y-axis) versus days after disease induction (in the x-axis) . Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • Figure 11 shows the effect of the dNP2-ctCTLA-4 fusions and dNP2-EGFP fusions of the invention on demyelination and immune cell infiltration in EAE-induced animal models against luxoph fast blue (LFB) Mattoxylin and eosin (H & E) staining.
  • the scale bar is 100 ⁇ .
  • FIG. 12 is a graph showing the number of infiltrated immune cells of spinal cord tissues counted by Image J software 1.48v according to each treatment group measured in FIG. Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • Figure 13 shows that IL-17A and / or IL-17A is produced by isolating spinal cord cells in an EAE animal model treated with Th1, Th17, Th2 and Treg cells (T cells) of dNP2-ctCTLA-4 fusions of the present invention and dNP2- IFN-y expressing CD4 T cells.
  • FIG. 14 is a graph showing the results of FIG. 13.
  • Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • 15 is a graph showing the results of measuring the inhibitory effect of 0.5, 1, 2 or 5 ⁇ M WT, 1YF, 2YF, and DYF on IL-2 expression. Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • FIG. 16 is a graph showing the measurement of introduction efficiency in primary mouse CD4-T cells treated with only PBS without adding any.
  • 17 is a graph showing the intracellular delivery efficiency of 0.1, 0.5, 1, 2 or 5 ⁇ M dNP2-TAMRA, dNP2-ctCTLA-4 fusions and dNP2-ctCTLA-4-fm3 fusions in primary mouse CD4-T cells to be.
  • 18 is a graph showing the results of measuring the inhibitory effect of IL-2 on 0.1, 0.5, 1, 2 or 5 ⁇ M dNP2-TAMRA fusions, dNP2-CTLA-4 fusions and dNP2-ctCTLA-4-fm3 fusions. Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • Central nervous system (CNS) -invasive effector T cells are known to play a pivotal role in the pathogenesis and progression of multiple sclerosis (MS).
  • MS Central nervous system
  • these MS-related drugs are currently very limited. This is because it is very difficult to control invasive T cells by transferring these drugs to the CNS.
  • the present invention overcomes the problems of the conventional medicines and attempts to develop a new protein related to the central nervous system disease while having excellent permeability of the tightly organized brain-blood barrier or brain-spinal cord barrier, .
  • An aspect of the present invention relates to a peptide having an IL-2 inhibitory activity comprising an amino acid sequence represented by SEQ ID NO: 1.
  • a feature of the present invention is that it has excellent permeability to brain blood or spinal cord blood which is not readily introduced into cells, and reduces the activity of T cells (particularly Th17), thereby reducing the cause of autoimmune disease, A new protein that can be prevented or treated has been developed.
  • the peptide having the IL-2 inhibitory activity comprising the amino acid sequence of SEQ ID NO: 1 used in the present invention encodes only a part of the full-length CTLA-4 protein 4 obtained from human or mouse.
  • the peptide having an inhibitory activity against IL-2 comprising the amino acid sequence represented by SEQ ID NO: 1 is a fragment of CTLA-4 that exhibits IL-2 expression inhibitory activity in CTLA-4 protein.
  • the peptide has a central nervous system, - It can be applied to all of them by using a fragment having 100% identical amino acid sequence in an animal (mouse) other than human and human.
  • the peptide having an IL-2 inhibitory activity comprising the amino acid sequence represented by SEQ ID NO: 1 comprises the 188th amino acid residue to the 213rd amino acid sequence of the cytotoxic T lymphocyte antigen type 4 (CTLA-4) protein (SEQ ID NO: 1), hereinafter also referred to as 'ctCTLA-4'.
  • CTLA-4' cytotoxic T lymphocyte antigen type 4
  • Another aspect of the present invention relates to a peptide having an IL-2 inhibitory activity comprising a fragment of the cytoplasmic domain of the CTLA-4 protein represented by SEQ ID NO: 2 or SEQ ID NO: 3, or a fusion peptide thereof.
  • the fragment of the ctCTLA-4 protein includes (from SEQ ID NO: 2) the amino acid sequence from the 201st amino acid residue to the 210th amino acid residue of the ctCTLA-4 protein, or the amino acid sequence from the 218th amino acid residue to the 223rd amino acid sequence May be a fragment of the above-mentioned polypeptide partially deleted at the N-terminus and C-terminus, which has a permeability of a brain-vascular barrier or a spinal-vascular barrier and a preventive or therapeutic effect.
  • the fusion peptide may be an amino acid sequence represented by SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • the peptide having the IL-2 inhibitory activity is a very small peptide, it has an advantage of minimizing possible biological interference.
  • the peptide having the IL-2 inhibitory activity may be extracted from natural sources, synthesized, or prepared by a DNA recombinant method.
  • the peptide having the IL-2 inhibitory activity derived from the CTLA-4 protein improves the migration to the central nervous system, the permeation of the blood-brain barrier and the blood-spinal cord barrier, . Therefore, the peptide having the IL-2 inhibitory activity of the present invention can be usefully used for prevention or treatment of diseases of the central nervous system.
  • Another aspect of the present invention is a peptide comprising the amino acid sequence of SEQ ID NO: 1, a fusion peptide thereof or a fragment thereof; And a cell permeable peptide.
  • the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the fragment thereof, or the fusion peptide thereof is the same as that described above, and will not be described here.
  • the peptide comprising the amino acid sequence of SEQ ID NO: 1, the fragment thereof, or the fusion peptide of the fragment may further include a cell permeable peptide at one or both ends thereof, , And such fusions may be referred to as " fusion proteins ".
  • fusion substance or " fusion protein” means a covalent bond complex formed by genetic fusion or chemical bonding of ctCTLA-4 peptide or a fragment or fusion peptide thereof and a cell permeable peptide.
  • genetic fusion means a link consisting of a linear, covalent bond formed through genetic expression of a DNA sequence encoding a protein.
  • the cell permeable peptide is HIV-1 tat (47-57), D-amino acid substituted HIV-1 tat (47-57), arginine substituted HIV-1 tat ), Drosophila Antennapaedia (43-58), viral RNA-binding peptides comprising 7 or more amino acids, DNA binding peptides comprising 7 or more arginines, polyarginine polypeptides having 6 to 8 arginines , Polylysine polypeptide having 7 to 11 lysines, dNP2 protein of the amino acid sequence of SEQ ID NO: 5, Hph-1 (SEQ ID NO: 6), Transportan (SEQ ID NO: 8), Pep-1 (SEQ ID No.
  • cell permeable peptides are known to have high in vitro permeability to various cell lines, and binding of cargo proteins thereto can be expected to increase cell permeability.
  • the above-mentioned cell permeable peptides have been found to have a much lower permeation efficiency to primary cells, and these cell permeable peptides have been greatly restricted in clinical applications in humans (Simon, MJ TAT-mediated intracellular protein delivery to primary brain cells is dependent on glycosaminoglycan expression. Biotechnology and bioengineering 104, 10-19, doi: 10.1002 /bit.22377 (2009)).
