JP7051687B2 - A novel fusion protein containing a transcriptional regulatory domain and a protein-carrying domain of P65 and their uses. - Google Patents
A novel fusion protein containing a transcriptional regulatory domain and a protein-carrying domain of P65 and their uses. Download PDFInfo
- Publication number
- JP7051687B2 JP7051687B2 JP2018536197A JP2018536197A JP7051687B2 JP 7051687 B2 JP7051687 B2 JP 7051687B2 JP 2018536197 A JP2018536197 A JP 2018536197A JP 2018536197 A JP2018536197 A JP 2018536197A JP 7051687 B2 JP7051687 B2 JP 7051687B2
- Authority
- JP
- Japan
- Prior art keywords
- fusion protein
- disease
- sequence
- domain
- protein
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
- 102000037865 fusion proteins Human genes 0.000 title claims description 56
- 108020001507 fusion proteins Proteins 0.000 title claims description 56
- 230000001105 regulatory effect Effects 0.000 title claims description 19
- 230000002103 transcriptional effect Effects 0.000 title claims description 19
- 108010057466 NF-kappa B Proteins 0.000 claims description 80
- 108090000623 proteins and genes Proteins 0.000 claims description 39
- 102000004169 proteins and genes Human genes 0.000 claims description 36
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 35
- 201000010099 disease Diseases 0.000 claims description 31
- 230000035897 transcription Effects 0.000 claims description 26
- 238000013518 transcription Methods 0.000 claims description 26
- 230000000694 effects Effects 0.000 claims description 20
- 239000000203 mixture Substances 0.000 claims description 19
- 208000013403 hyperactivity Diseases 0.000 claims description 13
- 239000008194 pharmaceutical composition Substances 0.000 claims description 13
- 102000003945 NF-kappa B Human genes 0.000 claims description 10
- 206010040070 Septic Shock Diseases 0.000 claims description 8
- 230000036303 septic shock Effects 0.000 claims description 8
- 230000006957 competitive inhibition Effects 0.000 claims description 7
- 206010003210 Arteriosclerosis Diseases 0.000 claims description 6
- 239000004480 active ingredient Substances 0.000 claims description 6
- 208000011775 arteriosclerosis disease Diseases 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 6
- 230000009885 systemic effect Effects 0.000 claims description 5
- 206010061218 Inflammation Diseases 0.000 claims description 4
- 208000035475 disorder Diseases 0.000 claims description 4
- 230000004054 inflammatory process Effects 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 208000011580 syndromic disease Diseases 0.000 claims description 4
- 208000023275 Autoimmune disease Diseases 0.000 claims description 3
- 230000002265 prevention Effects 0.000 claims description 3
- 230000002792 vascular Effects 0.000 claims description 3
- 208000024827 Alzheimer disease Diseases 0.000 claims description 2
- 208000035143 Bacterial infection Diseases 0.000 claims description 2
- 206010007572 Cardiac hypertrophy Diseases 0.000 claims description 2
- 208000006029 Cardiomegaly Diseases 0.000 claims description 2
- 208000015943 Coeliac disease Diseases 0.000 claims description 2
- 206010010741 Conjunctivitis Diseases 0.000 claims description 2
- 208000011231 Crohn disease Diseases 0.000 claims description 2
- 206010011732 Cyst Diseases 0.000 claims description 2
- 201000004624 Dermatitis Diseases 0.000 claims description 2
- 206010012438 Dermatitis atopic Diseases 0.000 claims description 2
- 206010012689 Diabetic retinopathy Diseases 0.000 claims description 2
- 206010015150 Erythema Diseases 0.000 claims description 2
- 201000005569 Gout Diseases 0.000 claims description 2
- 208000003250 Mixed connective tissue disease Diseases 0.000 claims description 2
- 206010028372 Muscular weakness Diseases 0.000 claims description 2
- 208000018737 Parkinson disease Diseases 0.000 claims description 2
- 206010036030 Polyarthritis Diseases 0.000 claims description 2
- 201000004681 Psoriasis Diseases 0.000 claims description 2
- 206010039085 Rhinitis allergic Diseases 0.000 claims description 2
- 208000006011 Stroke Diseases 0.000 claims description 2
- 206010043781 Thyroiditis chronic Diseases 0.000 claims description 2
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 claims description 2
- 208000024780 Urticaria Diseases 0.000 claims description 2
- 201000009961 allergic asthma Diseases 0.000 claims description 2
- 201000010105 allergic rhinitis Diseases 0.000 claims description 2
- 206010003246 arthritis Diseases 0.000 claims description 2
- 208000006673 asthma Diseases 0.000 claims description 2
- 201000008937 atopic dermatitis Diseases 0.000 claims description 2
- 208000022362 bacterial infectious disease Diseases 0.000 claims description 2
- 208000031513 cyst Diseases 0.000 claims description 2
- 231100000321 erythema Toxicity 0.000 claims description 2
- 206010016629 fibroma Diseases 0.000 claims description 2
- 201000011066 hemangioma Diseases 0.000 claims description 2
- 208000027866 inflammatory disease Diseases 0.000 claims description 2
- 201000003265 lymphadenitis Diseases 0.000 claims description 2
- 201000006417 multiple sclerosis Diseases 0.000 claims description 2
- 230000003387 muscular Effects 0.000 claims description 2
- 230000036473 myasthenia Effects 0.000 claims description 2
- 208000030428 polyarticular arthritis Diseases 0.000 claims description 2
- 208000005987 polymyositis Diseases 0.000 claims description 2
- 230000005855 radiation Effects 0.000 claims description 2
- 230000010410 reperfusion Effects 0.000 claims description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 claims description 2
- 230000003612 virological effect Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 206010009900 Colitis ulcerative Diseases 0.000 claims 1
- 208000031481 Pathologic Constriction Diseases 0.000 claims 1
- 201000006704 Ulcerative Colitis Diseases 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000036262 stenosis Effects 0.000 claims 1
- 208000037804 stenosis Diseases 0.000 claims 1
- 102100023050 Nuclear factor NF-kappa-B p105 subunit Human genes 0.000 description 72
- 208000028911 Temporomandibular Joint disease Diseases 0.000 description 39
- 208000008963 Transient myeloproliferative syndrome Diseases 0.000 description 39
- 235000018102 proteins Nutrition 0.000 description 34
- 210000004027 cell Anatomy 0.000 description 26
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 25
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 25
- 238000011160 research Methods 0.000 description 25
- 102100035100 Transcription factor p65 Human genes 0.000 description 20
- 108010002350 Interleukin-2 Proteins 0.000 description 15
- 239000002158 endotoxin Substances 0.000 description 15
- 102000000588 Interleukin-2 Human genes 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 12
- 102000004127 Cytokines Human genes 0.000 description 11
- 108090000695 Cytokines Proteins 0.000 description 11
- 210000001744 T-lymphocyte Anatomy 0.000 description 11
- 238000012790 confirmation Methods 0.000 description 9
- 230000002757 inflammatory effect Effects 0.000 description 9
- 230000028327 secretion Effects 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 8
- 210000004989 spleen cell Anatomy 0.000 description 8
- 102000013691 Interleukin-17 Human genes 0.000 description 7
- 108050003558 Interleukin-17 Proteins 0.000 description 7
- 108060001084 Luciferase Proteins 0.000 description 7
- 108091023040 Transcription factor Proteins 0.000 description 7
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 7
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 208000024891 symptom Diseases 0.000 description 7
- 230000001225 therapeutic effect Effects 0.000 description 7
- 230000032258 transport Effects 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 102100037850 Interferon gamma Human genes 0.000 description 6
- 108010074328 Interferon-gamma Proteins 0.000 description 6
- 102000003814 Interleukin-10 Human genes 0.000 description 6
- 108090000174 Interleukin-10 Proteins 0.000 description 6
- 102000004388 Interleukin-4 Human genes 0.000 description 6
- 108090000978 Interleukin-4 Proteins 0.000 description 6
- 239000005089 Luciferase Substances 0.000 description 6
- 102000040945 Transcription factor Human genes 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 102000004889 Interleukin-6 Human genes 0.000 description 5
- 108090001005 Interleukin-6 Proteins 0.000 description 5
- 102000008125 NF-kappa B p52 Subunit Human genes 0.000 description 5
- 108010074852 NF-kappa B p52 Subunit Proteins 0.000 description 5
- 230000005540 biological transmission Effects 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 230000002025 microglial effect Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 108010001859 Proto-Oncogene Proteins c-rel Proteins 0.000 description 4
- 102000000850 Proto-Oncogene Proteins c-rel Human genes 0.000 description 4
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 230000027455 binding Effects 0.000 description 4
- 230000002860 competitive effect Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 239000000539 dimer Substances 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 230000026731 phosphorylation Effects 0.000 description 4
- 238000006366 phosphorylation reaction Methods 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 3
- 230000004568 DNA-binding Effects 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 102000000589 Interleukin-1 Human genes 0.000 description 3
- 108010002352 Interleukin-1 Proteins 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 3
- 206010037211 Psychomotor hyperactivity Diseases 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 108010000134 Vascular Cell Adhesion Molecule-1 Proteins 0.000 description 3
- 102100023543 Vascular cell adhesion protein 1 Human genes 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000004891 communication Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 239000006188 syrup Substances 0.000 description 3
- 235000020357 syrup Nutrition 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 2
- 108010024212 E-Selectin Proteins 0.000 description 2
- 102100023471 E-selectin Human genes 0.000 description 2
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 2
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 2
- 206010021143 Hypoxia Diseases 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 102100038280 Prostaglandin G/H synthase 2 Human genes 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 230000006044 T cell activation Effects 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 238000010876 biochemical test Methods 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 230000003511 endothelial effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000007954 hypoxia Effects 0.000 description 2