  • the cytotoxic peptide is bound to the ctCTLA-4 protein or a fragment thereof having the central nervous system, particularly the brain-vascular barrier or spinal cord-vascular barrier permeability, I could confirm.
  • the cell permeable peptide is the dNP2 protein of the amino acid sequence shown in SEQ ID NO: 5, it was confirmed that the permeability to brain-vascular barrier or spinal-vascular barrier was significantly increased.
  • Yet another aspect of the invention relates to a recombinant expression vector comprising a gene that coat said fusion.
  • a recombinant expression vector comprising a gene encoding the peptide having the IL-2 inhibitory activity and a gene encoding the cell permeable peptide.
  • the recombinant expression vector may comprise a sequence of the cell permeable peptide and the peptide having the IL-2 inhibitory activity (SEQ ID NOS: 1, 2, 3 or 4) and a tag sequence facilitating purification of the fusion substance, A continuous histidine codon, a maltose binding protein codon, and a Myc codon, and may further include a fusion partner for increasing the solubility of the fusion, and the like.
  • a spacer amino acid or a base sequence may be further included for stability of the overall structure and function of the recombinant protein or flexibility of a protein encoded by each gene.
  • spacers examples include AAY (PM Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (RPM Sutmuller et al., J Immunol 2000, 165: 7308-7315) But are not limited to, lysine residues (S. Ota et al., Can Res. 62, 1471-1476, KS Kawamura et al., J Immunol. 2002, 168: 5709-5715). Also, it may include a sequence specifically cleaved by an enzyme to remove an unnecessary portion of the recombinant protein, an expression control sequence, and a marker or reporter gene sequence for confirming intracellular delivery. no.
  • the expression control sequence used in the recombinant expression vector may be a regulatory domain including a promoter specific to cells, tissues, or organs in which the target DNA and / or RNA is selectively transferred or expressed.
  • the fused substance according to the present invention can be used as a pharmaceutical composition for preventing or treating an effective ingredient.
  • This can be an effective therapeutic agent for treating central nervous system inflammatory diseases.
  • the central nervous system disease may be any one selected from the group consisting of spinal cord injury, stroke, cerebral infarction, cerebral ischemia, Alzheimer's disease, and multiple sclerosis, but is not particularly limited thereto.
  • compositions of the invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.
  • the pharmaceutical composition according to the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or other oral formulations, external preparation, suppository and sterilized injection solution, .
  • Suitable formulations known in the art are preferably those disclosed in Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton PA.
  • Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.
  • Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations are prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • examples of the non-aqueous solvent and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.
  • examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
  • administering as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.
  • the preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art.
  • the composition of the present invention can be administered at a daily dose of 0.001 to 1000 mg / kg.
  • the administration may be carried out once a day or divided into several times.
  • the dose is not intended to limit the scope of the invention in any way.
  • composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.
  • the present invention also provides a food composition for preventing or ameliorating a central nervous system disease comprising the fusion substance as an active ingredient.
  • the composition of the present invention When the composition of the present invention is used as a food additive, the composition can be added as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method.
  • the amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment).
  • the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage.
  • the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.
  • composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like.
  • composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
  • Another aspect of the present invention relates to a method for preventing or treating a central nervous system disease of an animal other than a human by administering the above pharmaceutical composition to an individual.
  • In vivo or intracellular infusion of the pharmaceutical composition may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intradermally, nasal, mucosal, By inhalation and oral routes.
  • ≪ RTI ID 0.0 > [0028] < / RTI >
  • the delivery method can be sufficiently extended not only for cultured cells but also for general in vivo delivery, that is, for delivery to animal cells, animal tissues and animals.
  • the pharmaceutical composition is non-immunogenic, non-infective, and is not limited in plasmid size because no DNA is packaged in vector organisms such as retroviruses or adenoviruses. Thus, it can be used for recombinant gene expression constructs of any practical size.
  • Jurkat (human leukemia cells) cells were purchased from the American Type Culture Collection (ATCC) and cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin antibiotics Respectively.
  • HeLa (human cervical cancer cell) cells were purchased from ATCC and cultured in Dulbecco's modified Eagle's media (DMEM) containing GlutaMAX supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin antibiotics. All cells were stored at 37 ° C in a 5% CO2 incubator. All of the above reagents were purchased from Thermo Scientific Hyclon.
  • mice Female C57BL / 6 mice, 6 to 8 weeks of age, were purchased from Orient Bio (Daejeon, Korea). The mice were kept and kept in a special aseptic facility in Hanyang University. The conditions were maintained at a constant temperature (21 ⁇ 1 °C), humidity (50 ⁇ 5%), and 12 hours / Lt; / RTI > All animal protocols used in the present invention were approved by the Animal Care and Use Committee of Hanyang University.
  • the Jurkat T cells at a concentration of 5.0 per well of RPMI 1640 in the medium ⁇ 10 5 cells were incubated in 24-well plates. After inoculating the cells, each protein was added at the indicated times. After incubation, cells were harvested and washed three times with phosphate-buffered saline (PBS). Intracellular fluorescence was analyzed on a fluorescence-activated cell sorting (FACS) Canto II flow cytometer (BD Bioscience) and the data were analyzed using FlowJo software (Tree Star, INC.). The spleen collected from 6-week-old C57BL / 6 mice was placed on a 60x15 mm cell culture dish containing 3 ml of PBS. The single cell suspension was physically prepared using a cell strainer with 0.45 ⁇ m pores, and 10 ml of fresh PBS was added followed by centrifugation.
  • FACS fluorescence-activated cell sorting
  • Red blood cells were lysed in ACK buffer (0.15 M NH 4 Cl, 10 mM KHCO 3 , 1 mM EDTA-2 Na, pH 7.2). 1.0 x 10 < 6 > spleen cells were inoculated per well, and then the delivery efficiency of proteins according to the present invention was analyzed.
  • Cells were incubated with anti-mouse CD4-PerCP-Cy5.5 and anti-mouse CD19-PE-Cy7 or anti-mouse F4 / 80 PerCP-Cy5.5, anti- mouse MHCII- And subdivided into various cell types by staining with anti-mouse CD11c-APC FACS antibodies. Antibodies were purchased from eBioscience.
  • Cell viability was measured using a water-soluble tetrazolium-8 based cell count kit-8 (CCK-8, Dojindo).
  • CCK-8 water-soluble tetrazolium-8 based cell count kit-8
  • a total of 5.0 x 10 3 HeLa cells were inoculated on 96-well plates and treated with ctCTLA-4 proteins or PBS for 10, 30, 50 or 100 ⁇ M, respectively, for 24 h. After incubation, cells were washed with PBS and further incubated with CCK-8 solution for 2 hours. Optical density values were then analyzed using a plate reader (Bio-Rad) at 450 nm.
  • CD4 + T cells and lymph nodes were isolated from the spleen using a CD4 + T cell negative selection kit (StemCell Technologies, INC). Separated CD4 + T cells in RPMI medium were inoculated onto anti-CD44 antibody-coated glass cover slips mounted in a Chamlide chamber. The protein solution was then added to the chamber and low-speed imaging was initiated. DIC and GFP images were recorded at 5 minute intervals for 2 hours. The obtained low - speed images were analyzed using MetaMorph or Image J software 1.48v.
  • Isolated splenocytes were cultured at various temperatures (4 ° C, 25 ° C, or 37 ° C) for 1 hour in the presence of respective ctCTLA-4 proteins.
  • Spleen cells or HeLa cells were incubated with heparin (0, 10, 20 or 50 ⁇ g / ml), methyl-beta-cyclodextrin (0, 3 or 5 mM), chlorpromazine (0, 10 or 30 ⁇ M) (0, 1, 2 or 5 mM) for 30 min at 37 ° C and then treated with each ctCTLA-4 protein and additionally incubated with each ctCTLA-4 protein at 37 ° C for 1 min Lt; / RTI > All cells were trypsinized (trypsin, Thermo Scientific Hyclone) and washed with FACS buffer (PBS containing 10% FBS, 5% sodium azide and 1% EDTA). Heparin, M? CD, chlorpromazine and amiloride were purchased from Sigma-
  • observation windows were introduced into the skull by performing surgical operations on male C57BL / 6 mice (23g-25g). Animals were anesthetized via isoflurane inhalation and maintained at body temperature (37 ° C - 38 ° C) using a warm blooded heating pad system controlled by rectal probes. The level of isoflurane was set at 3% to induce anesthesia and was maintained at 1.5% during cranial window surgery or multiphoton imaging. Animals were closely monitored throughout the process to confirm their physiological health. All surgical procedures were approved by Sungkyunkwan University's Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • sterile artificial cerebrospinal fluid 125 mM NaCl, 2.5 mM KCl , 25 mM NaHCO 3, 1.25 mM NaH 2 PO 4, 2 mM CaCl 2, 1 mM MgSO 4, 10 mM glucose, pH 7.4
  • sterile artificial cerebrospinal fluid 125 mM NaCl, 2.5 mM KCl , 25 mM NaHCO 3, 1.25 mM NaH 2 PO 4, 2 mM CaCl 2, 1 mM MgSO 4, 10 mM glucose, pH 7.4
  • the lenticular window was sealed using a cyanoacrylic adhesive and the animals were placed in a head-holding device (MAG-1, Narishige INC.) For multiphoton microscopy (TCS SP8 MP, Leica Microsystems CMS GmbH).
  • dNP2-dTomato The systemic delivery efficiency of dNP2-dTomato was analyzed by intraperitoneal injection of 5 mg of each ctCTLA-4 protein in 6-week-old C57BL / 6 mice. Mice were euthanized at 2 hours after injection. The tissues were then harvested, washed with PBS, and fixed with 4% paraformaldehyde. All collected tissues were obtained from O.C.T. Compound (WAKO Chemical) was used to freeze. The frozen blocks were cut into 6 ⁇ m thick flakes using a cryostat (Thermo Scientific) and examined through fluorescence microscopy (Leica Microsystems).
  • the frozen blocks were cut with a cryostat (Thermo Scientific, Logan, UT) into flakes 40 ⁇ m thick.
  • the flakes were incubated in cold acetone for 30 min at -20 < 0 > C and washed with PBS for 30 min at room temperature.
  • the washed samples were incubated with permeabilization buffer (0.5% Triton X-100 in PBS) for 10 minutes and further incubated with blocking buffer (3% BSA, 0.1% Tween-20) for 20 minutes.
  • mice Seven-week-old female C57BL / 6 mice were purchased from DBL. The protocol described in the present invention was approved by the Animal Experimental Ethics Committee of Hanyang University. EAE was induced by subcutaneous immunization with 100 ⁇ g of MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK) in Freund's adjuvant emulsion (adjuvant-incomplete Freund and 4 mg / ml Mycobacterium tuberculosis, BD Difco). The volume of total emulsion injected subcutaneously was 200 ⁇ l. At 0 hours and 48 hours after immunization, mice were treated with 200 ng pertussis toxin (List Biological Laboratories INC.) Intraperitoneally.
  • mice were euthanized and lymphocytes of the central nervous system were separated by Percoll (GE Healthcare) density-gradient centrifugation. The surface of the separated lymphocytes was stained with anti-mouse CD4 PerCP-Cy5.5 antibody (eBioscience). Lymphocytes were also stained with anti-mouse IFN-y-FITC and IL-17A-APC antibodies (eBioscience) using Fixation / Permeabilization Concentration and dilution kit (eBioscience).
  • mice 5 mg / kg of each ctCTLA-4 protein was injected repeatedly in three groups of C57BL / 6 mice at every other day for 14 days. The weight change of the mice was monitored daily. At the end of 15 days, the mice were sacrificed and the spleen, liver and brain morphology were carefully observed. Cytotoxicity of each protein to splenocytes and thymocytes was analyzed using Annexin V and 7-AAD staining kit (BD bioscience). The percentage of lymph node and spleen native CD4 T cells was stained with anti-mouse CD4-PerCP-Cy5.5, anti-mouse CD62L-FITC and anti-mouse CD44-PE FACS antibodies (eBioscience) And the lymphocytes were separated. The liver toxicity of the proteins was analyzed using an alanine aminotransferase (ALT) activity assay kit (BioVision) and an aspartate aminotransferase (AST) activity assay kit (BioVision).
  • ALT alanine amino
  • Peptides having the amino acid sequences of SEQ ID NOS: 1 to 7 were synthesized.
  • 'ctCTLA-4' a peptide fragment consisting of the amino acid sequence of SEQ ID NO: 2 (hereinafter also referred to as 'ctCTLA-4-fm1'), a peptide having the amino acid sequence of SEQ ID NO: (Hereinafter also referred to as 'ctCTLA-4-fm3') consisting of the amino acid sequence of SEQ ID NO: 4, a peptide fragment consisting of the amino acid sequence of SEQ ID NO: (Hereinafter also referred to as " Hph-1 ") consisting of the amino acid sequence of SEQ ID NO: 6, a cell permeable peptide consisting of the amino acid sequence of SEQ ID NO: 7 (hereinafter referred to as TAT ').
  • the sense and the antisense oligodeoxy nucleotide corresponding to the amino acid sequence were synthesized, respectively, and then allowed to stand at 95 ° C for 3 minutes to remove the secondary or tertiary structure formed (denaturation) at 50 ° C and Two strands of DNA were made by changing the temperature to 72 ° C. Restriction enzyme specific sequences other than sense and antisense oligodeoxynucleotides were inserted in 5 'and 3' to insert into the pRSET-b vector. Then, it was mass-amplified in E. coli. Afterwards, the integrity of the sequence was confirmed and expressed in E. coli. Each of the peptides expressed from each of the strains was purified.