- 239000005550 inflammation mediator Substances 0.000 description 2
- -1 macrophage-inflat Proteins 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 231100000956 nontoxicity Toxicity 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- 230000001718 repressive effect Effects 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- DHSSDEDRBUKTQY-UHFFFAOYSA-N 6-prop-2-enyl-4,5,7,8-tetrahydrothiazolo[4,5-d]azepin-2-amine Chemical compound C1CN(CC=C)CCC2=C1N=C(N)S2 DHSSDEDRBUKTQY-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 108700031308 Antennapedia Homeodomain Proteins 0.000 description 1
- 101150019028 Antp gene Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- OBMZMSLWNNWEJA-XNCRXQDQSA-N C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 Chemical compound C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 OBMZMSLWNNWEJA-XNCRXQDQSA-N 0.000 description 1
- 108700012434 CCL3 Proteins 0.000 description 1
- 101100189913 Caenorhabditis elegans pept-1 gene Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 108010072135 Cell Adhesion Molecule-1 Proteins 0.000 description 1
- 102100024649 Cell adhesion molecule 1 Human genes 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102000000013 Chemokine CCL3 Human genes 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 238000011537 Coomassie blue staining Methods 0.000 description 1
- 108010037462 Cyclooxygenase 2 Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100023688 Eotaxin Human genes 0.000 description 1
- 101710139422 Eotaxin Proteins 0.000 description 1
- 241001482630 Epinnula magistralis Species 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 108010021699 I-kappa B Proteins Proteins 0.000 description 1
- 102000008379 I-kappa B Proteins Human genes 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 description 1
- 102000015271 Intercellular Adhesion Molecule-1 Human genes 0.000 description 1
- 102000004890 Interleukin-8 Human genes 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000282560 Macaca mulatta Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 102100025748 Mothers against decapentaplegic homolog 3 Human genes 0.000 description 1
- 101710143111 Mothers against decapentaplegic homolog 3 Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 241000282520 Papio Species 0.000 description 1
- 101710176384 Peptide 1 Proteins 0.000 description 1
- 108050003267 Prostaglandin G/H synthase 2 Proteins 0.000 description 1
- 101710149951 Protein Tat Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 1
- 101710192266 Tegument protein VP22 Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 206010047115 Vasculitis Diseases 0.000 description 1
- 102000007624 ZAP-70 Protein-Tyrosine Kinase Human genes 0.000 description 1
- 108010046882 ZAP-70 Protein-Tyrosine Kinase Proteins 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 1
- 230000007059 acute toxicity Effects 0.000 description 1
- 231100000403 acute toxicity Toxicity 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 210000003484 anatomy Anatomy 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 235000010410 calcium alginate Nutrition 0.000 description 1
- 239000000648 calcium alginate Substances 0.000 description 1
- 229960002681 calcium alginate Drugs 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000004565 granule cell Anatomy 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 235000014304 histidine Nutrition 0.000 description 1
- 150000002411 histidines Chemical class 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 229940096397 interleukin-8 Drugs 0.000 description 1
- XKTZWUACRZHVAN-VADRZIEHSA-N interleukin-8 Chemical group C([C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](NC(C)=O)CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N1[C@H](CCC1)C(=O)N1[C@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC(O)=CC=1)C(=O)N[C@H](CO)C(=O)N1[C@H](CCC1)C(N)=O)C1=CC=CC=C1 XKTZWUACRZHVAN-VADRZIEHSA-N 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 201000010901 lateral sclerosis Diseases 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000023404 leukocyte cell-cell adhesion Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 231100000668 minimum lethal dose Toxicity 0.000 description 1
- 208000005264 motor neuron disease Diseases 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 230000030648 nucleus localization Effects 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 101150069487 p65 gene Proteins 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 208000037803 restenosis Diseases 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- VSIVTUIKYVGDCX-UHFFFAOYSA-M sodium;4-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].COC1=CC([N+]([O-])=O)=CC=C1[N+]1=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=NN1C1=CC=C([N+]([O-])=O)C=C1 VSIVTUIKYVGDCX-UHFFFAOYSA-M 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 229950008418 talipexole Drugs 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000035903 transrepression Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000001457 vasomotor Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Description
特許法第３０条第２項適用 平成２７年７月６日にウェブサイト（ＵＲＬ：ｈｔｔｐ：／／ｄｘ．ｄｏｉ．ｏｒｇ／１０．１０１６／ｊ．ｂｂｒｃ．２０１５．０７．００８）にて学術論文（Ｂｉｏｃｈｅｍｉｃａｌ ａｎｄ Ｂｉｏｐｈｙｓｉｃａｌ Ｒｅｓｅａｒｃｈ Ｃｏｍｍｕｎｉｃａｔｉｏｎｓ 第４６４巻、２０１５年、第３号、第７１１～７１７頁、エルゼビア社出版）を公開 平成２７年８月１日発行 ヨンセイ大学大学院 ディビジョン オブ ライフ サイエンス アンド バイオテクノロジー 博士論文 “Ｔｈｅｒａｐｅｕｔｉｃ ｅｆｆｅｃｔ ｏｆ ｔｒａｎｓｄｕｃｉｂｌｅ Ｓｍａｄ３ ａｎｄ ｐ６５ ｔｒａｎｓｃｒｉｐｔｉｏｎ ｍｏｄｕｌａｔｉｏｎ ｄｏｍａｉｎ ｉｎ ｉｎｆｌａｍｍａｔｏｒｙ ｄｉｓｅａｓｅｓ” パク、ソン トン著Application of
本発明は、大韓民国未来創造科学部支援下の課題番号ＮＲＦ－２０１４Ｒ１Ａ２Ａ１Ａ１００５２４６６、２００９－００８３５２２（ＥＲＣ）、１００４４４９４、および延世大学支援下の２０１５－２２－００６５によって行われたもので、前記課題番号ＮＲＦ－２０１４Ｒ１Ａ２Ａ１Ａ１００５２４６６の研究管理専門機関は「韓国研究財団」、研究事業名は「中堅研究者支援事業」、研究課題名は「免疫活性化制御細胞のＴｒｅｇ合わせ型免疫疾患新素材開発」、主管機関は「延世大学産学協力団」、研究期間は２０１４年１１月１日～２０１７年１０月３１日であり、前記課題番号２００９－００８３５２２（ＥＲＣ）の研究管理専門機関は「韓国研究財団」、研究事業名は「韓国研究財団－理工学分野（ＳＲＣ／ＥＲＣ）」、研究課題名は「ＥＲＣ／蛋白質機能制御履行研究センター（３／３、２段階）」、主管機関は「延世大学産学協力団」、研究期間は２００９年９月１日～２０１６年２月２９日であり、前記課題番号１００４４４９４の研究管理専門機関は「情報通信技術研究振興センター」、研究事業名は「ＳＷコンピューティング産業源泉技術開発事業」、研究課題名は「ＷｉｓｅＫＢ：ビッグデータ理解基盤自己学習型知識ベースおよび推論技術開発」、主管機関は「（株）ソルトルックス」、研究期間は２０１３年７月１日～２０１６年２月２８日であり、前記課題番号２０１５－２２－００６５の研究管理専門機関は「延世大学」、研究事業名は「校内研究支援」、研究課題名は「［未来先導－国際］自己免疫疾患の病因性Ｔ細胞ｓｕｂｓｅｔ遺伝子発現を制御できる転写因子機能制御用の個人合わせ型蛋白質新薬開発」、主管機関は「延世大学産学協力団」、研究期間は２０１５年９月１日～２０１６年８月３１日である。 The present invention was made by Task No. NRF-2014R1A2A1A100522666, 2009-0083522 (ERC), 10041494 with the support of the Faculty of Future Creation Science of the Republic of Korea, and 2015-22-0065 with the support of Ensei University. The research management specialized institution of 2014R1A2A1A10052466 is "Korean Research Foundation", the research project name is "Mid-level Researcher Support Project", the research project name is "Treg-combined immune disease new material development of immune activation control cells", and the main institution is " Ensei University Industry-Academia Cooperation Group ”, the research period is from November 1, 2014 to October 31, 2017, and the research management specialized institution of the above-mentioned project number 2009-0083522 (ERC) is“ Korea Research Foundation ”, research project name. Is "Korean Research Foundation-Science and Engineering Field (SRC / ERC)", the research project name is "ERC / Protein Function Control Implementation Research Center (3/3, 2nd stage)", and the governing body is "Ensei University Industry-Academia Cooperation Group". The research period is from September 1, 2009 to February 29, 2016. The research management specialist organization of the above-mentioned subject number 10041494 is "Information and Communication Technology Research Promotion Center", and the research project name is "SW Computing Industry Source Technology Development". "Business", the research project name is "WiseKB: Big Data Understanding Infrastructure Self-learning Knowledge Base and Inference Technology Development", the main institution is "Soltorx Co., Ltd.", the research period is from July 1, 2013 to February 2016. On the 28th, the research management specialized institution of the above-mentioned subject number 2015-22-0065 is "Ensei University", the research project name is "School Research Support", and the research subject name is "[Future Leading-International] Pathogenesis of Autoimmune Diseases". Development of a new personalized protein for controlling transcription factor function that can control the expression of sex T cell subset gene ”, the governing body is“ Ensei University Industry-Academia Cooperation Group ”, the research period is from September 1, 2015 to August 31, 2016. Is.
本発明は、転写因子ＮＦ－κＢのサブユニットであるｐ６５の転写調節ドメイン（ｔｒａｎｓｃｒｉｐｔｉｏｎ ｍｏｄｕｌａｔｉｏｎ ｄｏｍａｉｎ、ＴＭＤ）と蛋白質運搬ドメイン（ｐｒｏｔｅｉｎ ｔｒａｎｓｄｕｃｔｉｏｎ ｄｏｍａｉｎ、ＰＴＤ）とを含む新規融合蛋白質およびその用途に関する。 The present invention relates to a novel fusion protein comprising a transcription regulation domain (TMD) and a protein transport domain (PTD) of p65, which is a subunit of the transcription factor NF-κB, and its use.
転写因子ＮＦ－κＢによって調節されるサイトカイン（ｃｙｔｏｋｉｎｅ）は、腫瘍壊死因子－α（ｔｕｍｏｒ ｎｅｃｒｏｓｉｓ ｆａｃｔｏｒ－α、ＴＮＦ－α）、インターロイキン－１（ｉｎｔｅｒｌｅｕｋｉｎ－１、ＩＬ－１）、インターロイキン－６および顆粒球大食細胞コロニー刺激因子（ｇｒａｎｕｌｏｃｙｔｅ－ｍａｃｒｏｐｈａｇｅ ｃｏｌｏｎｙ ｓｔｉｍｕｌａｔｉｎｇ ｆａｃｔｏｒ、ＧＭ－ＣＳＦ）などがあり、ケモカイン（ｃｈｅｍｏｋｉｎｅ）は、インターロイキン－８、大食細胞炎症蛋白質－１α（ｍａｃｒｏｐｈａｇｅ－ｉｎｆｌａｍｍａｔｏｒｙ ｐｒｏｔｅｉｎ－１α、ＭＩＰ－１α）、走化性調節蛋白質－１（ｍｅｔｈｙｌ ａｃｃｅｐｔｉｎｇ ｃｈｅｍｏｔａｘｉｓ ｐｒｏｔｅｉｎ－１、ＭＣＰ－１）およびエオタキシン（ｅｏｔａｘｉｎ）などがある。また、ＮＦ－κＢによって調節される付着分子（ａｄｈｅｓｉｏｎ ｍｏｌｅｃｕｌｅ）は、Ｅ－セレクチン（Ｅ－ｓｅｌｅｃｔｉｎ）、血管細胞付着分子－１（ｖａｓｃｕｌａｒ ｃｅｌｌ ａｄｈｅｓｉｏｎ ｍｏｌｅｃｕｌｅ－１、ＶＣＡＭ－１）、内皮白血球付着分子－１（ｅｎｄｏｔｈｅｌｉａｌ ｌｅｕｋｏｃｙｔｅ ａｄｈｅｓｉｏｎ ｍｏｌｅｃｕｌｅ－１、ＥＬＡＭ－１）および細胞内付着分子－１（ｉｎｔｅｒｃｅｌｌｕｌａｒ ｃｅｌｌ ａｄｈｅｓｉｏｎ ｍｏｌｅｃｕｌｅ－１、ＩＣＡＭ－１）があり、誘導酵素（ｉｎｄｕｃｉｂｌｅ ｅｎｚｙｍｅ）は、シクロオキシゲナーゼ－２（ｃｙｃｌｏｏｘｙｇｅｎａｓｅ－２、ＣＯＸ－２）などがあり、ＮＦ－κＢは、体内のほぼすべての生理反応に関与している。 Cytokines regulated by the transcription factor NF-κB are tumor necrosis factor-α (tumor inflammation factor-α, TNF-α), interleukin-1 (interleukin-1, IL-1), interleukin-6. And granulocyte-macrophage colony stimulating factor (GM-CSF), etc. Chemokine is interleukin-8, macrophage-inflat , MIP-1α), methyl-accepting cytokine-1 (MCP-1) and eotaxin. The adhesion molecules regulated by NF-κB are E-selectin, vascular cell adhesion molecule-1 (vascular cell adhesion molecule-1, VCAM-1), and endothelial leukocyte adhesion molecule-. There are 1 (endothelial endothelium molecule-1, ELAM-1) and intracellular adhesion molecule-1 (intercellular cell adhesion molecule-1, ICAM-1), and the inducible enzyme (inducible enzyme-2) is cyclooxygenase-2. , COX-2), etc., and NF-κB is involved in almost all physiological reactions in the body.