  • Peptides having the amino acid sequences of SEQ ID NOS: 8 to 10 were synthesized.
  • the peptide variants of SEQ ID NOS: 8 to 10 are those obtained by replacing the Y amino acid residues of 1Y and 2Y shown in SEQ ID NO: 1 with F shown in FIG. 1A.
  • the peptide mutant having the amino acid sequence of SEQ ID NO: 8 is represented by "1YF" in which the Y amino acid residue in the portion denoted by 1Y is substituted with F and the peptide mutant having the amino acid sequence of SEQ ID NO: 9 is represented by 2Y
  • the peptide mutant having the amino acid sequence of SEQ ID NO: 10 is a peptide mutant in which the Y amino acid residues in the 1Y and 2Y regions are all substituted with F.
  • the peptide mutant having the amino acid sequence of SEQ ID NO: 10 is represented by 'DYF' Respectively.
  • the peptide variants were synthesized and purified in the same manner as in Preparation Example 1, except that the above amino acid sequence was used.
  • the cell permeable peptide represented by SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 at the N-terminus of the ctCTLA- was constructed by preparing a primer which allows the dNP2-ctCTLA-4, Hph-1-ctCTLA-4 or TAT-ctCTLA- After the protein was expressed and purified, an experiment was conducted to confirm the delivery efficiency in the cells. Specific experimental procedures are described below.
  • Each primer, sequence number and restriction enzyme recognition site are shown in Table 1.
  • the primers of SEQ ID NOS: 18 to 21 were used as a template for the pRSETb vector containing the gene coding for the peptide of SEQ ID NO: 1.
  • thermo-denaturation reaction of the template After an initial thermal denaturation reaction at 95 ° C for 3 minutes, a thermo-denaturation reaction of the template at 95 ° C for 20 seconds, a polymerization reaction for binding the primer and the template at 50 ° C for 20 seconds, and an extension reaction at 72 ° C for 30 seconds And 30 cycles were performed using a PCR reactor (Biorad).
  • dNP2-ctCTLA-4 among the forward primers, the sequence of dNP2 (KIKKVKKKGRKGSKIKKKKKKGRK) was very long, and PCR was carried out in duplicate.
  • the gene (DNA) fragment prepared in 1) of Production Example 3 was digested with restriction enzyme into pRSETb, which is a protein expression vector And inserted into the vector using a ligase.
  • the DNA fragment amplified in Production Example 3, 1) was subjected to enzymatic reaction using NheI and HindIII (NEB) so that the 5 '/ 3' end of the DNA became a sticky end.
  • NEB NheI and HindIII
  • pRSETb was enzymatically reacted using the same two restriction enzymes to construct a linear pRSETb vector having NheI and HindIII insertion sites. After each enzyme reaction, PCR was carried out using a purification kit (Kosomogin Tech).
  • the double-stranded DNA fragment of the dNP2-ctCTLA-4, Hph-1-ctCTLA-4 or TAT-ctCTLA-4 fused duplex and the pRSET-b vector were ligated with the T4 ligase (NEB) .
  • a circular pRSETb vector into which dNP2-ctCTLA-4, Hph-1-ctCTLA-4 or TAT-ctCTLA-4 was inserted was transformed into DH5a E. coli strain, and LB plate medium containing 50 ⁇ g / ml of antibiotic ampicillin To thereby select a transformed E. coli which forms a colony.
  • the selected E. coli colonies were inoculated again on a liquid LB medium containing 50 ⁇ g / ml of ampicillin, and then plasmid vectors were isolated using the plasmid Mini Preparation Kit (Cosmogin Tech).
  • the plasmid vector isolated by the above procedure is a pRSETb vector into which dNP2-ctCTLA-4, Hph-1-ctCTLA-4 or TAT-ctCTLA-4 was inserted, an enzyme reaction was first performed using NheI and HindIII restriction enzymes , And DNA sequencing (Bionics).
  • the E. coli-expressing protein contains a 6X-His tag coded in the pRSET-b vector in the front. Proteins were purified by the following method.
  • the cultures were collected by centrifugation and resuspended in native solution (0.5 M NaCl, 5 mM imidazole, 20 mM Tris-HCl, pH 8.0).
  • native solution 0.5 M NaCl, 5 mM imidazole, 20 mM Tris-HCl, pH 8.0.
  • the cell wall and membrane of E. coli were allowed to float in the dissolution solution for 10 minutes to break.
  • Cells were crushed and centrifuged using VCX-130 (Sonics & Materials), an ultrasound cell crusher, to separate the supernatant.
  • the separated supernatant was filtered once with 0.45 ⁇ m filter (Advantec) and allowed to bind with Ni-NTA agarose (Qiagen) for 1 hour at room temperature.
  • ctCTLA-4-fm3 comprising the amino acid sequence of SEQ ID NO: 4 prepared in Preparation Example 1 and a cell permeable peptide (dNP2) comprising the amino acid sequence of SEQ ID NO: quot; dNP2-ctCTLA-4-fm3 fusion ") was synthesized by commissioning Cosmosintech, Inc.
  • a primer that allows EGFP to be connected to the N-terminus of dNP2 was prepared, and the dNP2- And inserted into a vector (pRSET-b) to express and purify the protein in the E. coli strain.
  • the overall procedure was the same as that of Preparation Example 3 except for the primer.
  • the primers used were as follows.
  • the cell permeable peptide of SEQ ID NO: 5 prepared in Preparation Example 1 was fused with the fluorescence labeling compound TARMA to prepare a fusion product, which was synthesized by asking Cosmosintech Co.,
  • Mouse spleen cells were incubated with 1 ⁇ M ctCTLA-4 peptide, dNP2-ctCTLA-4 or Hph-1-ctCTLA-4 fused for 1 hour and ctCTLA-4 peptide or CPP conjugated ctCTLA- Were stained with anti-HA antibodies and the signals were amplified with PE-conjugated anti-rabbit IgG antibodies. The cells were collected and the intracellular fluorescence was measured using a flow cytometer to measure the efficiency of introduction of ctCTLA-4 protein in primary mouse CD4-T cells.
  • FIG. 3 is a graph showing the intracellular delivery efficiency of ctCTLA-4 peptide, dNP2-ctCTLA-4, and Hph-1-ctCTLA-4 fusions in primary mouse CD4-T cells.
  • the intracellular delivery efficiency is further improved and preferably the intracellular delivery efficiency of the dNP2-ctCTLA- The highest was confirmed.
  • the dNP2-ctCTLA-4 fusions show about 10 times higher intracellular delivery efficiency than the Hph-1-ctCTLA-4 fusion and ctCTLA-4 peptide.
  • dNP2-ctCTLA-4 fusions with the highest transfer efficiency among the fusions in which CPP and ctCTLA-4 were combined were selected through the above experiments.
  • dNP2-ctCTLA-4 fusions using dNP2 as a representative among conventional CPPs were prepared, Respectively.
  • anti-CD3 and anti-CD28 monoclonal antibodies were coated on a 96-well plate at a concentration of 0.1 ⁇ g / well at 37 ° C. for 5 hours, and then 7-week-old C57BL / 6, and the isolated splenocytes are suspended in a single cell.
  • 2.5 x 10 5 cells of the spleen cells suspended in the above procedure were dispensed into the wells coated with the anti-CD3 and anti-mouse CD28 monoclonal antibodies and washed with 1 ⁇ M PBS, dNP2-ctCTLA-4 fusion and TAT-ctCTLA-4 fusion, followed by activation for 24 hours.
  • FIG. 4 is a graph showing the results of measuring the inhibitory effect of IL-2 on 1 ⁇ M PBS, dNP2-ct CTLA-4 fusions and TAT-ct CTLA-4 fusions. Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • the ctCTLA-4 protein according to the present invention has a 40% to 70% reduction effect on IL-2 expression compared to PBS treatment by binding to a conventional cell permeable peptide (TAT, dNP2, Hph-1) .