転写因子ＮＦ－κＢは、同種またはヘテロ二量体で構成された、互いに異なるサブユニットから構成されている。ＮＦ－κＢ蛋白質は、ＲｅｌＡ（ｐ６５）、ｃ－Ｒｅｌ、Ｒｅｌ－Ｂ、ＮＦ－κＢ１（ｐ５０）およびＮＦ－κＢ２（ｐ５２）が含まれ、前記ｐ５０およびｐ５２は、それらの前駆体であるＮＦ－κＢ１（ｐ１０５）およびＮＦ－κＢ２（ｐ１００）からそれぞれ生成される。ＮＦ－κＢ蛋白質は、すべてＮ末端のＲＨＤ（Ｒｅｌ－ｈｏｍｏｌｏｇｙ ｄｏｍａｉｎ）と呼ばれる３００個のアミノ酸を共通して有しているが、これらは、二量体形成、特定ＤＮＡとの結合およびＩκＢ蛋白質との反応に関与する。また、核内に移動して転写因子として作用するための核位置信号（ｎｕｃｌｅａｒ ｌｏｃａｌｉｚａｔｉｏｎ ｓｉｇｎａｌ、ＮＬＳ）も含んでいる。そして、ＮＦ－κＢの構成蛋白質は、Ｃ末端のトランスアクチベーションドメイン（ｔｒａｎｓａｃｔｉｖａｔｉｏｎ ｄｏｍａｉｎ、ＴＡＤ）の存在の有無によってｃｌａｓｓＩ（ＮＦ－κＢ１、ＮＦ－κＢ２）とｃｌａｓｓＩＩ（ＲｅｌＡ／ｐ６５、ＲｅｌＢ、ｃ－Ｒｅｌ）に区分することができる。ｃｌａｓｓＩＩは、他のＮＦ－κＢ構成蛋白質なくても転写因子として作用できるトランスアクチベーションドメインを有しており、ｃｌａｓｓＩにはトランスアクチベーションドメインがない。この２種類のＮＦ－κＢの間で二量体が形成されてＤＮＡ転写因子として作用し、二量体の中でｐ５０／ｐ６５の形態が最もありふれて存在する。ＲｅｌＡ（ｐ６５）、ＲｅｌＢおよびｃ－Ｒｅｌは、Ｃ末端にトランスアクチベーションドメインを有していて、標的遺伝子の発現を活性化することができる。逆に、ＮＦ－κＢ１とＮＦ－κＢ２であるｐ５０とｐ５２は、Ｃ末端にトランスアクチベーションドメインを有しておらず、ｐ５０とｐ５２の同種二量体は、トランスアクチベーションドメインを有している補助因子（ｃｏ－ａｃｔｉｖａｔｏｒ）のような蛋白質の結合なしには転写をすることができない。 The transcription factor NF-κB is composed of different subunits composed of homologous or heterodimers. NF-κB proteins include RelA (p65), c-Rel, Rel-B, NF-κB1 (p50) and NF-κB2 (p52), the p50 and p52 being their precursors, NF-. It is produced from κB1 (p105) and NF-κB2 (p100), respectively. All NF-κB proteins share 300 amino acids called N-terminal RHD (Rel-homology domin), which are dimer-forming, binding to specific DNA, and IκB proteins. Involved in the reaction of. It also contains a nuclear localization signal (NLS) to move into the nucleus and act as a transcription factor. The constituent proteins of NF-κB are classI (NF-κB1, NF-κB2) and classII (RelA / p65, RelB, c-Rel) depending on the presence or absence of the C-terminal transactivation domain (TAD). It can be divided into. ClassII has a transactivation domain that can act as a transcription factor without other NF-κB constituent proteins, and classI does not have a transactivation domain. A dimer is formed between these two types of NF-κB and acts as a DNA transcription factor, and the p50 / p65 form is the most common dimer among the dimers. RelA (p65), RelB and c-Rel have a transactivation domain at the C-terminus and can activate the expression of the target gene. Conversely, p50 and p52, which are NF-κB1 and NF-κB2, do not have a transactivation domain at the C-terminus, and homologous dimers of p50 and p52 are cofactors that have a transactivation domain. Transcription is not possible without binding of proteins such as (co-activator).
蛋白質運搬ドメイン（Ｐｒｏｔｅｉｎ Ｔｒａｎｓｄｕｃｔｉｏｎ Ｄｏｍａｉｎ、ＰＴＤ）は、疎水性の強い短いペプチドで、共に融合した蛋白質やＤＮＡおよびＲＮＡなどの生理活性分子を細胞内に効果的に伝達することが知られている。本発明者らは、現在まで２種のＰＴＤを開発し、前記ＰＴＤは、ＷＯ２００３０５９９４０およびＷＯ２００３０５９９４１に詳しく開示されている。蛋白質運搬ドメインは、細胞質のみならず、核内にも生理活性分子を運搬できるため、本発明の核心物質である変形した転写因子を核内に伝達するのに適した特性を有している。 The protein transport domain (PTD) is a short peptide with strong hydrophobicity, and it is known that it effectively transfers bioactive molecules such as proteins and DNA and RNA fused together into cells. The present inventors have developed two types of PTDs to date, and the PTDs are disclosed in detail in WO2003059940 and WO2003059941. Since the protein transport domain can transport bioactive molecules not only in the cytoplasm but also in the nucleus, it has a property suitable for transmitting a modified transcription factor, which is the core substance of the present invention, into the nucleus.
したがって、本発明者らは、ＮＦ－κＢのサブユニットであるｐ６５の転写調節ドメインと蛋白質運搬ドメインとを含む融合蛋白質を用いて、競合阻害によってＮＦ－κＢの転写および活性を抑制することにより、ＮＦ－κＢの過度の活性によって引き起こされる疾患を効果的に治療しようとした。 Therefore, we use a fusion protein containing a transcriptional regulatory domain and a protein-carrying domain of p65, which is a subunit of NF-κB, by inhibiting the transcription and activity of NF-κB by competitive inhibition. Attempts have been made to effectively treat diseases caused by excessive activity of NF-κB.
本明細書全体にわたって多数の論文および特許文献が参照され、その引用が表示されている。引用された論文および特許文献の開示内容はその全体として本明細書に参照として組み込まれ、本発明の属する技術分野における水準および本発明の内容がより明確に説明される。 Numerous articles and patent documents have been referenced and cited throughout the specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the standards in the art to which the invention belongs and the content of the invention.
本発明者らは、ＮＦ－κＢの過度の活性によって引き起こされる疾患を効果的に予防、改善または治療できる物質を開発すべく、鋭意研究努力した。その結果、ＮＦ－κＢのサブユニットであるｐ６５の転写調節ドメインと蛋白質運搬ドメインとを含む融合蛋白質を用いる場合、競合阻害によってＮＦ－κＢの転写および活性を抑制できることを確認することによって、本発明を完成した。 The present inventors have made diligent research efforts to develop a substance capable of effectively preventing, ameliorating or treating a disease caused by excessive activity of NF-κB. As a result, the present invention confirms that when a fusion protein containing the transcriptional regulatory domain of p65, which is a subunit of NF-κB, and the protein transport domain is used, the transcription and activity of NF-κB can be suppressed by competitive inhibition. Was completed.
したがって、本発明の目的は、ＮＦ－κＢのサブユニットであるｐ６５の転写調節ドメインと蛋白質運搬ドメインとを含む融合蛋白質を提供することである。 Therefore, an object of the present invention is to provide a fusion protein containing a transcriptional regulatory domain and a protein carrying domain of p65, which is a subunit of NF-κB.
本発明の他の目的は、本発明の融合蛋白質を含むＮＦ－κＢの転写または活性抑制剤を提供することである。 Another object of the present invention is to provide a transcription or activity inhibitor of NF-κB containing the fusion protein of the present invention.
本発明のさらに他の目的は、本発明の融合蛋白質を有効成分として含むＮＦ－κＢの過活性－関連疾患の予防または治療用薬剤学的組成物を提供することである。 Yet another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of hyperactivity-related diseases of NF-κB containing the fusion protein of the present invention as an active ingredient.
本発明のさらに他の目的は、ＮＦ－κＢの転写または活性抑制方法およびＮＦ－κＢの過活性－関連疾患の予防、改善または治療方法を提供することである。 Yet another object of the present invention is to provide a method for suppressing transcription or activity of NF-κB and a method for preventing, ameliorating or treating a hyperactivity-related disease of NF-κB.
本発明の他の目的および利点は、下記の発明の詳細な説明、請求の範囲および図面によってより明確になる。 Other objects and advantages of the invention will be further clarified by the detailed description, claims and drawings of the invention below.
本発明の一態様によれば、本発明は、ＮＦ－κＢのサブユニットであるｐ６５の転写調節ドメインと蛋白質運搬ドメインとを含む融合蛋白質を提供する。本発明の融合蛋白質は、競合阻害によってＮＦ－κＢの転写を抑制する。 According to one aspect of the invention, the invention provides a fusion protein comprising a transcriptional regulatory domain and a protein carrying domain of p65, which is a subunit of NF-κB. The fusion protein of the present invention suppresses transcription of NF-κB by competitive inhibition.
本発明者らは、ＮＦ－κＢの過度の活性によって引き起こされる疾患を効果的に予防、改善または治療できる物質を開発すべく、鋭意研究努力した。その結果、ＮＦ－κＢのサブユニットであるｐ６５の転写調節ドメインと蛋白質運搬ドメインとを含む融合蛋白質を用いる場合、競合阻害によってＮＦ－κＢの転写および活性を抑制できることを確認した。 The present inventors have made diligent research efforts to develop a substance capable of effectively preventing, ameliorating or treating a disease caused by excessive activity of NF-κB. As a result, it was confirmed that when a fusion protein containing the transcriptional regulatory domain of p65, which is a subunit of NF-κB, and the protein transport domain is used, the transcription and activity of NF-κB can be suppressed by competitive inhibition.