  • TAT cell permeable peptide
  • the dNP2-ctCTLA-4 fusions showed the most excellent effect with a 70% reduction effect (specifically, about twice as much as the TAT-ctCTLA-4 fusions).
  • the dNP2-ctCTLA-4 fusion and dNP2-EGFP fusions were treated with splenocytes activated with anti-CD3 and anti-CD28 antibodies, respectively, and the effect of inhibiting IL-2 expression was measured by ELISA, Respectively.
  • the ELISA was performed using a kit commercially available from Biolegend, and its standard protocol was used.
  • splenocytes activated by PMA / inomycin were treated with dNP2-ctCTLA-4 fusion and dNP2-EGFP fusion, respectively, and the inhibitory effect of IL-2 on IL-2 expression was measured by ELISA.
  • the activated splenocytes were activated for 24 hours with anti-CD3 / CD28 antibody or PMA / ionomycin in the presence of 1 ⁇ M PBS, dNP2-ctCTLA-4 fusions or dNP2-EGFP fusions.
  • FIGS. 5A and 5B are graphs showing the results of measuring the inhibitory effect of IL-2 on dNP2-ctCTLA-4 fused dNP2-EGFP fusions. Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • the dNP2-ctCTLA-4 fusions of the present invention showed no effect on splenocytes activated by PMA and ionomycin stimulation, confirming that the target of dNP2-ctCTLA-4 fusions are proximal TcR signaling molecules . That is, the dNP2-ctCTLA-4 fusion according to the present invention has a specific target directivity.
  • the splenocytes activated by anti-CD3 and anti-CD28 antibodies were treated with dNP2-ctCTLA-4 and dNP2-EGFP fusions, respectively, and IFN-y and IL-17A The expression inhibition effect was measured and is shown in Fig.
  • FIG. 6A is a graph showing the results of measuring the inhibitory effect of dNP2-ctCTLA-4 fusion and dNP2-EGFP fusion on the expression of IFN-y.
  • FIG. 6B is a graph showing the inhibitory effect of dNP2-ctCTLA-4 fusion and dNP2- As shown in FIG. Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • the expression level of interferon-gamma (IFN-gamma) and interleukin-17A (IL-17A) in the activated splenocytes is remarkably decreased by the dNP2-ctCTLA-4 fusion.
  • the dNP2-ctCTLA-4 fusions according to the present invention had a value three times or more lower than that of dNP2-EGFP fusions.
  • FIG. 7 is a graph showing the experimental results of Experiment 3 (1), which graph shows the clinical scores for EAE animal models (in the y-axis) versus days after disease induction (in the x-axis) .
  • Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • mice treated with dNP2-ctCTLA-4 fusions were found to be lower than those of mice treated with dNP2-EGFP fusions or PBS.
  • mice treated with dNP2-EGFP fusions or PBS started clinical symptoms on the 9th day, and then became very suddenly depressed and confirmed that the hind limbs were paralyzed or the limbs were paralyzed. .
  • the mice treated with the dNP2-ctCTLA-4 fusions developed clinical symptoms from the 11th day, and then the symptoms were very gentle and the maximal tail was paralyzed.
  • Suggesting that the dNP2-ctCTLA-4 fusions in accordance with the present invention have excellent preventive effects against diseases of the central nervous system, particularly, multiple sclerosis.
  • mice were treated with PBS or 25 ⁇ g dNP2-ctCTLA-4 fusion and Hph-1-ctCTLA-4 fusion by intraperitoneal injection, respectively, 15). The mice were observed daily after EAE induction and the EAE clinical signs were scored and shown in Figure 8.
  • FIG. 8 is a graph showing the experimental results of Experiment 3 (2), which shows the clinical score for the EAE animal model (in the y-axis) on days after disease induction (in the x-axis) .
  • Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • the mice treated with the Hph-1-ct CTLA-4 fusions started clinical symptoms from the late 9th day compared to the mice treated with PBS alone, .
  • the Hph-1-ctCTLA-4 fusion also has excellent permeability to the blood-brain barrier or blood-spinal cord barrier, exhibits in vivo brain tissue segmentation characteristics, and substantially prevents central nervous system diseases, Or delayed effect.
  • mice treated with the dNP2-ctCTLA-4 fusions developed clinical symptoms from the 11th day, and then the symptoms were very gentle and the maximal tail was paralyzed.
  • the dNP2-ctCTLA-4 fusions were most effective for prevention of central nervous system diseases, especially multiple sclerosis.
  • FIG. 9 is a graph showing the experimental results from Experiment 3 (3), which shows the clinical scores for EAE animal models (in the y-axis) versus days after disease induction (in the x-axis) .
  • Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • mice treated with the dNP2-ctCTLA-4 fusions were significantly reduced compared to mice treated with dNP2-EGFP fusions or PBS.
  • mice immunized with MOG 35-55 peptide and treated with pertussis toxin were used as a standard EAE model, and EAE was induced in C57BL / 6 mice at 7 weeks of age as described above.
  • FIG. 10 is a graph showing experimental results from Experimental Example 3, 4). This graph shows the clinical scores for EAE animal models (in the y-axis) versus days after disease induction (in the x-axis) . Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • the data of the present invention suggests that the dNP2-ctCTLA-4 fusions can regulate the activated T cell response in in vitro and in vivo, and prevent central nervous system disorders such as multiple sclerosis Can be used as an immunomodulating protein to regulate the function of the immune system.
  • Figure 11 shows the effect of the dNP2-ctCTLA-4 fusions and dNP2-EGFP fusions of the invention on demyelination and immune cell infiltration in EAE-induced animal models against luxoph fast blue (LFB) Mattoxylin and eosin (H & E) staining.
  • the scale bar is 100 ⁇ .
  • FIG. 12 is a graph showing the number of infiltrated immune cells of spinal cord tissues counted by Image J software 1.48v according to each treatment group measured in FIG. Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • Figure 13 shows that IL-17A and / or IL-17A is produced by isolating spinal cord cells in an EAE animal model treated with Th1, Th17, Th2 and Treg cells (T cells) of dNP2-ctCTLA-4 fusions of the present invention and dNP2- IFN-y expressing CD4 T cells.
  • FIG. 14 is a graph showing the results of FIG. 13.
  • Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • IL-2 expression inhibition activity was measured by ELISA after 0.5, 1, 2 or 5 ⁇ M WT, 1YF, 2YF, and DYF were treated with spleen cells activated with anti-CD3 and anti- This is shown in Fig.
  • the activated splenocytes were activated with anti-CD3 / CD28 antibody for 24 hours in the presence of 1 ⁇ M PBS, 0.5, 1, 2 or 5 ⁇ M WT, 1YF, 2YF, DYF.
  • 15 is a graph showing the results of measuring the inhibitory effect of 0.5, 1, 2 or 5 ⁇ M WT, 1YF, 2YF, and DYF on IL-2 expression. Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • the ctCTLA-4 fragment peptide (SEQ ID NOS: 2 and 3) containing the amino acid sequence of the region containing the 1Y and 2Y amino acid residues described above can obtain an excellent IL-2 expression inhibitory effect as in the present invention.
  • CD25 is an activation marker that increases the expression level when T cells are activated.
  • CD4 T cell activation through the expression amount of CD25 was analyzed.
  • 0.1 ⁇ g of anti-CD3 and anti-CD28 antibody were coated on a 96-well plate at 37 ° C in a 0.5% carbon dioxide cell incubator for 5 hours. After splitting, splenocytes of the mice are dispensed at 2.5 ⁇ 10 5 per well. The cells were treated with PBS or 0.1, 0.5, 1, 2 or 5 ⁇ M of dNP2-TAMRA, dNP2-ctCTLA-4 or dNP2-ctCTLA-4-Fm for 24 hours at 37 ° C in a 0.5% do. The cells were then stained with anti-CD4 mAb fused with APC fluorescence and anti-CD25 mAb fused with PE fluorescence at 4 ° C for 20 minutes. Then, the cells prepared through the above procedure were analyzed by flow cytometry (FACS) to compare the expression levels of CD25.
  • FACS flow cytometry
  • FIG. 16 is a graph showing the introduction efficiency in primary mouse CD4-T cells (NA & PBS) treated with NA or PBS
  • FIG. 17 is a graph showing the effect of 0.1, 0.5, 1, 2 or 5 ⁇ M dNP2 -TAMRA, dNP2-ctCTLA-4 fusion, and dNP2-ctCTLA-4-fm3 fusion.
  • NA is a negative control that does not stimulate T cells to activate
  • PBS stimulates anti-CD3 and anti-CD28 monoclonal antibodies to activate T cells, which is a positive control .
  • NA is a red graph
  • PBS is a blue graph.
  • CD3 is a receptor for T cells
  • CD28 is a co-receptor, which stimulates T cell receptors using an nmonoclonal antibody targeting them.
  • the PBS graph treated with the T cell receptor shows a significant increase in the CD25 expression level, whereas the NA graph does not include any stimulus, so that the CD25 expression level remains unchanged have.
  • dNP2-ctCTLA-4 inhibits CD25 expression in proportion to the concentration when treated at each concentration, and dNP2-ctCTLA-4-fm3 shows a similar effect to that of dNP2-ctCTLA-4 Able to know.
  • dNP2-TAMRA can not inhibit CD25 expression because it is a non-active substance such as ctCTLA-4.
  • dNP2-ctCTLA-4-fm fusions showed a significant effect only when dNP2-CTLA-4 fusions were used at a concentration of 2 ⁇ M or more, whereas dNP2-ctCTLA-4-fm fusions showed 0.1 ⁇ M Was also excellent in the intracellular delivery efficiency.
  • Splenocytes activated by anti-CD3 and anti-CD28 antibodies were treated with 0.1, 0.5, 1, 2 or 5 ⁇ M dNP2-TAMRA fusions, dNP2-CTLA-4 fusions and dNP2-ctCTLA-4-fm3 fices, respectively After that, the inhibitory effect of IL-2 on ELISA was measured and shown in Fig.
  • the activated splenocytes were incubated with anti-CD3 / CD28 antibody in the presence of 1 ⁇ M PBS, 0.1, 0.5, 1, 2 or 5 ⁇ M dNP2-TAMRA fusions, dNP2-CTLA-4 fusions and dNP2-ctCTLA- For 24 hours.
  • 18 is a graph showing the results of measuring the inhibitory effect of IL-2 on 0.1, 0.5, 1, 2 or 5 ⁇ M dNP2-TAMRA fusions, dNP2-CTLA-4 fusions and dNP2-ctCTLA-4-fm3 fusions. Values represent mean ⁇ sem, * is p ⁇ 0.05; ** is p <0.01; *** represents p <0.001;Student's t- test.
  • the dNP2-ctCTLA-4-fm fusant using the fragment of ctCTLA-4 of the present invention has an excellent IL-2 expression inhibitory effect similar to that of the dNP2-ctCTLA-4 fusion protein of Example 2 Respectively.
  • the ctCTLA-4 peptide of Example 1 according to the present invention showed immunomodulatory functions in NOD mice transplanted with the gene regardless of B7 binding.
  • the present invention has revealed a B7-independent function of CTLA-4 (ctCTLA-4) encoding only a portion of the exon 4 of the CTLA-4 gene.
  • a fusion protein comprising a ctCTLA-4 protein and a cell permeable peptide according to the present invention was also found, which inhibited T cell activation and showed an efficacious therapeutic effect in an autoimmune encephalomyelitis model.
  • dNP2-ctCTLA-4 fusions could be delivered into macrophages and dendritic cells, no significant changes in the production of inflammatory cytokines by Toll-like receptor (TLR) ligand stimulation in such cells were observed, The production of IL-2 and IFN-y was successfully inhibited by activated CD4 and CD8 T cells.
  • TLR Toll-like receptor
  • the fusion according to the present invention is not mediated by the inhibition of innate immune cells but rather by inhibiting effector T cell functions.
  • the in vivo tests of the present invention improve clinical symptoms, even when treated with the dNP2-ctCTLA-4 fusion, even after treatment of the tail paralysis in animal models, Indicating that they have very strong preventive or therapeutic effects.
  • the present invention has developed a fusion of a novel potent ctCTLA-4 peptide and a ctCTLA-4 peptide with a cell permeable peptide, and clearly shows that the peptide or its fragment or fusion can be delivered to the brain and spinal cord .
  • New therapies based on the technology of transferring ctCTLA-4 peptide and ctCTLA-4 peptide and cell permeable peptide-conjugated fusions according to the present invention into CNS-infiltrating T cells may be effective in controlling multiple sclerosis, especially dNP2- The efficiency and stability of the ctCTLA-4 fusions were optimized.
  • the peptides prepared according to the present invention have a function of transmitting the brain-vascular barrier and the spinal-vascular barrier of the central nervous system which can not pass significantly, with excellent efficiency, so that an effective therapeutic effect can be obtained from a low content, It is expected to be applicable to various substances that can prevent or treat central nervous system diseases.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Toxicology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention a pour fonction de permettre la pénétration de la barrière hémato-encéphalique et la barrière sang-moelle épinière du système nerveux central, qui n'ont pas été significativement pénétrées, avec une excellente efficacité, ce qui permet d'obtenir un effet thérapeutique rapide, prompt et plus efficace par une administration à faible dose. En outre, la présente invention permet une administration locale à la différence des agents thérapeutiques classiques, ce qui permet de réduire les effets secondaires, et permet l'administration locale d'un agent thérapeutique à une concentration élevée, ce qui permet potentiellement de nouveaux traitements et de nouvelles prescriptions.
PCT/KR2016/009201 2015-08-21 2016-08-19 Peptide ayant pour effet de prévenir ou traiter des maladies du système nerveux central et composition pharmaceutique pour la prévention et le traitement de maladies du système nerveux central, contenant ce dernier en tant que principe actif WO2017034244A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP16839533.3A EP3336100B1 (fr) 2015-08-21 2016-08-19 Peptide ayant pour effet de prévenir ou traiter des maladies du système nerveux central et composition pharmaceutique pour la prévention et le traitement de maladies du système nerveux central, contenant ce dernier en tant que principe actif
CN201680056710.3A CN108137659B (zh) 2015-08-21 2016-08-19 具有预防或治疗中枢神经系统疾病作用的肽和包含其作为活性成分的药物组合物
US15/753,915 US11053295B2 (en) 2015-08-21 2016-08-19 Peptides having effects of preventing or treating central nervous system diseases and pharmaceutical compositions for preventing or treating central nervous system diseases containing same as active ingredient
US16/544,671 US11351224B2 (en) 2015-08-21 2019-08-19 Pharmaceutical composition for preventing and treating transplant rejection