本明細書で使われる用語「転写調節ドメイン」は、転写因子を構成するドメインで、トランスアクチベーションドメインなしにＤＮＡ結合部位だけで構成されたドメインを意味する。下記の実施例で立証したように、本発明の融合蛋白質は、トランスアクチベーションドメインはないが、ＤＮＡ結合部位を有しているため、目的のプロモーターに結合することはできるが、転写を促進することができない。したがって、本発明の融合蛋白質は、ＮＦ－κＢ遺伝子に対する優性ネガティブ変異体（ｄｏｍｉｎａｎｔ ｎｅｇａｔｉｖｅ ｍｕｔａｎｔ）であるため、細胞内の野生型ＮＦ－κＢに対する競合阻害剤として作用して、ＮＦ－κＢの転写および活性を抑制することができる。 As used herein, the term "transcriptional regulatory domain" is a domain that constitutes a transcription factor and means a domain that is composed solely of DNA binding sites without a transactivation domain. As demonstrated in the examples below, the fusion protein of the invention does not have a transactivation domain but has a DNA binding site, so that it can bind to the promoter of interest but promote transcription. I can't. Therefore, since the fusion protein of the present invention is a dominant negative mutant for the NF-κB gene, it acts as a competitive inhibitor against the intracellular wild-type NF-κB, and is responsible for transcription of NF-κB and transcription of NF-κB. The activity can be suppressed.
本発明の一実施形態によれば、本発明のＮＦ－κＢは、ＲｅｌＡ（ｐ６５）、ｃ－Ｒｅｌ、Ｒｅｌ－Ｂ、ＮＦ－κＢ１（ｐ５０）およびＮＦ－κＢ２（ｐ５２）から構成された群より選択されたＮＦ－κＢである。本発明の特定の実施形態によれば、本発明のＮＦ－κＢは、ＲｅｌＡ（ｐ６５）である。 According to one embodiment of the present invention, NF-κB of the present invention is composed of a group composed of RelA (p65), c-Rel, Rel-B, NF-κB1 (p50) and NF-κB2 (p52). The selected NF-κB. According to a particular embodiment of the invention, the NF-κB of the invention is RelA (p65).
本発明の一実施形態によれば、本発明のＮＦ－κＢの転写調節ドメインは、配列リスト第１配列のアミノ酸配列からなる。本発明の他の実施形態によれば、本発明の配列リスト第１配列のアミノ酸配列からなるＮＦ－κＢの転写調節ドメインは、配列リスト第３配列のヌクレオチド配列によってコードされる。 According to one embodiment of the present invention, the transcriptional regulatory domain of NF-κB of the present invention consists of the amino acid sequence of the first sequence of the sequence list. According to another embodiment of the present invention, the transcriptional regulatory domain of NF-κB consisting of the amino acid sequence of the first sequence of the sequence list of the present invention is encoded by the nucleotide sequence of the third sequence of the sequence list.
本明細書で使われる用語「蛋白質運搬ドメイン」は、７－５０個のアミノ酸からなる、疎水性の強い短いペプチドで、１２０ｋＤａ以上の分子量を、蛋白質のみならず、ＤＮＡまたはＲＮＡを細胞内に伝達できるドメインを意味する。下記の実施例で立証したように、本発明の蛋白質運搬ドメインが付着しない蛋白質（つまり、転写調節ドメインだけで構成されたｐ６５－ＴＭＤ）の場合、本発明の融合蛋白質とは異なり、ＮＦ－κＢおよびＩＬ－２の転写抑制、ＬＰＳによる炎症性サイトカインの分泌抑制、脾臓細胞におけるＩＬ－２、ＩＦＮ－γ、ＩＬ－４、ＩＬ－１７ＡおよびＩＬ－１０の生成抑制効果がないことを確認することができた。 As used herein, the term "protein-carrying domain" is a short, highly hydrophobic peptide consisting of 7-50 amino acids that transfers a molecular weight of 120 kDa or more, not only protein, but also DNA or RNA into the cell. Means a domain that can be. As demonstrated in the examples below, in the case of a protein to which the protein-carrying domain of the present invention does not adhere (that is, p65-TMD composed only of the transrepression regulatory domain), unlike the fusion protein of the present invention, NF-κB And to confirm that there is no effect of suppressing the transcription of IL-2, suppressing the secretion of inflammatory cytokines by LPS, and suppressing the production of IL-2, IFN-γ, IL-4, IL-17A and IL-10 in spleen cells. Was done.
本発明の一実施形態によれば、本発明の蛋白質運搬ドメインは、Ｈｐｈ－１、Ｓｉｍ－２、Ｔａｔ、ＶＰ２２、Ａｎｔｐ（ａｎｔｅｎｎａｐｅｄｉａ）、Ｐｅｐ－１（ｐｅｐｔｉｄｅ－１）、ＰＴＤ－５（ｐｒｏｔｅｉｎ ｔｒａｎｓｄｕｃｔｉｏｎ ｄｏｍａｉｎ－５）、７Ｒ、９Ｒ、１１ＲおよびＣＴＰ（ｃｙｔｏｐｌａｍｉｃ ｔｒａｎｓｄｕｃｔｉｏｎ ｐｅｐｔｉｄｅ）から構成された群より選択される。本明細書の用語「７Ｒ」、「９Ｒ」および「１１Ｒ」は、アルギニン（ａｒｇｉｎｉｎｅ）がそれぞれ７個、９個および１１個から構成されたペプチドを意味する。本発明の特定の実施形態によれば、本発明の蛋白質運搬ドメインは、Ｈｐｈ－１である。 According to one embodiment of the invention, the protein transport domains of the invention are Hph-1, Sim-2, Tat, VP22, Antp (antennapedia), Pept-1 (peptide-1), PTD-5 (protein transduction). It is selected from the group composed of domain-5), 7R, 9R, 11R and CTP (cytoplastic transduction peptide). As used herein, the terms "7R", "9R" and "11R" mean peptides composed of 7, 9 and 11 arginine, respectively. According to a particular embodiment of the invention, the protein carrier domain of the invention is Hph-1.
本発明の一実施形態によれば、本発明の蛋白質運搬ドメインは、配列リスト第２配列のアミノ酸配列からなる。本発明の他の実施形態によれば、本発明の配列リスト第２配列のアミノ酸配列からなる蛋白質運搬ドメインは、配列リスト第４配列のヌクレオチド配列によってコードされる。 According to one embodiment of the present invention, the protein carrying domain of the present invention consists of the amino acid sequence of the second sequence of the sequence list. According to another embodiment of the present invention, the protein carrying domain consisting of the amino acid sequence of the second sequence of the sequence list of the present invention is encoded by the nucleotide sequence of the fourth sequence of the sequence list.
本発明の一実施形態によれば、本発明の融合蛋白質は、配列リスト第５配列のアミノ酸配列を含む。本発明の特定の実施形態によれば、本発明の配列リスト第５配列のアミノ酸配列を含む融合蛋白質は、配列リスト第６配列のヌクレオチド配列によってコードされる。
According to one embodiment of the invention, the fusion protein of the invention comprises the amino acid sequence of
本発明の他の態様によれば、本発明は、本発明の融合蛋白質を含むＮＦ－κＢの転写または活性抑制剤を提供する。下記の実施例で立証したように、本発明の融合蛋白質は、ＮＦ－κＢおよびＩＬ－２の転写および活性を抑制し（図６）、ＬＰＳによる炎症性サイトカインの分泌抑制し（図７）、脾臓細胞における抗－ＣＤ３／ＣＤ２８で刺激されたＴ細胞の活性化によって発現するＩＬ－２、ＩＦＮ－γ、ＩＬ－４、ＩＬ－１７ＡおよびＩＬ－１０の生成を抑制して（図９）、ＮＦ－κＢによるＴ細胞の増殖、分化および炎症性サイトカインの分泌誘導活性を抑制できることを確認し、したがって、ＮＦ－κＢの過度の活性によって引き起こされる疾患を効果的に予防、改善または治療できることを確認することができた。 According to another aspect of the invention, the invention provides a transcription or activity inhibitor of NF-κB comprising the fusion protein of the invention. As demonstrated in the examples below, the fusion proteins of the invention suppress the transcription and activity of NF-κB and IL-2 (FIG. 6) and suppress the secretion of inflammatory cytokines by LPS (FIG. 7). Suppressing the production of IL-2, IFN-γ, IL-4, IL-17A and IL-10 expressed by anti-CD3 / CD28-stimulated T cell activation in spleen cells (FIG. 9). Confirmed that NF-κB can suppress T cell proliferation, differentiation and inflammatory cytokine secretion-inducing activity, and therefore can effectively prevent, ameliorate or treat diseases caused by excessive activity of NF-κB. We were able to.
発明のさらに他の態様によれば、本発明は、本発明の融合蛋白質を有効成分として含む、ＮＦ－κＢの過活性－関連疾患の予防または治療用薬剤学的組成物を提供する。 According to still another aspect of the invention, the invention provides a pharmaceutical composition for the prevention or treatment of overactivity-related diseases of NF-κB comprising the fusion protein of the invention as an active ingredient.
本明細書で使われた用語、「治療」は、（ａ）疾患、疾病または症状の進行の抑制；（ｂ）疾患、疾病または症状の軽減；または（ｃ）疾患、疾病または症状を除去することを意味する。本発明の組成物は、代謝疾患の症状の進行を抑制するか、これを除去または軽減させる役割を果たす。したがって、本発明の組成物は、それ自体でＮＦ－κＢの過活性－関連疾患の治療用組成物になってもよく、あるいは他のＮＦ－κＢの過活性－関連疾患の治療用組成物と共に投与され、これら疾患に対する治療補助剤として適用されてもよい。そこで、本明細書において、用語「治療」または「治療剤」は、「治療補助」または「治療補助剤」の意味を含む。 As used herein, the term "treatment" is used to (a) suppress the progression of a disease, disease or symptom; (b) alleviate the disease, illness or symptom; or (c) eliminate the disease, illness or symptom. Means that. The compositions of the present invention serve to suppress, eliminate or alleviate the progression of symptoms of metabolic disorders. Accordingly, the compositions of the invention may themselves be NF-κB hyperactivity-therapeutic compositions of related diseases, or together with other NF-κB hyperactivity-therapeutic compositions of related diseases. It may be administered and applied as a therapeutic adjunct to these diseases. Therefore, in the present specification, the term "treatment" or "therapeutic agent" includes the meaning of "therapeutic aid" or "therapeutic agent".
下記の実施例で立証したように、本発明の融合蛋白質は、ＮＦ－κＢおよびＩＬ－２の転写を抑制し、ＬＰＳによる炎症性サイトカインの分泌を抑制し、脾臓細胞におけるＩＬ－２、ＩＦＮ－γ、ＩＬ－４、ＩＬ－１７ＡおよびＩＬ－１０の生成を抑制する。したがって、本発明の融合蛋白質は、多様なＮＦ－κＢの過活性－関連疾患の効率的な予防または治療組成物として有用に利用可能である。 As demonstrated in the examples below, the fusion proteins of the invention suppress the transcription of NF-κB and IL-2, suppress the secretion of inflammatory cytokines by LPS, and IL-2, IFN- in spleen cells. It suppresses the production of γ, IL-4, IL-17A and IL-10. Therefore, the fusion proteins of the present invention can be usefully used as an efficient prophylactic or therapeutic composition for various NF-κB hyperactivity-related diseases.