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20150117966 2015-08-21
KR10-2015-0117966 2015-08-21
KR1020160105642A KR101969513B1 (ko) 2015-08-21 2016-08-19 중추신경계 질환에 대한 예방 또는 치료효과를 갖는 펩타이드 및 이를 유효성분으로 하는 중추신경계 질환 예방 및 치료용 약학적 조성물
KR10-2016-0105642 2016-08-19

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US15/753,915 A-371-Of-International US11053295B2 (en) 2015-08-21 2016-08-19 Peptides having effects of preventing or treating central nervous system diseases and pharmaceutical compositions for preventing or treating central nervous system diseases containing same as active ingredient
US16/544,671 Continuation-In-Part US11351224B2 (en) 2015-08-21 2019-08-19 Pharmaceutical composition for preventing and treating transplant rejection

Publications (1)

Publication Number Publication Date
WO2017034244A1 true WO2017034244A1 (fr) 2017-03-02

Family

ID=58100436

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2016/009201 WO2017034244A1 (fr) 2015-08-21 2016-08-19 Peptide ayant pour effet de prévenir ou traiter des maladies du système nerveux central et composition pharmaceutique pour la prévention et le traitement de maladies du système nerveux central, contenant ce dernier en tant que principe actif

Country Status (1)

Country Link
WO (1) WO2017034244A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110366422A (zh) * 2017-03-27 2019-10-22 汉阳大学校产学协力团 包含其中细胞穿透肽与ctctla4肽融合的融合蛋白作为活性成分的用于预防或治疗炎性呼吸系统疾病的药物组合物
US11053295B2 (en) 2015-08-21 2021-07-06 Iucf-Hyu (Industry-University Cooperation Foundation Hanyang University) Peptides having effects of preventing or treating central nervous system diseases and pharmaceutical compositions for preventing or treating central nervous system diseases containing same as active ingredient
US11351224B2 (en) 2015-08-21 2022-06-07 Iucf-Hyu (Industry-University Cooperation Foundation Hanyang University) Pharmaceutical composition for preventing and treating transplant rejection
US11566055B2 (en) * 2020-07-09 2023-01-31 Iucf-Hyu (Industry-University Cooperation Foundation Hanyang University) Composition for preventing or treating autoimmune disease based on LRR domain of NLRX1 protein