本発明の一実施形態によれば、本発明のＮＦ－κＢの過活性－関連疾患は、炎症疾患または自己免疫疾患である。本発明の他の実施形態によれば、本発明のＮＦ－κＢの過活性－関連疾患は、敗血症性ショック、アレルギー性喘息、アレルギー性鼻炎、アトピー性皮膚炎、全身性紅斑性狼瘡、リウマチ関節炎、潰瘍性大腸炎、涙腺炎、アルツハイマー疾患、脳卒中、動脈硬化症、血管再狭窄、Ｉ型糖尿病、ＩＩ型糖尿病、蕁麻疹、結膜炎、乾癬、全身性炎症反応症候群、多発性筋炎、皮膚筋炎、結節性多発関節炎、混合結合組織症、シェーグレン症候群、痛風、パーキンソン病、筋萎縮性側索硬化症、糖尿性網膜症、多発性硬化症、クローン病、慢性甲状腺炎、セリアック病、重症筋無力症、尋常性天疱瘡、ウイルス疾患、細菌性疾患、放射線による障害、動脈硬化、血管腫、血管線維腫、再潅流障害および心臓肥大症から構成される群より選択される疾患である。本発明の特定の実施形態によれば、本発明のＮＦ－κＢの過活性－関連疾患は、敗血症性ショックである。微生物に感染して全身に深刻な炎症反応が現れる敗血症性ショックは、血管を循環する内毒素と炎症媒介物質によって心脈管系と血管運動因子の異常症状が現れる。これによって脱水と血管内液体の流出による低血量症が発生し、毛細管上に血管の収縮と弛緩が起こって血液の供給に異常が生じ、血管炎と血栓は組織への還流をさらに困難にして、最終的に、組織に低酸素症と代謝性酸症を誘発させる。特に、内毒素は、兔疫細胞（大食細胞、顆粒細胞、樹枝状細胞、リンパ球）でＮＦ－κＢを活性化させて、腫瘍壊死因子、インターロイキン１と６のようなサイトカインを分泌させ、このようなサイトカインは、好中球、内皮細胞、血小板、または炎症媒介因子を放出する細胞を刺激して全身的な反応が現れる。下記の実施例で立証したように、本発明の融合蛋白質は、ＬＰＳによって誘導した敗血症ショック動物モデルにおいて炎症性サイトカインの分泌を抑制し、生存率を増加させることを確認した（図１０）。
According to one embodiment of the invention, the hyperactivity-related disease of NF-κB of the present invention is an inflammatory disease or an autoimmune disease. According to other embodiments of the invention, the hyperactivity-related diseases of NF-κB of the invention are septic shock, allergic asthma, allergic rhinitis, atopic dermatitis, systemic erythema cyst, rheumatoid arthritis. , Ulcering colitis, lacrimal adenitis, Alzheimer's disease, stroke, arteriosclerosis, vascular restenosis, type I diabetes, type II diabetes, urticaria, conjunctivitis, psoriasis, systemic inflammatory reaction syndrome, polymyositis, dermatitis, Nodular polyarthritis, mixed connective tissue disease, Schegren's syndrome, gout, Parkinson's disease, muscular atrophic lateral sclerosis, diabetic retinopathy, multiple sclerosis, Crohn's disease, chronic thyroiditis, Celiac's disease, severe myasthenia , Vulgaris vulgaris, viral disease, bacterial disease, radiation disorder, arteriosclerosis, hemangiomas, vascular fibroma, reperfusion disorder and cardiac hypertrophy. According to a particular embodiment of the invention, the hyperactivity-related disease of NF-κB of the invention is septic shock. In septic shock, which is infected with microorganisms and causes a serious inflammatory reaction throughout the body, abnormal symptoms of the cardiovascular system and vasomotor factors appear due to endotoxins circulating in blood vessels and inflammation mediators. This causes hypoxia due to dehydration and outflow of intravascular fluid, causing constriction and relaxation of blood vessels on the capillaries, resulting in abnormal blood supply, and vasculitis and thrombi make it more difficult to return to tissue. Finally, it induces hypoxia and metabolic acid disease in the tissue. In particular, endotoxin activates NF-κB in epidemic cells (large phagocytic cells, granule cells, dendritic cells, lymphocytes) to secrete cytokines such as tumor necrosis factor,
したがって、本発明によれば、本発明の融合蛋白質は、ＮＦ－κＢの過活性－関連疾患を予防または治療する効果がある。 Therefore, according to the present invention, the fusion protein of the present invention has the effect of preventing or treating the hyperactivity-related disease of NF-κB.
本発明の組成物が薬剤学的組成物として製造される場合、本発明の薬剤学的組成物は、薬剤学的に許容される担体を含む。本発明の薬剤学的組成物に含まれる薬剤学的に許容される担体は、製剤時に通常用いられるものであって、ラクトース、デキストロース、スクロース、ソルビトール、マンニトール、デンプン、アカシアガム、リン酸カルシウム、アルギネート、ゼラチン、ケイ酸カルシウム、微細結晶性セルロース、ポリビニルピロリドン、セルロース、水、シロップ、メチルセルロース、メチルヒドロキシベンゾエート、プロピルヒドロキシベンゾエート、滑石、ステアリン酸マグネシウムおよびミネラルオイルなどを含むが、これらに限定されるものではない。本発明の薬剤学的組成物は、前記成分のほか、潤滑剤、湿潤剤、甘味剤、香味剤、乳化剤、懸濁剤、保存剤などを追加的に含んでもよい。好適な薬剤学的に許容される担体および製剤は、Ｒｅｍｉｎｇｔｏｎ’ｓ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｓｃｉｅｎｃｅｓ（１９ｔｈ ｅｄ．，１９９５）に詳しく記載されている。 When the composition of the invention is produced as a pharmaceutical composition, the pharmaceutical composition of the invention comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers contained in the pharmaceutical composition of the present invention are those usually used at the time of formulation, and are usually used at the time of formulation, such as lactose, dextrose, syrup, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, and the like. Includes, but is not limited to, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. not. In addition to the above-mentioned components, the pharmaceutical composition of the present invention may additionally contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
本発明の薬剤学的組成物は、経口または非経口投与することができ、非経口投与の場合には、静脈内注入、皮下注入、筋肉注入、腹腔注入、経皮投与などで投与することができる。本発明の一実施形態によれば、本発明の薬剤学的組成物は、腹腔注入によって投与される。 The pharmaceutical composition of the present invention can 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. can. According to one embodiment of the invention, the pharmaceutical composition of the invention is administered by peritoneal infusion.
本発明の薬剤学的組成物の好適な投与量は、製剤化方法、投与方式、患者の年齢、体重、性、病的状態、飲食、投与時間、投与経路、排泄速度および反応感応性のような要因によって多様に処方可能である。本発明の薬剤学的組成物の１日投与量は、例えば、０．０００１－１０００ｍｇ／ｋｇである。 Suitable doses of the pharmaceutical composition of the present invention include formulation method, administration method, patient age, body weight, sex, pathological condition, eating and drinking, administration time, administration route, excretion rate and reaction sensitivity. It can be prescribed in various ways depending on various factors. The daily dose of the pharmaceutical composition of the present invention is, for example, 0.0001-1000 mg / kg.
本発明の薬剤学的組成物は、当該発明の属する技術分野における通常の知識を有する者が容易に実施できる方法により、薬剤学的に許容される担体および／または賦形剤を用いて製剤化することにより、単位用量の形態で製造されるか、または多用量容器内に入れられて製造される。この時、剤形は、オイルまたは水性媒質中の溶液、懸濁液、シロップ剤または乳化液の形態であるか、エキス剤、散剤、粉末剤、顆粒剤、錠剤またはカプセル剤の形態であってもよいし、分散剤または安定化剤を追加的に含んでもよい。 The pharmaceutical composition of the present invention is formulated with a pharmaceutically acceptable carrier and / or excipient by a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the invention belongs. By doing so, it is manufactured in the form of a unit dose or placed in a multi-dose container. At this time, the dosage form is in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an extract, powder, powder, granule, tablet or capsule. It may be added with a dispersant or a stabilizer.
本発明のＮＦ－κＢの転写または活性抑制剤、そしてＮＦ－κＢの過活性－関連疾患の予防または治療用薬剤学的組成物は、上述した融合蛋白質と共通するため、前記融合蛋白質との関係において共通する内容は、本発明の過度の複雑性を回避するためにその記載を省略する。 Since the transcriptional or activity inhibitor of NF-κB of the present invention and the pharmaceutical composition for preventing or treating overactivity of NF-κB-related diseases are common to the above-mentioned fusion proteins, the relationship with the fusion proteins. In order to avoid excessive complexity of the present invention, the contents common to the above are omitted.
本発明のさらに他の態様によれば、本発明は、本発明の融合蛋白質を有効成分として含む組成物を投与する段階を含む、ＮＦ－κＢの転写または活性抑制方法を提供する。 According to still another aspect of the invention, the invention provides a method of inhibiting transcription or activity of NF-κB comprising administering a composition comprising the fusion protein of the invention as an active ingredient.
本発明のさらに他の態様によれば、本発明は、本発明の融合蛋白質を有効成分として含む組成物を、これを必要とする個体に投与する段階を含む、ＮＦ－κＢの過活性－関連疾患の予防、改善または治療方法を提供する。 According to still another aspect of the invention, the invention comprises the step of administering a composition comprising the fusion protein of the invention as an active ingredient to an individual in need thereof-related to overactivity of NF-κB. Provide methods for preventing, ameliorating or treating diseases.
本明細書で使われた用語、「投与」または「投与する」は、本発明の組成物の治療的有効量を、前記組成物を必要とする個体（つまり、対象体）に直接的に投与することにより、対象の体内で同量が形成されるようにすることをいう。したがって、用語「投与」は、本発明の有効成分（本発明の融合蛋白質）を病変部位に注入することを含むので、用語「投与する」は、「注入する」と同じ意味で使われる。 As used herein, the term "administer" or "administer" is a therapeutically effective amount of the composition of the invention administered directly to an individual (ie, subject) in need of said composition. By doing so, it means that the same amount is formed in the body of the subject. Therefore, the term "administer" includes injecting the active ingredient of the invention (the fusion protein of the invention) into the lesion site, so the term "administer" is used interchangeably with "inject".
組成物の「治療的有効量」は、組成物を投与しようとする個体に治療的または予防的効果を提供するのに十分な抽出物の含有量を意味し、よって、「予防的有効量」を含む意味である。本明細書で使われた用語、「個体」は、制限なく、ヒト、マウス、ラット、ギニーピッグ、イヌ、ネコ、ウマ、ウシ、ブタ、サル、チンパンジー、ヒヒまたはアカゲザルを含む。具体的には、本発明の個体は、ヒトである。 A "therapeutically effective amount" of a composition means the content of an extract sufficient to provide a therapeutic or prophylactic effect to the individual to whom the composition is to be administered, and thus a "prophylactically effective amount". It means to include. As used herein, the term "individual" includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Specifically, the individual of the present invention is a human.
本発明のＮＦ－κＢの転写または活性抑制方法、そしてＮＦ－κＢの過活性－関連疾患の予防、改善または治療方法は、上述した融合蛋白質およびその用途を共通するため、前記融合蛋白質、ＮＦ－κＢの転写または活性抑制およびＮＦ－κＢの過活性－関連疾患と共通する内容は、本発明の過度の複雑性を回避するためにその記載を省略する。 The fusion protein, NF- Content in common with κB transcription or activity suppression and NF-κB hyperactivity-related diseases is omitted to avoid excessive complexity of the invention.