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20080084937A (ko) * 2005-11-04 2008-09-22 포휴먼텍(주) 융합 폴리펩타이드를 세포로 전달하는 방법
KR20100105551A (ko) * 2007-11-01 2010-09-29 퍼시이드 쎄라퓨틱스 엘엘씨 면역억제 폴리펩티드 및 핵산
US20100322893A1 (en) * 2006-10-20 2010-12-23 Medlmmune Limited Ctla-4 protein variants
KR20150014443A (ko) * 2012-05-11 2015-02-06 메디뮨 엘엘씨 Ｃｔｌａ－４ 변이체

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20080084937A (ko) * 2005-11-04 2008-09-22 포휴먼텍(주) 융합 폴리펩타이드를 세포로 전달하는 방법
US20100322893A1 (en) * 2006-10-20 2010-12-23 Medlmmune Limited Ctla-4 protein variants
KR20100105551A (ko) * 2007-11-01 2010-09-29 퍼시이드 쎄라퓨틱스 엘엘씨 면역억제 폴리펩티드 및 핵산
KR20150014443A (ko) * 2012-05-11 2015-02-06 메디뮨 엘엘씨 Ｃｔｌａ－４ 변이체

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE protein [O] 16 October 1999 (1999-10-16), "cytotoxic T-lymphocyte activated protein 4, partial [Homo sapiens]", XP055368587, retrieved from ncbi Database accession no. AAF02499.1 *
LIM ET AL.: "dNP2 is a Blood-brain Barrier-permeable Peptide Enabling ctCTLA-4 Protein Delivery to Ameliorate Experimental Autoimmune Encephalomyelitis", NATURE COMMUNICATIONS, vol. 6, no. 8244, 15 September 2015 (2015-09-15), pages 1 - 13, XP055368583 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11053295B2 (en) 2015-08-21 2021-07-06 Iucf-Hyu (Industry-University Cooperation Foundation Hanyang University) Peptides having effects of preventing or treating central nervous system diseases and pharmaceutical compositions for preventing or treating central nervous system diseases containing same as active ingredient
US11351224B2 (en) 2015-08-21 2022-06-07 Iucf-Hyu (Industry-University Cooperation Foundation Hanyang University) Pharmaceutical composition for preventing and treating transplant rejection
CN110366422A (zh) * 2017-03-27 2019-10-22 汉阳大学校产学协力团 包含其中细胞穿透肽与ctctla4肽融合的融合蛋白作为活性成分的用于预防或治疗炎性呼吸系统疾病的药物组合物
US11566055B2 (en) * 2020-07-09 2023-01-31 Iucf-Hyu (Industry-University Cooperation Foundation Hanyang University) Composition for preventing or treating autoimmune disease based on LRR domain of NLRX1 protein

Similar Documents

Publication Publication Date Title
WO2017034244A1 (fr) Peptide ayant pour effet de prévenir ou traiter des maladies du système nerveux central et composition pharmaceutique pour la prévention et le traitement de maladies du système nerveux central, contenant ce dernier en tant que principe actif
WO2017078440A1 (fr) Peptide présentant des effets de prévention et de régénérescence de la perte neuronale, et composition le contenant
WO2016200219A1 (fr) Protéine d'interleukine-7 modifiée et ses utilisations
WO2013169077A1 (fr) Composition pour la prévention ou le traitement de la cachexie
WO2015156649A1 (fr) Peptide présentant une activité inhibitrice contre la fibrose, et composition le contenant
KR101969513B1 (ko) 중추신경계 질환에 대한 예방 또는 치료효과를 갖는 펩타이드 및 이를 유효성분으로 하는 중추신경계 질환 예방 및 치료용 약학적 조성물
WO2018097540A9 (fr) Kit à ajout de milieu de culture de cellules immunitaires sans sérum, méthode de culture de cellules immunitaires utilisant ledit kit, culture de cellules immunitaires sans sérum obtenue au moyen dudit kit ou de ladite méthode de culture, et composition cosmétique comprenant ladite culture
WO2017023138A1 (fr) Récepteur d'antigènes chimère et lymphocytes t dans lesquels le récepteur d'antigènes chimère est exprimé
WO2019212324A1 (fr) Ciblage de macrophages associés à une tumeur de type m2 par l'utilisation d'un peptide pro-apoptotique à base de mélittine
WO2019182425A1 (fr) Lignée de cellules nk génétiquement modifiée ayant un nouveau gène codant pour le récepteur chimérique de l'antigène et son utilisation
WO2013129739A1 (fr) Composition pharmaceutique comprenant une phospholipase a2 (bv-pla2) de venin d'abeille pour traiter ou prévenir des maladies associées à la dégradation d'une activité anormale de lymphocytes t régulateurs
WO2011142514A1 (fr) Composition contenant du pias3 comme ingrédient actif pour la prévention ou le traitement d'un cancer ou d'une maladie immune
WO2021096275A1 (fr) Protéine de fusion comprenant l'interleukine -7 modifiée et le récepteur bêta ii du tgf et son utilisation
WO2011052883A2 (fr) Procédé d'activation d'une cellule tueuse naturelle par l'ajustement de l'expression du gène socs2
WO2020101361A1 (fr) Procédé de culture de cellules tueuses naturelles dérivées de sang de cordon ombilical au moyen de lymphocytes t transformés
WO2021107689A1 (fr) Composition pharmaceutique pour le traitement d'un cancer, comprenant un inhibiteur de point de contrôle immunitaire et une protéine de fusion constituée de la protéine il-2 et de la protéine cd80
WO2015167243A1 (fr) Nouveau composé ayant un effet thérapeutique sur les maladies immunitaires et utilisation de ce composé
WO2022025638A1 (fr) Lymphocyte t de récepteur antigénique chimérique (car) stabilisant la synapse immunologique
WO2020226438A1 (fr) Peptide pour la prévention ou le traitement de maladies intestinales inflammatoires
WO2011115458A2 (fr) Antagoniste double pour le tnf-α et l'il-21 dans la prévention et le traitement de maladies auto-immunes
WO2016027990A1 (fr) Composition pharmaceutique comprenant dusp5 en tant que substance active pour prévenir ou traiter des maladies métaboliques osseuses
WO2021107635A1 (fr) Composition de traitement anticancéreux, comprenant des cellules nk et une protéine de fusion qui comporte une protéine il-2 et une protéine cd80
WO2018203613A1 (fr) Peptide pour l'inhibition du récepteur de type toll (tlr) et composition pharmaceutique le comprenant
WO2021149971A1 (fr) Nouveau composé et son utilisation
WO2018182301A1 (fr) Composition pharmaceutique de prévention ou traitement de maladie respiratoire inflammatoire, à protéine de fusion de peptide perméable aux cellules et de peptide ctctla4, contenue en tant que principe actif dans cette composition

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16839533

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2016839533

Country of ref document: EP