本発明の特徴および利点をまとめると、次の通りである：
（ａ）本発明は、ＮＦ－κＢのサブユニットであるｐ６５の転写調節ドメインと蛋白質運搬ドメインとを含む融合蛋白質およびその用途を提供する。
（ｂ）本発明の融合蛋白質は、競合阻害によってＮＦ－κＢおよびＩＬ－２の転写を抑制し、ＬＰＳによる炎症性サイトカインの分泌を抑制し、脾臓細胞におけるＩＬ－２、ＩＦＮ－γ、ＩＬ－４、ＩＬ－１７ＡおよびＩＬ－１０の生成を抑制する効果があることから、ＮＦ－κＢの過活性－関連疾患の予防または治療組成物として有用に利用可能である。
The features and advantages of the present invention can be summarized as follows:
(A) The present invention provides a fusion protein containing a transcriptional regulatory domain and a protein-carrying domain of p65, which is a subunit of NF-κB, and uses thereof.
(B) The fusion protein of the present invention suppresses the transcription of NF-κB and IL-2 by competitive inhibition, suppresses the secretion of inflammatory cytokines by LPS, and IL-2, IFN-γ, IL- in spleen cells. 4. Since it has an effect of suppressing the production of IL-17A and IL-10, it can be usefully used as a preventive or therapeutic composition for NF-κB hyperactivity-related diseases.
以下、実施例を通じて本発明をより詳細に説明する。これらの実施例は単に本発明をより具体的に説明するためのものであり、本発明の要旨により本発明の範囲がこれらの実施例によって制限されないことは、当業界における通常の知識を有する者にとって自明であろう。 Hereinafter, the present invention will be described in more detail through examples. These examples are merely for the purpose of explaining the present invention more specifically, and it is a person having ordinary knowledge in the art that the scope of the present invention is not limited by these examples by the gist of the present invention. Will be self-explanatory.
［実施例１：ｐ６５の転写調節ドメインと蛋白質運搬ドメインとを含む組換え融合蛋白質の製造］
蛋白質運搬ドメイン（ＰＴＤ）のＨｐｈ－１（配列リスト第４配列）を、ｐ６５の転写調節ドメインのｐ６５－ＴＭＤ（配列リスト第２配列）とｐＥＴ－２８ａ（＋）ベクター（Ｎｏｖａｇｅｎ）にクローニングして、組換え融合ＤＮＡ（ｎｔ－ｐ６５－ＴＭＤ）（配列リスト第６配列）を製作した。前記組換え融合ＤＮＡをＢＬ２１ＣｏｄｏｎＰｌｕｓ（ＤＥ３）－ＲＩＰＬ大腸菌菌株（Ｉｎｖｉｔｏｇｅｎ）に形質転換させた。前記形質転換菌株を培養した後、１ｍＭ ＩＰＴＧ（ｉｓｏｐｒｏｐｙｌ－β－Ｄ－ｔｈｉｏｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅ、Ｄｕｃｈｅｆａ）を入れて、３７℃で５時間蛋白質を発現するように誘導した。その後、細胞のみを集めて分解用緩衝液（１０ｍＭ イミダゾール、５０ｍＭ ＮａＨ２ＰＯ４、３００ｍＭ ＮａＣｌおよびｐＨ８．０）に溶解させた後、粉砕機で細胞を分解させた。融合蛋白質は、蛋白質の前部分に人為的に結合させておいた６個のヒスチジン（Ｈｉｓｔｉｄｉｎｅ）を用いてＮｉ－ＮＴＡビーズ（Ｑｉａｇｅｎ）と結合させた。カラム（ＨｉｓＴｒａｐ ｃｈｒｏｍａｔｏｇｒａｐｈｙ ｃｏｌｕｍｎｓ、Ｂｉｏ－Ｒａｄ）に蛋白質を入れて洗浄用緩衝液（３０ｍＭ イミダゾール、５０ｍＭ ＮａＨ２ＰＯ４、３００ｍＭ ＮａＣｌおよびｐＨ８．０）で十分に洗浄し、溶出用緩衝液（２５０ｍＭ イミダゾール、５０ｍＭ ＮａＨ２ＰＯ４、３００ｍＭ ＮａＣｌおよびｐＨ８．０）で蛋白質を分離した。ＰＤ－１０Ｓｅｐｈａｄｅｘ Ｇ－２５（ＧＥ Ｈｅａｌｔｈｃａｒｅ）を用いて緩衝液を１０％グリセロールＰＢＳに切り替えながらイミダゾールとＮａＣｌを除去した。得られた蛋白質にはＬＰＳのようなエンドトキシンが存在するので、これを除去するために、ＳＰビーズ（ＳＰ ＳｅｐｈａｒｏｓｅＴＭ Ｆａｓｔ Ｆｌｏｗ、ＧＥ Ｈｅａｌｔｈｃａｒｅ）によりもう一度精製をした。これを再び結合用緩衝液（５０ｍＭ ＮａＨ２ＰＯ４、３００ｍＭ ＮａＣｌ、ｐＨ６．０）で結合させた後、これをカラムに入れて溶出用緩衝液（５０ｍＭ ＮａＨ２ＰＯ４、２Ｍ ＮａＣｌ、ｐＨ６．０）で蛋白質を分離した。最後に、ＰＤ－１０Ｓｅｐｈａｄｅｘ Ｇ－２５によりＮａＣｌを除去し、緩衝液を１０％グリセロールＰＢＳに切り替えた後、最終的に得られた蛋白質（配列リスト第５配列、組換え融合蛋白質）は実験前まで８０℃で保管した（図１および２参照）。
[Example 1: Production of a recombinant fusion protein containing a transcriptional regulatory domain and a protein-carrying domain of p65]
Hph-1 (sequence 4th sequence) of the protein carrier domain (PTD) was cloned into p65-TMD (sequence list 2nd sequence) and pET-28a (+) vector (Novagen) of the transcriptional regulatory domain of p65. , Recombinant fusion DNA (nt-p65-TMD) (Sequence list 6th sequence) was produced. The recombinant fusion DNA was transformed into BL21CodonPlus (DE3) -RIPL E. coli strain (Invitogen). After culturing the transformed strain, 1 mM IPTG (isopropanol-β-D-thiogalactopylanoside, Duchefa) was added to induce protein expression at 37 ° C. for 5 hours. Then, only the cells were collected and dissolved in a degradation buffer (10 mM imidazole, 50 mM NaH 2 PO 4 , 300 mM NaCl and pH 8.0), and then the cells were degraded by a pulverizer. The fusion protein was bound to Ni-NTA beads (Qiagen) using 6 histidines that had been artificially bound to the anterior portion of the protein. Put the protein in a column (HisTrap chromatography colons, Bio-Rad), wash thoroughly with wash buffer (30 mM imidazole, 50 mM NaH 2 PO 4 , 300 mM NaCl and pH 8.0), and wash thoroughly with elution buffer (250 mM imidazole, Bio-Rad). Proteins were separated with 50 mM NaH 2 PO 4 , 300 mM NaCl and pH 8.0). Imidazole and NaCl were removed while switching the buffer to 10% glycerol PBS using PD-10 Sephadex G-25 (GE Healthcare). Since endotoxin such as LPS is present in the obtained protein, it was purified again with SP beads (SP Sepharose TM Fast Flow, GE Healthcare) in order to remove it. This was bound again with a binding buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, pH 6.0), then placed in a column and eluted with an elution buffer (50 mM NaH 2 PO 4 , 2M NaCl, pH 6.0). ) Separated the protein. Finally, after removing NaCl with PD-10 Sephadex G-25 and switching the buffer to 10% glycerol PBS, the finally obtained protein (
［実施例２：ｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質の細胞内伝達の確認］
（２－１．ウェスタンブロットを利用した細胞内伝達の確認）
ＢＶ２ミクログリア細胞とジャーカット（Ｊｕｒｋａｔ）Ｔ細胞を用いて、実施例１のｎｔ－ｐ６５－ＴＭＤを濃度別（０、０．１、０．５、１、２および５μＭ）または時間別（０、１、２、４、６、１２、２４および４８ｈ）に組換え融合蛋白質と共に培養し、ウェスタンブロットで蛋白質伝達の有無を確認した。
その結果、濃度に比例してよく伝達されることを確認し、４８時間後にも細胞培養液内で蛋白質が構造をよく維持しつつ持続的に伝達されることを確認した（図３参照）。
[Example 2: Confirmation of intracellular transmission of nt-p65-TMD recombinant fusion protein]
(2-1. Confirmation of intracellular transmission using Western blotting)
Using BV2 microglial cells and Jurkat T cells, nt-p65-TMD of Example 1 was subjected to concentration (0, 0.1, 0.5, 1, 2 and 5 μM) or time (0, 0, 1, 2, 4, 6, 12, 24 and 48h) were cultured with the recombinant fusion protein, and the presence or absence of protein transmission was confirmed by Western blot.
As a result, it was confirmed that the protein was transmitted well in proportion to the concentration, and it was confirmed that the protein was continuously transmitted in the cell culture medium while maintaining the structure well even after 48 hours (see FIG. 3).
（２－２．抗体を用いて細胞の核まで伝達されるか否かの確認）
実施例１のｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質５μＭを、１時間、ＢＶ２ミクログリア細胞およびＨｅＬａ細胞と共に培養をし、ＰＢＳで洗った後、０．２％Ｔｒｉｔｏｎ Ｘ－１００（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）を用いて細胞に隙間を設けた後、その間に蛍光標識抗体を前記組換え融合蛋白質に結合させた。次に、ＤＡＰＩ染色剤（Ｉｎｖｉｔｒｏｇｅｎ）を用いて細胞の核を染色した後、蛍光顕微鏡により蛍光の位置を確認して、組換え融合蛋白質が伝達された位置を確認した。
(2-2. Confirmation of whether or not it is transmitted to the nucleus of the cell using an antibody)
5 μM of the nt-p65-TMD recombinant fusion protein of Example 1 was cultured with BV2 microglial cells and HeLa cells for 1 hour, washed with PBS, and then 0.2% Triton X-100 (Sigma-Aldrich) was added. After making a gap in the cells using the cell, a fluorescently labeled antibody was bound to the recombinant fusion protein between them. Next, after staining the nucleus of the cell with a DAPI stain (Invitrogen), the position of fluorescence was confirmed with a fluorescence microscope to confirm the position where the recombinant fusion protein was transmitted.
その結果、融合蛋白質はＰＴＤの性質によって細胞の核までよく伝達されたことを確認した（図４参照）。 As a result, it was confirmed that the fusion protein was well transmitted to the nucleus of the cell due to the nature of PTD (see FIG. 4).
［実施例３：ｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質の細胞毒性の確認］
大腸菌菌株から発現して得た蛋白質からＬＰＳが完全に除去されて細胞や動物で毒性を示さないことを確認するために、細胞毒性テストを行った。ＢＶ２ミクログリア細胞および脾臓細胞に多様な濃度の蛋白質を伝達した後に、生きている細胞に存在するジヒドロゲナーゼによって色を示す基質のＷＳＴ－８を入れて共に培養した。
[Example 3: Confirmation of cytotoxicity of nt-p65-TMD recombinant fusion protein]
A cytotoxicity test was performed to confirm that LPS was completely removed from the protein expressed from the E. coli strain and showed no toxicity in cells or animals. After transmitting various concentrations of protein to BV2 microglial cells and spleen cells, WST-8, a substrate colored by dihydrogenase present in living cells, was added and cultured together.
その結果、処理した融合蛋白質の濃度に関係なく、蛋白質を処理しない細胞と比較して全く毒性を示さないことを確認することができた（図５参照）。 As a result, it was confirmed that regardless of the concentration of the treated fusion protein, it showed no toxicity as compared with the cells not treated with the protein (see FIG. 5).
［実施例４：ｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質の転写因子の特異的抑制効果の確認］
（４－１．ＨＥＫ２９３Ｔ細胞におけるＮＦ－κＢおよびＩＬ－２の転写抑制効果の確認）
実施例１のｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質がＮＦ－κＢおよびＩＬ－２サイトカイン遺伝子のプロモーターに野生型ｐ６５の代わりに結合して発現を抑制するかを直接的に確認するために、ルシフェラーゼレポーター遺伝子（ｌｕｃｉｆｅｒａｓｅ ｒｅｐｏｒｔｅｒ ｇｅｎｅ）を用いた。まず、ＨＥＫ２９３Ｔ細胞に、下位にルシフェラーゼを有するＮＦ－κＢおよびＩＬ－２プロモーターと野生型ｐ６５遺伝子を核内注入（ｔｒａｎｓｆｅｃｔｉｏｎ）した後、ｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質で処理した。
[Example 4: Confirmation of specific inhibitory effect of transcription factor of nt-p65-TMD recombinant fusion protein]
(4-1. Confirmation of transcriptional repressive effect of NF-κB and IL-2 on HEK293T cells)
To directly confirm whether the nt-p65-TMD recombinant fusion protein of Example 1 binds to the promoters of the NF-κB and IL-2 cytokine genes instead of wild-type p65 and suppresses its expression, luciferase. A reporter gene (luciferase promoter gene) was used. First, HEK293T cells were transfected with the NF-κB and IL-2 promoters having luciferase at the lower level and the wild-type p65 gene, and then treated with the nt-p65-TMD recombinant fusion protein.
その結果、組換え融合蛋白質で処理した場合に、ルシフェラーゼの発現が効果的に抑制されたことを確認することができた。これは、ｎｔ－ｐ６５－ＴＭＤが野生型ｐ６５の競合阻害剤として作用してＮＦ－κＢおよびＩＬ－２プロモーターのｐ６５の結合サイトを塞いだことを意味する（図６参照）。 As a result, it was confirmed that the expression of luciferase was effectively suppressed when treated with the recombinant fusion protein. This means that nt-p65-TMD acted as a competitive inhibitor of wild-type p65 and blocked the binding site of p65 of the NF-κB and IL-2 promoters (see FIG. 6).
（４－２．ＢＶ２ミクログリア細胞における炎症性サイトカインの分泌抑制効果の確認）
ＢＶ２ミクログリア細胞に、実施例１のｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質を処理した後、１時間後にＬＰＳ（１μｇ／ｍｌ、Ｅ．ｃｏｌｉ ｓｅｒｏｔｙｐｅ Ｏ５５：Ｂ５、Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）を処理して２４時間培養した。
(4-2. Confirmation of inflammatory cytokine secretion inhibitory effect on BV2 microglial cells)
BV2 microglial cells were treated with the nt-p65-TMD recombinant fusion protein of Example 1 and then treated with LPS (1 μg / ml, E. coli serotype O55: B5, Sigma-Aldrich) 1 hour later for 24 hours. It was cultured.
その結果、ＬＰＳによって活性化されて発現するＴＮＦ－α、ＩＬ－１βおよびＩＬ－６がｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質によって発現が抑制されたことを確認することができた（図７参照）。 As a result, it was confirmed that the expression of TNF-α, IL-1β and IL-6 activated and expressed by LPS was suppressed by the nt-p65-TMD recombinant fusion protein (see FIG. 7). ).
（４－３．ジャーカットＴ細胞におけるＮＦ－κＢの転写因子の特異的抑制効果の確認）
実施例１のｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質が抗ＣＤ３（１μｇ／ｍｌ、ＢＤ Ｐｈａｒｍｉｎｇｅｎ）および抗ＣＤ２８（１μｇ／ｍｌ、ＢＤ Ｐｈａｒｍｉｎｇｅｎ）で刺激されたジャーカットＴ細胞の活性化によって活性化されたＮＦ－κＢの転写を特異的に抑制するか否かを確認した。Ｔ細胞が活性化されると、ＮＦ－κＢのみならず、ＮＦＡＴの転写も活性化されるため、ｎｔ－ｐ６５－ＴＭＤ融合蛋白質がＮＦＡＴの転写には影響を及ぼさずにＮＦ－κＢだけを抑制する場合、ｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質がＮＦ－κＢに特異的な競合阻害剤として作用することを確認することができる。したがって、これを確認するために、ルシフェラーゼレポーター遺伝子を用いた。まず、ジャーカットＴ細胞に、下位にルシフェラーゼを有するＮＦ－κＢおよびＮＦＡＴレポーター遺伝子を、電気穿孔法（ｅｌｃｔｒｏｐｏｒａｔｉｏｎ）を利用して核内注入した後、抗ＣＤ３および抗ＣＤ２８で刺激されたジャーカットＴ細胞をｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質で処理した。
(4-3. Confirmation of specific inhibitory effect of NF-κB transcription factor on Jurkat T cells)
The nt-p65-TMD recombinant fusion protein of Example 1 was activated by activation of Jurkat T cells stimulated with anti-CD3 (1 μg / ml, BD Pharmingen) and anti-CD28 (1 μg / ml, BD Pharmingen). It was confirmed whether or not the transcription of NF-κB was specifically suppressed. When T cells are activated, not only NF-κB but also NFAT transcription is activated, so that the nt-p65-TMD fusion protein suppresses only NF-κB without affecting the transcription of NFAT. If so, it can be confirmed that the nt-p65-TMD recombinant fusion protein acts as a competitive inhibitor specific to NF-κB. Therefore, a luciferase reporter gene was used to confirm this. First, NF-κB and NFAT reporter genes having lower luciferases were intranuclearly injected into Jurkat T cells using an electric perforation method, and then Jurkat T stimulated with anti-CD3 and anti-CD28. Cells were treated with nt-p65-TMD recombinant fusion protein.
その結果、組換え融合蛋白質を処理したＮＦ－κＢの場合に、ルシフェラーゼの発現が効果的に抑制されたが、ＮＦＡＴには影響を及ぼさないことを確認することができた（図８Ａ参照）。 As a result, it was confirmed that the expression of luciferase was effectively suppressed in the case of NF-κB treated with the recombinant fusion protein, but it did not affect NFAT (see FIG. 8A).
（４－４．細胞内信号伝達蛋白質のリン酸化）
実施例１のｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質が細胞内の多様な信号伝達体系に関連する蛋白質のチロシンリン酸化に関与するかを確認するために、ウェスタンブロットを利用した。ジャーカットＴ細胞にｎｔ－ｐ６５－ＴＭＤ ２μＭを１時間処理した後、抗ＣＤ３（２．５μｇ／ｍｌ）および抗ＣＤ２８（２．５μｇ／ｍｌ）で刺激を与えて、ＺＡＰ－７０、ｐ３８、ＪＮＫおよびＥＲＫのチロシンリン酸化の有無を観察した。
(4-4. Phosphorylation of intracellular signal transduction protein)
Western blotting was used to confirm whether the nt-p65-TMD recombinant fusion protein of Example 1 is involved in tyrosine phosphorylation of proteins associated with various intracellular signaling systems. Jurkat T cells were treated with nt-p65-
その結果、ｎｔ－ｐ６５－ＴＭＤはこれらのリン酸化には影響を及ぼさないことを確認することができた（図８Ｂ参照）。 As a result, it was confirmed that nt-p65-TMD did not affect these phosphorylations (see FIG. 8B).
（４－５．脾臓細胞におけるサイトカインＩＬ－２、ＩＦＮ－γ、ＩＬ－４、ＩＬ－１７ＡおよびＩＬ－１０の生成抑制）
６－８週齢の雌Ｃ５７ＢＬ／６マウスの脾臓から脾臓細胞を分離し、実施例１のｎｔ－ｐ６５－ＴＭＤを１時間処理して、組換え融合蛋白質を細胞内に伝達した。この細胞を抗ＣＤ３（１μｇ／ｍｌ）および抗ＣＤ２８（１μｇ／ｍｌ）で刺激した後、７２時間培養した。その後、培養液に存在するサイトカインの量を、ＥＬＩＳＡを利用して測定した。
(4-5. Suppression of production of cytokines IL-2, IFN-γ, IL-4, IL-17A and IL-10 in spleen cells)
Spleen cells were isolated from the spleen of 6-8 week old female C57BL / 6 mice and treated with nt-p65-TMD of Example 1 for 1 hour to transfer the recombinant fusion protein into the cells. The cells were stimulated with anti-CD3 (1 μg / ml) and anti-CD28 (1 μg / ml) and then cultured for 72 hours. Then, the amount of cytokines present in the culture medium was measured using ELISA.
その結果、ｎｔ－ｐ６５－ＴＭＤを処理した場合、ＩＬ－２、ＩＦＮ－γ、ＩＬ－４、ＩＬ－１７ＡおよびＩＬ－１０の発現量が大きく抑制されることを確認することができた。また、Ｔ細胞活性化の指標になるＣＤ６９の発現量をＦＡＣＳ Ｃａｌｉｂｕｒ（ＢＤ Ｂｉｏｓｃｉｅｎｃｅｓ）を用いて確認した結果、ｎｔ－ｐ６５－ＴＭＤはＴ細胞におけるＣＤ６９の発現には影響を及ぼさないことを確認することができた（図９参照）。 As a result, it was confirmed that when nt-p65-TMD was treated, the expression levels of IL-2, IFN-γ, IL-4, IL-17A and IL-10 were significantly suppressed. In addition, as a result of confirming the expression level of CD69, which is an index of T cell activation, using FACS Calibur (BD Biosciences), it is confirmed that nt-p65-TMD does not affect the expression of CD69 in T cells. I was able to do it (see Fig. 9).
［実施例５：敗血症ショック動物モデルにおけるｎｔ－ｐ６５－ＴＭＤの治療効果の確認］
６－８週齢の雄ＢＡＬＢ／ｃマウスにＬＰＳ（２０ｍｇ／ｋｇ）を腹腔注入して、敗血症ショック動物モデルにした。その後、２時間、１４時間経過時、それぞれ実施例１のｎｔ－ｐ６５－ＴＭＤ組換え融合蛋白質を腹腔内注入し、６日間観察した。
[Example 5: Confirmation of therapeutic effect of nt-p65-TMD in a septic shock animal model]
LPS (20 mg / kg) was intraperitoneally injected into 6-8 week old male BALB / c mice to create a septic shock animal model. Then, after 2 hours and 14 hours, the nt-p65-TMD recombinant fusion protein of Example 1 was intraperitoneally injected and observed for 6 days.
その結果、ｎｔ－ｐ６５－ＴＭＤを処理した場合、炎症性サイトカインであるＴＮＦ－α、ＩＬ－１βおよびＩＬ－６の分泌を抑制して、それによる生存率が大きく増加したことを確認することができた（図１０参照）。 As a result, it can be confirmed that when nt-p65-TMD was treated, the secretion of the inflammatory cytokines TNF-α, IL-1β and IL-6 was suppressed, and the survival rate was greatly increased. It was done (see Fig. 10).
［実施例６：組換え融合蛋白質の急性毒性実験］
大韓実験供給センターから供給された６週齢の特定病原体不在（ｓｐｅｃｉｆｉｃ ｐａｔｈｏｇｅｎ－ｆｒｅｅ、ＳＰＦ）ＳＤ系ラットを用いて、急性毒性実験を下記のように行った：各グループあたり２匹ずつの動物に、本発明の実施例１の組換え融合蛋白質を１ｇ／ｋｇの用量で１回経口投与後、動物のへい死の有無、臨床症状および体重変化を観察し、血液学的検査と血液生化学的検査を実施し、解剖して、肉眼で腔臓器と胸腔臓器の異常の有無を観察した。
[Example 6: Acute toxicity experiment of recombinant fusion protein]
Acute toxicity studies were performed using 6-week-old specific pathogen-free (SPF) SD-series rats supplied by the Korea Experimental Supply Center as follows: 2 animals per group. After oral administration of the recombinant fusion protein of Example 1 of the present invention at a dose of 1 g / kg once, the presence or absence of mortality of animals, clinical symptoms and changes in body weight were observed, and hematological and biochemical tests were performed. Was performed and dissected, and the presence or absence of abnormalities in the cavity and thoracic organs was observed with the naked eye.
その結果、実験物質を投与したすべての動物で特異的な臨床症状やへい死した動物はおらず、体重変化、血液検査、血液生化学検査および解剖と所見などからも毒性変化は観察されなかった。したがって、本発明の実施例１の組換え融合蛋白質はラットでそれぞれ１ｇ／ｋｇまでも毒性を示しておらず、経口投与の最小致死量（ＬＤ５０）が１ｇ／ｋｇ以上である安全な物質と判断されることを確認することができた。 As a result, none of the animals treated with the experimental substance had specific clinical symptoms or died, and no toxic changes were observed from body weight changes, blood tests, blood biochemical tests and anatomy and findings. Therefore, the recombinant fusion protein of Example 1 of the present invention is not toxic to rats up to 1 g / kg, respectively, and is a safe substance having a minimum lethal dose (LD 50 ) of 1 g / kg or more for oral administration. I was able to confirm that it was judged.
（参考文献）
Park et al., Intranuclear interactomic inhibition of NF-kB suppresses LPS-induced severe sepsis, Biochemical and Biophysical Research Communications, 2015;464:711-717.
(Reference)
Park et al., Intranuclear interactomic inhibition of NF-kB suppresses LPS-induced severe sepsis, Biochemical and Biophysical Research Communications, 2015; 464: 711-717.
以上、本発明の特定部分を詳細に述べたが、当業界における通常の知識を有する者にとってこのような具体的な記述は単に好ましい実施形態に過ぎず、これによって本発明の範囲が制限されない点は明らかである。したがって、本発明の実質的な範囲は、添付した請求項とその等価物によって定義されるべきであろう。 Although the specific parts of the present invention have been described in detail above, such a specific description is merely a preferred embodiment for a person having ordinary knowledge in the art, and this does not limit the scope of the present invention. Is clear. Therefore, the substantial scope of the invention should be defined by the appended claims and their equivalents.
Claims (5)
前記融合蛋白質は、ＮＦ－κＢの転写調節ドメインおよび蛋白質運搬ドメインを含み、
前記融合蛋白質は、競合阻害によってＮＦ－κＢの転写を抑制し、
前記融合蛋白質は、配列リスト第５配列のアミノ酸配列からなり、
前記ＮＦ－κＢの過活性－関連疾患は、炎症疾患または自己免疫疾患であることを特徴とする、組成物。 A pharmaceutical composition comprising a fusion protein as an active ingredient for the prevention or treatment of NF-κB hyperactivity-related diseases.
The fusion protein comprises a transcriptional regulatory domain and a protein transport domain of NF-κB.
The fusion protein suppresses the transcription of NF-κB by competitive inhibition,
The fusion protein consists of the amino acid sequence of the 5th sequence in the sequence list.
The composition, wherein the overactive-related disease of NF-κB is an inflammatory disease or an autoimmune disease.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/KR2016/000104 WO2017119521A1 (en) | 2016-01-06 | 2016-01-06 | Novel fusion protein comprising transcription modulation domain of p65 and protein transport domain and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2019507117A JP2019507117A (en) | 2019-03-14 |
| JP7051687B2 true JP7051687B2 (en) | 2022-04-11 |
Family
ID=59274225
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2018536197A Active JP7051687B2 (en) | 2016-01-06 | 2016-01-06 | A novel fusion protein containing a transcriptional regulatory domain and a protein-carrying domain of P65 and their uses. |
Country Status (4)
| Country | Link |
|---|---|
| JP (1) | JP7051687B2 (en) |
| AU (1) | AU2016385177B2 (en) |
| CA (1) | CA3010811A1 (en) |
| WO (1) | WO2017119521A1 (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080084937A (en) * | 2005-11-04 | 2008-09-22 | 포휴먼텍(주) | Methods for fusion polypeptide delivery into a cell |
| CN103314012B (en) * | 2010-08-20 | 2018-07-10 | 李尚揆 | Fusion protein with transcriptional regulatory domain and protein transduction domain and the functional transcription factor inhibitor containing it |
| KR101525856B1 (en) * | 2010-08-20 | 2015-06-04 | 연세대학교 산학협력단 | A inhibitor for transcription factor of fusion protein comprising DNA binding domain of transcription factor and protein transduction domain |
-
2016
- 2016-01-06 JP JP2018536197A patent/JP7051687B2/en active Active
- 2016-01-06 AU AU2016385177A patent/AU2016385177B2/en active Active
- 2016-01-06 CA CA3010811A patent/CA3010811A1/en active Pending
- 2016-01-06 WO PCT/KR2016/000104 patent/WO2017119521A1/en active Application Filing
Non-Patent Citations (2)
| Title |
|---|
| Ｄａｔａｂａｓｅ ＧｅｎＰｅｐｔ［ｏｎｌｉｎｅ］，Ａｃｃｅｓｓｉｏｎ Ｎｏ．ＮＰ＿０３３０７１ |
| Ｓｃｉｅｎｔｉｆｉｃ Ｒｅｐｏｒｔｓ，２０１５年，５：８１３３，ｐｐ．１－１３ |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2016385177A1 (en) | 2018-08-09 |
| JP2019507117A (en) | 2019-03-14 |
| CA3010811A1 (en) | 2017-07-13 |
| AU2016385177B2 (en) | 2023-10-19 |
| WO2017119521A1 (en) | 2017-07-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Van Hauwermeiren et al. | Treatment of TNF mediated diseases by selective inhibition of soluble TNF or TNFR1 | |
| Tesch et al. | ASK1: a new therapeutic target for kidney disease | |
| Wakamatsu et al. | Effect of a small molecule inhibitor of nuclear factor-κB nuclear translocation in a murine model of arthritis and cultured human synovial cells | |
| Sandner et al. | The potential of sGC modulators for the treatment of age-related fibrosis: a mini-review | |
| Liu et al. | MLIF modulates microglia polarization in ischemic stroke by targeting eEF1A1 | |
| ES2922824T3 (en) | HMGB1 mutants | |
| CA3017689A1 (en) | Use of pten-long leader sequence for transmembrane delivery of molecules | |
| Tanudji et al. | The nonclassic secretion of thioredoxin is not sensitive to redox state | |
| JP7051687B2 (en) | A novel fusion protein containing a transcriptional regulatory domain and a protein-carrying domain of P65 and their uses. | |
| US20240041988A1 (en) | Use of elapidae postsynaptic neurotoxin in the treatment of over expression of inflammatory cytokines related diseases | |
| Namai et al. | Recombinant mouse calcitonin gene‐related peptide secreted by Lactococcus lactis inhibits lipopolysaccharide‐induced inflammatory response in macrophages | |
| US11414467B2 (en) | Fusion protein comprising transcription modulation domain of p65 and protein transduction domain, and uses thereof | |
| CN108690123B (en) | Application of short peptide in preparation of immunoregulation medicine | |
| EP4046648A1 (en) | Application of elapidae snake postsynaptic neurotoxin monomer molecule in treatment of alzheimer's disease | |
| Ma et al. | A novel fusion protein consisting of anti-ANGPTL3 antibody and interleukin-22 ameliorates diabetic nephropathy in mice | |
| JPH1052282A (en) | Angiogenin suppressing agent | |
| JP2023538013A (en) | Mutants of bone morphogenetic protein-9, 10 with reduced ectopic osteogenic side effects and enhanced therapeutic efficacy and pharmaceutical compositions containing the same | |
| US20190315820A1 (en) | A Composition Containing SMAD Protein for Treatment of Autoimmune Diseases, a Fusion Protein Comprising SMAD Protein, an Expression Vector and a Method for Preparing the Same | |
| US9321820B2 (en) | Compositions and methods for treating bladder cancer | |
| Avlas et al. | CD300b regulates intestinal inflammation and promotes repair in colitis | |
| CN103113467A (en) | PirB extracellular polypeptide and application | |
| RU2772733C2 (en) | Hmgb1 mutants | |
| CN112390855B (en) | Application of Pretide-146a in preparation of medicine for relieving or treating autoimmune diseases | |
| Nguyen et al. | Production of recombinant human tumor necrosis factor receptor-IgG1 Fc domain fusion protein expressed by genetically CHO-DG44 cells | |
| US20210085754A1 (en) | Molecular design of recombinant protein drug |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A80 | Written request to apply exceptions to lack of novelty of invention |
Free format text: JAPANESE INTERMEDIATE CODE: A801 Effective date: 20180828 Free format text: JAPANESE INTERMEDIATE CODE: A80 Effective date: 20180828 |
|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20181106 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20181106 |
|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20181130 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20181130 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20181227 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20191031 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20191113 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200207 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20200825 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20201218 |
|
| C60 | Trial request (containing other claim documents, opposition documents) |
Free format text: JAPANESE INTERMEDIATE CODE: C60 Effective date: 20201218 |
|
| A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20201228 |
|
| C21 | Notice of transfer of a case for reconsideration by examiners before appeal proceedings |
Free format text: JAPANESE INTERMEDIATE CODE: C21 Effective date: 20210105 |
|
| A912 | Re-examination (zenchi) completed and case transferred to appeal board |
Free format text: JAPANESE INTERMEDIATE CODE: A912 Effective date: 20210305 |
|
| C211 | Notice of termination of reconsideration by examiners before appeal proceedings |
Free format text: JAPANESE INTERMEDIATE CODE: C211 Effective date: 20210309 |
|
| C22 | Notice of designation (change) of administrative judge |
Free format text: JAPANESE INTERMEDIATE CODE: C22 Effective date: 20210902 |
|
| C13 | Notice of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: C13 Effective date: 20211109 |
|
| C22 | Notice of designation (change) of administrative judge |
Free format text: JAPANESE INTERMEDIATE CODE: C22 Effective date: 20220105 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220127 |
|
| C23 | Notice of termination of proceedings |
Free format text: JAPANESE INTERMEDIATE CODE: C23 Effective date: 20220209 |
|
| C03 | Trial/appeal decision taken |
Free format text: JAPANESE INTERMEDIATE CODE: C03 Effective date: 20220311 |
|
| C30A | Notification sent |
Free format text: JAPANESE INTERMEDIATE CODE: C3012 Effective date: 20220311 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20220330 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7051687 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |