WO2017074118A1 - Pharmaceutical composition containing dendritic cell expressing foxp3 for regulating immunity - Google Patents
Pharmaceutical composition containing dendritic cell expressing foxp3 for regulating immunity Download PDFInfo
- Publication number
- WO2017074118A1 WO2017074118A1 PCT/KR2016/012289 KR2016012289W WO2017074118A1 WO 2017074118 A1 WO2017074118 A1 WO 2017074118A1 KR 2016012289 W KR2016012289 W KR 2016012289W WO 2017074118 A1 WO2017074118 A1 WO 2017074118A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- foxp3
- fxdc
- present
- blood
- Prior art date
Links
- 210000004443 dendritic cell Anatomy 0.000 title claims abstract description 71
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 30
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 7
- 230000036039 immunity Effects 0.000 title abstract description 7
- 210000003289 regulatory T cell Anatomy 0.000 claims abstract description 51
- 210000004369 blood Anatomy 0.000 claims abstract description 43
- 239000008280 blood Substances 0.000 claims abstract description 43
- 230000013632 homeostatic process Effects 0.000 claims abstract description 19
- 210000004027 cell Anatomy 0.000 claims description 33
- 210000004985 myeloid-derived suppressor cell Anatomy 0.000 claims description 23
- 208000027866 inflammatory disease Diseases 0.000 claims description 15
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 claims description 13
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 13
- 239000004480 active ingredient Substances 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 11
- 210000001185 bone marrow Anatomy 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 229940124597 therapeutic agent Drugs 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000001902 propagating effect Effects 0.000 claims 1
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 27
- 230000035755 proliferation Effects 0.000 abstract description 19
- 238000011282 treatment Methods 0.000 abstract description 16
- 208000026278 immune system disease Diseases 0.000 abstract description 8
- 230000001900 immune effect Effects 0.000 abstract description 4
- 230000003213 activating effect Effects 0.000 abstract description 2
- 230000003466 anti-cipated effect Effects 0.000 abstract 1
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 34
- 102100022297 Integrin alpha-X Human genes 0.000 description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 16
- 201000010099 disease Diseases 0.000 description 14
- 210000001165 lymph node Anatomy 0.000 description 14
- 230000000694 effects Effects 0.000 description 13
- 230000004069 differentiation Effects 0.000 description 12
- 238000001565 modulated differential scanning calorimetry Methods 0.000 description 12
- 239000000427 antigen Substances 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 10
- 102000036639 antigens Human genes 0.000 description 10
- 210000005210 lymphoid organ Anatomy 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 9
- 210000002865 immune cell Anatomy 0.000 description 9
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 8
- 102100022338 Integrin alpha-M Human genes 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 210000002540 macrophage Anatomy 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 210000003068 cdc Anatomy 0.000 description 7
- 201000010276 collecting duct carcinoma Diseases 0.000 description 7
- 230000028993 immune response Effects 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- 210000000130 stem cell Anatomy 0.000 description 7
- 230000007246 mechanism Effects 0.000 description 6
- 230000002265 prevention Effects 0.000 description 6
- 208000037581 Persistent Infection Diseases 0.000 description 5
- 230000006052 T cell proliferation Effects 0.000 description 5
- 230000033228 biological regulation Effects 0.000 description 5
- 238000012790 confirmation Methods 0.000 description 5
- 230000002519 immonomodulatory effect Effects 0.000 description 5
- 210000000987 immune system Anatomy 0.000 description 5
- 210000003563 lymphoid tissue Anatomy 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 208000023275 Autoimmune disease Diseases 0.000 description 4
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 4
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 4
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 4
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 230000001684 chronic effect Effects 0.000 description 4
- 238000003501 co-culture Methods 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 230000006058 immune tolerance Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 210000004981 tumor-associated macrophage Anatomy 0.000 description 4
- 108700031361 Brachyury Proteins 0.000 description 3
- 102100036842 C-C motif chemokine 19 Human genes 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 description 3
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 3
- 206010020751 Hypersensitivity Diseases 0.000 description 3
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 3
- 208000026935 allergic disease Diseases 0.000 description 3
- 210000000612 antigen-presenting cell Anatomy 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 210000003162 effector t lymphocyte Anatomy 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000003018 immunosuppressive agent Substances 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 210000005236 CD8+ effector T cell Anatomy 0.000 description 2
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 2
- 208000021386 Sjogren Syndrome Diseases 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 230000007815 allergy Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000007969 cellular immunity Effects 0.000 description 2
- 208000037976 chronic inflammation Diseases 0.000 description 2
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 2
- 230000003832 immune regulation Effects 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 229960003444 immunosuppressant agent Drugs 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 206010025135 lupus erythematosus Diseases 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
- 238000012423 maintenance Methods 0.000 description 2
- 210000001986 peyer's patch Anatomy 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 230000009758 senescence Effects 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- 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
- 241000220479 Acacia Species 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
- 206010002556 Ankylosing Spondylitis Diseases 0.000 description 1
- 206010003645 Atopy Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- PTHCMJGKKRQCBF-UHFFFAOYSA-N Cellulose, microcrystalline Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC)C(CO)O1 PTHCMJGKKRQCBF-UHFFFAOYSA-N 0.000 description 1
- 206010010741 Conjunctivitis Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 229930105110 Cyclosporin A Natural products 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine 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
- 241000283086 Equidae Species 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 208000001640 Fibromyalgia Diseases 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 208000007882 Gastritis Diseases 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
- 201000005569 Gout Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 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
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 210000004322 M2 macrophage Anatomy 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 201000002481 Myositis Diseases 0.000 description 1
- 206010068319 Oropharyngeal pain Diseases 0.000 description 1
- 206010033078 Otitis media Diseases 0.000 description 1
- 206010034464 Periarthritis Diseases 0.000 description 1
- 201000007100 Pharyngitis Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 201000001263 Psoriatic Arthritis Diseases 0.000 description 1
- 208000036824 Psoriatic arthropathy Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 206010042674 Swelling Diseases 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 208000000491 Tendinopathy Diseases 0.000 description 1
- 206010043255 Tendonitis Diseases 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 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
- 239000011230 binding agent Substances 0.000 description 1
- 210000005208 blood dendritic cell Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 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
- 239000002771 cell marker Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular effect 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
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 210000003690 classically activated macrophage Anatomy 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 201000003146 cystitis Diseases 0.000 description 1
- 210000004544 dc2 Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 201000005917 gastric ulcer Diseases 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
- 208000014617 hemorrhoid Diseases 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 230000008629 immune suppression Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 229940125721 immunosuppressive agent Drugs 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000845 maltitol Substances 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 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
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 210000004479 myeloid suppressor cell Anatomy 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 201000008383 nephritis Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 201000001245 periodontitis Diseases 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 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
- 239000002994 raw material Substances 0.000 description 1
- 238000012950 reanalysis Methods 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 206010039083 rhinitis Diseases 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 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
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 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
- 201000004415 tendinitis Diseases 0.000 description 1
- 206010044008 tonsillitis Diseases 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 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
- 230000003612 virological effect Effects 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
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4615—Dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
Definitions
- the present invention relates to a pharmaceutical composition for immunomodulation, More specifically, the present invention relates to a pharmaceutical composition for immunomodulation comprising dendritic cells expressing Foxp3.
- the immune system consists of immune tolerance, which is a mechanism for suppressing and regulating immunity, and an immune response that promotes immunity.
- the immune system maintains immune homeostasis by properly balancing these two immune actions. have. This maintained immunological balance can lead to imbalances for a variety of causes, which eventually lead to various diseases. Immunological imbalance can result if the immune tolerant function is stronger than the immune response or vice versa. For example, when immune tolerance is stronger than that of the immune response, the human immune system may easily develop cancer, invade foreign viruses, and pathogenic bacteria, and may cause cancer or viral and bacterial diseases.
- Immunosuppressants refer to a variety of substances that are used to reduce or block the host's ability to produce antibodies (humidary immune response) or cellular immune response to the action of an antigen.
- it is also usefully used for skin hypersensitivity reactions, such as autoimmune diseases such as lupus, rheumatoid arthritis, atopy, allergy (see Korean Patent Publication No. 10-2015-0026839).
- immunosuppressive agents such as cyclosporin A and FK506 are compounds derived from natural products having a complicated chemical structure and are expensive in terms of supply and demand of raw materials, and thus are inexpensive and carry a risk that various side effects may be caused by long-term administration. Therefore, there is an urgent need for the development of new immunomodulators that exhibit low toxicity and are capable of economic production.
- dendritic cells (Dendritic Cell, DC) has a variety of characteristics depending on the origin, form, phenotype, function, maturation process, etc., in particular, can promote or inhibit the T cell response depending on the situation, the homeostasis of the immune system Plays an important role in regulating
- the basic study of conventional dendritic cells was performed by using bone marrow-derived dendritic cells (BMDC) made by artificially differentiating mouse bone marrow cells or by separating DCs residing in secondary lymphoid tissues such as mouse spleen and lymph nodes.
- BMDC bone marrow-derived dendritic cells
- secondary lymphoid tissues such as mouse spleen and lymph nodes.
- the present inventors have found for the first time the dendritic cells expressing Foxp3 present only in the blood, and tried to provide the basic data for the development of disease treatments according to immune imbalance by identifying their immune regulation and immune homeostasis maintenance effect.
- the present invention has been made to solve the above-mentioned conventional problems, the present inventors have confirmed the immune homeostasis maintenance effect of the dendritic cells expressing Foxp3 present in the blood, and completed the present invention based on this.
- an object of the present invention is to provide a pharmaceutical composition for immunomodulation comprising dendritic cells expressing Foxp3 as an active ingredient.
- Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases, including dendritic cells expressing Foxp3 as an active ingredient.
- Another object of the present invention is to isolate the myeloid-derived suppressor cells (MDSC) present in the blood; And culturing the isolated bone marrow-derived suppressor cells in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF).
- MDSC myeloid-derived suppressor cells
- GM-CSF granulocyte-macrophage colony-stimulating factor
- the present invention provides a pharmaceutical composition for immunomodulation, comprising dendritic cells expressing Foxp3 as an active ingredient.
- Foxp3 may be composed of the amino acid sequence set forth in SEQ ID NO: 1.
- the dendritic cells may be isolated from blood.
- the composition can modulate immune homeostasis in the blood.
- the composition can propagate CD8 + regulatory T cells (CD8 + Tregs).
- the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, including dendritic cells expressing Foxp3 as an active ingredient.
- the present invention comprises the steps of separating the myeloid-derived suppressor cells (MDSC) present in the blood; And it provides a method for producing dendritic cells expressing Foxp3 comprising the step of culturing the isolated bone marrow-derived suppressor cells in a medium containing GM-CSF.
- MDSC myeloid-derived suppressor cells
- the myeloid-derived suppressor cells may be monocytic myeloid-derived suppressor cells (M-MDSC).
- the present invention provides a method for treating an inflammatory disease comprising administering the pharmaceutical composition to a subject.
- the present invention provides a novel use of dendritic cells expressing Foxp3 for the preparation of a therapeutic agent for an inflammatory disease.
- the composition according to the present invention comprises a dendritic cell (fxDC) expressing Foxp3 as an active ingredient
- fxDC dendritic cell
- the fxDC is a key immune cell involved in the immune homeostasis of the blood itself, the number of individuals in an imbalanced state such as an infectious disease It is possible to strongly inhibit the proliferation of effector T cells in the blood through the regulation of CD8 + regulatory T cell (CD8 + Treg) proliferation, which is useful as a pharmaceutical composition for the prevention or treatment of chronic inflammatory immune diseases. It is expected to be able to be used.
- the reduction of CD8 + Treg cells is one of the characteristics of immune senescence, and in the elderly, the CD8 + Treg cells decrease the immune homeostasis and increase the number of patients with chronic inflammatory diseases or autoimmune diseases. No cause of decline was found.
- fxDC in the blood is a causative immune cell that induces the proliferation of CD8 + Tregs.
- dendritic cells expressing Foxp3 can be used as a pharmaceutical composition for the treatment of immune aging.
- Figure 2a is the result of re-analysis by reverse gating the presence of fxDC with a T cell marker (CD3) in mouse peripheral blood mononuclear cells (mPBMC).
- CD3 T cell marker
- Figure 2b is a result confirming that fxDC is not pDC using plasmacytoid DC (pDC) specific surface antigen (CD11c, and B220).
- pDC plasmacytoid DC
- Figure 2c is a result of investigating the expression of Macrophage (macrophage) specific surface antigens (CD11b, CD14, F4 / 80 and CD64) in fxDC present in peripheral blood to confirm that fxDC is not monocytes or macrophages.
- Macrophage macrophage
- CD11b, CD14, F4 / 80 and CD64 specific surface antigens
- Figure 3 shows the results of the change in the presence and population of fxDC in the normal and acute (LCMV armstrong) and chronic (LCML clone13) virus infected mouse blood.
- Figure 4 shows the results confirmed by FACS after analyzing the mobility of the fxDC CCL19 by transwell experiments.
- 5 is a result of dividing mPBMC into CD11c + cell group and CD11c- cell group in order to identify progenitor cells of fxDC, and confirming the differentiation into fxDC in each group.
- Figure 6 shows the results of FACS analysis of differentiation of fxDC after culturing MDSCs subgroups (DP-MDSC, G-MDSC, M-MDSC, and DN cells) in the presence of GM-CSF for 3 days.
- T cells 7 is a result of co-culture of CFSF-labeled syngeneic T cells with mDC, G-MDSC, M-MDSC, DP-MDSC, and fxDC for 3 days to confirm the proliferation of T cells.
- FIG. 8 shows CD4 + regulatory T cells that are generated when Foxp3-GFP mouse T cells are treated with CD3 and CD28 antibodies and co-cultured with mDC, G-MDSS, M-MDSC, DP-MDSC, or fxDC. (CD4 + Tregs) and CD8 + regulatory T cells (CD8 + Tregs).
- FIG. 9 shows the inhibition of proliferation of CD8 + T cells of fxDC by fxDC and coculture with fxDC and coculture with a transwell plate (cell binding block) after activation and activation of CD3 and CD28 antibodies to normal mouse T cells. The results were confirmed to be due to direct contact and not by cain.
- FIG. 10 shows CD4 + regulatory T cells generated when Foxp3-GFP mouse T cells are treated with CD3 and CD28 antibodies and then co-cultured with mDC, G-MDSS, M-MDSC, DP-MDSC, or fxDC. (CD4 + Tregs) and CD8 + regulatory T cells (CD8 + Tregs).
- fxDC Foxp3 + dendritic cells
- MDSC bone marrow-derived suppressor cells
- the fxDCs were differentiated from the cells (M-MDSCs).
- blood fxDC unlike other immunoregulatory DCs, confirms the novel fact that it induces proliferation of CD8 + regulatory T cells (CD8 + Tregs) rather than CD4 + regulatory T cells (CD4 + Tregs) to regulate T cell activity and completes the present invention It was.
- the present invention provides a pharmaceutical composition for immunomodulation comprising dendritic cells expressing Foxp3 as an active ingredient.
- the Foxp3 protein preferably has an amino acid sequence as set forth in SEQ ID NO: 1, but is not limited thereto, and 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 70% or more of the amino acid sequence. May comprise a protein represented by an amino acid sequence having at least 95% homology.
- dendritic cells (Dendritic Cells, DC) is one of the important antigen presenting cells (APC) that initiates an antigen specific adaptive immune response, which may be derived from human or mouse, preferably dendritic from human blood Cells may be isolated or differentiated from monocytes isolated from blood into dendritic cells, but are not limited thereto.
- APC antigen presenting cells
- dendritic cells (fxDC) expressing Foxp3 are essential immune cells involved in the immune homeostasis of the blood itself, and do not exist in a normal state, but are newly generated only in an imbalanced state, thereby generating CD8 + regulatory T cells ( CD8 + Tregs) can be strongly inhibited the proliferation of effector T cells in the blood through the regulation of proliferation, it is expected to be useful as a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
- CD8 Tregs are induced, CD4 Tregs are not induced, and CD8 T cell activity is specifically inhibited, which is different from the existing recombinant FoxP3 expressing dendritic cells.
- the recombinant Foxp3 expressing dendritic cells treated with the anti-TGF- ⁇ antibody disappeared the immunosuppressive activity, and showed immunosuppressive activity by cytokines, but Foxp3 expressing dendritic cells in the body or prepared through in vitro differentiation.
- Dendritic cells were found to be different from recombinant dendritic cells because they exhibited the property of inhibiting T cell immunity by direct contact rather than indirect immune suppression by cytokines.
- the term "immunoregulation" used in the present invention means to alleviate immune imbalance in the blood and maintain immune homeostasis. Maintaining immune homeostasis refers to a condition in which a balance between immune tolerance and immune response-immunity-promoting mechanisms is maintained. Is an essential element.
- the present invention is the first to reveal that when the imbalance in the blood caused fxDC present in the blood increases, they induce CD8 + regulatory T cells (CD8 + Tregs) proliferation, maintaining immune homeostasis in the blood There is a characteristic.
- the present invention also provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, including dendritic cells expressing Foxp3 as an active ingredient.
- prevention means any action that inhibits or delays the development of an inflammatory disease by administration of a pharmaceutical composition according to the present invention.
- treatment means any action in which symptoms for inflammatory diseases are improved or beneficially altered by administration of the pharmaceutical composition according to the invention.
- CD11c + Foxp3 + dendritic cells fxDC
- various lymphoid organs Spleen, inguinal lymph node, mesenteric lymph node, peyer's patch and thymus, Lung, Bone marrow
- Foxp3 was highly expressed in regulatory T cells (Treg cells) of lymphoid organs or tissues, whereas Foxp3 was not expressed in immune cells isolated from lymphoid organs (see Example 1).
- monocyte-derived myeloid-derived suppressor cells M-MDSCs
- fxDC monocyte-derived myeloid-derived suppressor cells
- CD8 + regulatory T cells CD8 + Tregs
- the dendritic cells expressing Foxp3 may be used as an active ingredient of a pharmaceutical composition for regulating cellular immune and / or preventing or treating inflammatory diseases. Suggests that it can (see Example 6).
- composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. It may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions according to conventional methods.
- Carriers, excipients and diluents which may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.
- diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
- the pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount.
- pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts.
- the pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
- the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general It may be administered in an amount of 0.1 mg / kg to 100 mg / kg, preferably in an amount of 1 to 30 mg / kg per day, it may be administered once or several times a day.
- the pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, by subcutaneous, intravenous, intramuscular or intrauterine dural or cerebrovascular injections.
- the pharmaceutical composition of the present invention is determined according to the type of drug that is the active ingredient, along with various related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient and the severity of the disease.
- the invention provides a method of treating an inflammatory disease comprising administering the pharmaceutical composition to a subject.
- subject means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses and cattle, etc. Mean mammal.
- the present invention comprises the steps of separating the myeloid-derived suppressor cells (MDSC) present in the blood; And culturing the isolated bone marrow-derived suppressor cells in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF). to provide.
- MDSC myeloid-derived suppressor cells
- GM-CSF granulocyte-macrophage colony-stimulating factor
- CD11c + Foxp3 + dendritic cells in various lymphoid organs and mucosal tissues (Spleen, inguinal lymph node, mesenteric lymph node, peyer's patch, thymus, lung, bone marrow) in the mouse were analyzed by FACS (Fluorescence activated cell sorter). .
- Foxp3 was highly expressed in regulatory T cells of lymphoid organs or tissues, whereas Foxp3 was not expressed in immune cells isolated from lymphoid organs.
- Foxp3 was reported to be expressed only in CD4 + regulatory T cells, and reanalysis by CD3 reverse gating confirmed that Foxp3 + expressing dendritic cells were not caused by contamination of Foxp3 + regulatory T cells (FIG. 2A).
- Dendritic cells present in the blood are composed of plasmacytoid DC (pDC) directly differentiated from common DC precursor (CDP) and pre-conventional DC (pre-cDC) in the form of progenitor cells.
- pDC plasmacytoid DC
- pre-cDC pre-conventional DC
- dendritic cells in the blood may be either pDCs differentiated directly from CDP, or pre-DCs of differentiation stages.
- the present inventors examined pDC specific surface antigen expression to determine whether the fxDC is derived from pDC.
- the cells gated with B220 + did not express GFP (Foxp3) in any case irrespective of the expression of CD11c, and even when gated with CD11c, B220 + cells expressed GFP (Foxp3). It was confirmed not to.
- macrophages have M1, M2, and TAM (Tumor associated macrophage) that induce or inhibit immunity to the same antigen presenting cells as dendritic cells.
- TAM Tumor associated macrophage
- dendritic cells (Foxp3 + dendritic cells) expressing Foxp3 were named fxDC.
- Blood cDCs are activated by capturing and breaking down invading pathogens or foreign antigens, and migrate to the spleen or local lymph nodes to deliver antigens to T cells, thereby activating acquired immunity.
- the present inventors expect fxDC to play an important role in intractable immune disease, and therefore, fxDC in the blood of acute infection model (LCMV Arm), and chronic infection model (LCMV C13) using lymphocytic chriomeningitis virus (LCMV). Population changes were investigated.
- fxDC Foxp3 + dendritic cells
- PBMC peripheral blood mononuclear cells
- CD11c bead were filled in the upper chambers of a 24-well transwell plate (8 ⁇ M pore size polycarbonate filter, Corning Costar, Cambridge, Mass.) And 0.6 ml in the lower chambers.
- PBMC peripheral blood mononuclear cells
- the trans well plate was left in a CO 2 incubator at 37 ° C. for 3 hours. Foxp3 + cells (Foxp3-GFP) migrated to the upper chamber and the lower chamber were analyzed by FACS.
- Example 2 it was confirmed that the fxDC of the present invention was not derived from plasmacytoid DC (pDC), and it was intended to confirm whether it is derived from CD11c + pre-conventional DC (pre-cDC). Specifically, mouse blood PBMCs were divided into CD11c + cells and CD11c ⁇ cells, followed by further differentiation with GM-CSF for one day, and the expression of each Foxp3 (GFP) was examined.
- pDC plasmacytoid DC
- pre-cDC pre-conventional DC
- the fxDC of the present invention is not derived from pre-cDC, that is, DC precursor cells, but differentiated into CD11c + fxDC in CD11c (-) precursor cells. In other words, fxDC progenitor cells are present in the CD11c- cell population.
- the present inventors confirmed that the fxDC population increased significantly in the blood of the existing tumor model, and fxDC was not derived from the existing dendritic cell progenitor cells (pre-cDC) through the above experiments.
- Myeloid derived suppressor cells (MDSCs), on the other hand, have two main subtypes: CD11b + Ly6G + Ly6C low granulocytic MDSCs (G-MDSCs), and mononuclear myeloid suppressor cells (MDSCs).
- M-MDSCs CD11b + Ly6G - Ly6C high monocytic MDSCs
- TAM tumor-associated macrophage
- GM-CSF dendritic cells involved in immunity by GM-CSF
- GM-CSF treatment is an essential factor in the differentiation of DCs from blood progenitor cells
- dendritic cells in GM-CSF medium for 3 days were treated with MDSCs subgroups (DP-MDSC, G-MDSC, M-MSDC, DN cells) isolated from blood. After differentiation, the expression of Foxp3 (GFP) in subdivision differentiated cells was examined. In addition, MDSCs subgroups were labeled with CFSF prior to the incubation process, and FACS was confirmed for cell proliferation during differentiation.
- MDSCs subgroups DP-MDSC, G-MDSC, M-MSDC, DN cells
- fxDC of the present invention was differentiated into fxDC by GM-CSF secreted in an inflammatory environment, a part of M-MDSC present in a large amount in blood.
- T cell proliferation was confirmed by co-culture of fxDC differentiated from T cells and M-MDSC.
- CD4 + Tregs CD4 + regulatory T cells
- CD8 + Tregs CD8 + regulatory T cells
- M-MDSC a precursor of G-MDSC or fxDC
- mDC mature BMDC
- DP-MDSC or fxDC Confirmed little induction of T cell proliferation
- T cells activated / proliferated by CD3 / CD28 were co-cultured with fxDC
- CD4 + T cell proliferation was not inhibited at all, whereas only CD8 + T cells selectively inhibited proliferation, and Foxp3 / DTR mice were suppressed.
- fxDC was removed from blood DC, it was confirmed that the proliferation of CD8 + T cells was recovered (FIG. 8).
- fxDC directly contacts T cells to regulate T cell immunity (FIG. 9).
- MDSC subgroups induce CD4 + regulatory T cell (CD4 + Tregs) proliferation to regulate T cell activity, whereas fxDC significantly increases CD8 + regulatory T cell (CD8 + Tregs) population and CD4 + regulatory T (CD4 + Tregs) at all. It was confirmed that no adjustment was made (FIG. 10).
- fxDC strongly regulates the proliferative activity of CD8 + effector T cells (CTL) antigen-specifically through the proliferation of CD8 + Tregs.
- the composition according to the present invention comprises a dendritic cell (fxDC) expressing Foxp3 as an active ingredient
- fxDC dendritic cell
- the fxDC is a key immune cell involved in the immune homeostasis of the blood itself, the number of individuals in an imbalanced state such as an infectious disease It is possible to strongly inhibit the proliferation of effector T cells in the blood through the regulation of CD8 + regulatory T cell (CD8 + Treg) proliferation, which is useful as a pharmaceutical composition for the prevention or treatment of chronic inflammatory immune diseases. It is expected to be able to be used.
- the reduction of CD8 + Treg cells is one of the characteristics of immune senescence, and in the elderly, the CD8 + Treg cells decrease the immune homeostasis and increase the number of patients with chronic inflammatory diseases or autoimmune diseases. No cause of decline was found.
- fxDC in the blood is a causative immune cell that induces the proliferation of CD8 + Tregs.
- dendritic cells expressing Foxp3 can be used as a pharmaceutical composition for the treatment of immune aging.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a pharmaceutical composition containing dendritic cells expressing Foxp3 for regulating immunity. The present invention has confirmed the efficacy, in a state of immune imbalance, of using Foxp3+ dendritic cells (fxDC) present only in the blood to inhibit proliferation of T cells and restore and maintain immunological homeostasis by activating CD8+ regulatory T cells (CD8+ Tregs), and as such, the present invention is anticipated to allow, with respect to preventing and treating immune disorders, a targeted treatment by employing a more fundamental approach.
Description
본 발명은 면역 조절용 약학적 조성물에 관한 것으로서, 보다 구체적으로는 Foxp3를 발현하는 수지상 세포를 포함하는 면역 조절용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for immunomodulation, More specifically, the present invention relates to a pharmaceutical composition for immunomodulation comprising dendritic cells expressing Foxp3.
박테리아, 바이러스, 독소, 암세포, 다른 사람 혹은 동물의 혈액, 및 조직과 같은 외부 물질인 항원으로부터 신체를 보호하기 위하여 면역반응이 일어난다. 이러한 면역계는 크게 면역을 억제, 조절하는 메카니즘인 면역관용 (tolerance)과 면역을 증진하는 면역반응 (immunity)으로 구성되며, 면역계는 이러한 두 가지 면역 작용이 적절한 균형을 이룸으로써, 면역 항상성을 유지하고 있다. 이렇게 유지되는 면역학적 균형은 다양한 원인에 의해 불균형이 초래될 수 있으며, 이러한 불균형은 결국, 다양한 질병을 발생시킨다. 면역관용 기능이 면역반응에 비해 상대적으로 강하거나 이와 반대로 면역반응 기능이 면역관용에 비해 강해질 경우, 면역학적 불균형이 초래될 수 있다. 예를 들어, 면역반응에 비해 면역관용이 강해질 경우, 인체 면역계는 암의 발생이나 외부 바이러스, 및 병원성 세균 등의 침입이 용이하게 되어 암 또는 바이러스 및 세균성 질환을 발생시키게 되며, 이와 반대로 면역반응이 면역관용보다 강해질 경우, 자가 면역질환, 강력한 이식 거부 반응, 및 알레르기성 질환과 같은 염증성 질환을 초래하게 된다. 따라서 면역학적 관점에서 바라본 질병은 면역 시스템 항상성의 불균형에 의해 나타나는 결과물이며, 이러한 불균형의 조절을 통해 질병의 치료가 가능하다고 할 수 있다. An immune response occurs to protect the body from antigens that are foreign substances such as bacteria, viruses, toxins, cancer cells, blood of other people or animals, and tissues. The immune system consists of immune tolerance, which is a mechanism for suppressing and regulating immunity, and an immune response that promotes immunity. The immune system maintains immune homeostasis by properly balancing these two immune actions. have. This maintained immunological balance can lead to imbalances for a variety of causes, which eventually lead to various diseases. Immunological imbalance can result if the immune tolerant function is stronger than the immune response or vice versa. For example, when immune tolerance is stronger than that of the immune response, the human immune system may easily develop cancer, invade foreign viruses, and pathogenic bacteria, and may cause cancer or viral and bacterial diseases. Stronger immune tolerance leads to inflammatory diseases such as autoimmune diseases, potent transplant rejection, and allergic diseases. Therefore, the disease seen from the immunological point of view is a result of imbalance of immune system homeostasis, and it can be said that the treatment of the disease is possible through the regulation of this imbalance.
일례로서, 현재 과도한 면역반응에 의한 질환의 치료방법으로, 면역억제제를 단독 또는 병용 투여함으로써 상기 질환에 의해 야기되는 각종 증상을 완화 내지 감소시키는 방법이 이용되고 있다. 면역억제제란 항원의 작용에 대하여 숙주가 항체를 만드는 능력 (체액성 면역반응) 또는 세포성 면역반응을 일으키는 능력을 저하시키거나 차단하기 위해 사용되는 다양한 물질들을 일컫는 것으로서, 이러한 면역억제제는 장기 이식뿐만 아니라 루푸스, 류머티스성 관절염 등과 같은 자가면역질환, 아토피, 알레르기 등의 피부 과민반응에도 유용하게 사용되고 있다 (국내공개특허 10-2015-0026839 참조). 다만 현재 사용되고 있는 면역억제제인 사이클로스포린 A, FK506 등은 복잡한 화학구조를 가진 천연물 유래의 화합물로서 원료 수급 측면에서 고비용이므로 비경제적이고, 장기 투여로 인해 각종 부작용이 야기될 수 있다는 위험성을 내포하고 있다. 따라서 낮은 독성을 나타내며, 경제적인 생산이 가능한 새로운 면역 조절 물질의 개발이 절실히 요구되고 있는 실정이다.As one example, as a method for treating a disease caused by an excessive immune response, a method of alleviating or reducing various symptoms caused by the disease by using an immunosuppressant alone or in combination is used. Immunosuppressants refer to a variety of substances that are used to reduce or block the host's ability to produce antibodies (humidary immune response) or cellular immune response to the action of an antigen. In addition, it is also usefully used for skin hypersensitivity reactions, such as autoimmune diseases such as lupus, rheumatoid arthritis, atopy, allergy (see Korean Patent Publication No. 10-2015-0026839). However, currently used immunosuppressive agents such as cyclosporin A and FK506 are compounds derived from natural products having a complicated chemical structure and are expensive in terms of supply and demand of raw materials, and thus are inexpensive and carry a risk that various side effects may be caused by long-term administration. Therefore, there is an urgent need for the development of new immunomodulators that exhibit low toxicity and are capable of economic production.
한편, 수지상 세포 (Dendritic Cell, DC)는 기원, 형태, 표현형, 기능, 성숙화 과정 등에 따라 다양한 특성을 지니게 되며, 특히, 상황에 따라 T 세포 반응을 촉진 또는 억제할 수 있는바, 체내 면역의 항상성을 조절하는 중요한 역할을 한다. 종래 수지상 세포의 기초 연구는, 마우스 골수세포를 인위적으로 분화시켜 만든 골수유래 수지상 세포 (BMDC)를 사용하거나, 마우스 비장 (spleen), 림프절 등 2차 림프조직에 상주하는 DC를 분리하여 수행하였다. 혈액 내 수지상 세포 연구는 그 중요성에도 불구하고, 마우스에서는, 그 양적 한계와 주위 림프절에 머물던 미성숙 수지상 세포가 혈액으로 흘러 들어온 것으로 간주되어져 별다른 주목을 받지 못하고 있다.On the other hand, dendritic cells (Dendritic Cell, DC) has a variety of characteristics depending on the origin, form, phenotype, function, maturation process, etc., in particular, can promote or inhibit the T cell response depending on the situation, the homeostasis of the immune system Plays an important role in regulating The basic study of conventional dendritic cells was performed by using bone marrow-derived dendritic cells (BMDC) made by artificially differentiating mouse bone marrow cells or by separating DCs residing in secondary lymphoid tissues such as mouse spleen and lymph nodes. Despite the importance of dendritic cell research in the blood, in mice, immature dendritic cells that have stayed in the quantitative limits and surrounding lymph nodes are considered to have flowed into the blood and have received little attention.
이에, 본 발명자들은 혈액에만 존재하는 Foxp3를 발현하는 수지상 세포를 최초로 발견하였으며, 이들의 면역 조절 및 면역 항상성 유지 효과를 규명하여 면역 불균형에 따른 질환 치료제 개발의 기초 자료를 제공하고자 하였다.Therefore, the present inventors have found for the first time the dendritic cells expressing Foxp3 present only in the blood, and tried to provide the basic data for the development of disease treatments according to immune imbalance by identifying their immune regulation and immune homeostasis maintenance effect.
본 발명은 상기와 같은 종래의 문제점을 해결하기 위해 안출된 것으로서, 본 발명자들은 혈액 내 존재하는, Foxp3를 발현하는 수지상 세포의 면역 항상성 유지 효과를 확인하고 이에 기초하여 본 발명을 완성하게 되었다.The present invention has been made to solve the above-mentioned conventional problems, the present inventors have confirmed the immune homeostasis maintenance effect of the dendritic cells expressing Foxp3 present in the blood, and completed the present invention based on this.
이에, 본 발명의 목적은 Foxp3를 발현하는 수지상 세포를 유효성분으로 포함하는, 면역 조절용 약학적 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for immunomodulation comprising dendritic cells expressing Foxp3 as an active ingredient.
또한, 본 발명의 다른 목적은 Foxp3를 발현하는 수지상 세포를 유효성분으로 포함하는, 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다. In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases, including dendritic cells expressing Foxp3 as an active ingredient.
또한, 본 발명의 또 다른 목적은 혈액 내 존재하는 골수유래억제세포 (myeloid-derived suppressor cell; MDSC)를 분리하는 단계; 및 상기 분리한 골수유래억제세포를 과립구-대식세포 콜로니 자극인자 (Granulocyte-macrophage colony-stimulating factor; GM-CSF)가 포함된 배지에서 배양하는 단계를 포함하는, Foxp3를 발현하는 수지상 세포의 제조방법을 제공하는 것이다.In addition, another object of the present invention is to isolate the myeloid-derived suppressor cells (MDSC) present in the blood; And culturing the isolated bone marrow-derived suppressor cells in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF). To provide.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 Foxp3를 발현하는 수지상 세포를 유효성분으로 포함하는, 면역 조절용 약학적 조성물을 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for immunomodulation, comprising dendritic cells expressing Foxp3 as an active ingredient.
본 발명의 일 구현예로서, 상기 Foxp3는 서열번호 1로 기재된 아미노산 서열로 이루어질 수 있다.In one embodiment of the present invention, Foxp3 may be composed of the amino acid sequence set forth in SEQ ID NO: 1.
본 발명의 다른 구현예로서, 상기 수지상 세포는 혈액에서 분리된 것일 수 있다. In another embodiment of the present invention, the dendritic cells may be isolated from blood.
본 발명의 또 다른 구현예로서, 상기 조성물은 혈액 내 면역 항상성을 조절할 수 있다. In another embodiment of the invention, the composition can modulate immune homeostasis in the blood.
본 발명의 또 다른 구현예로서, 상기 조성물은 CD8+ 조절 T 세포 (CD8+ Treg)를 증식시킬 수 있다.In another embodiment of the present invention, the composition can propagate CD8 + regulatory T cells (CD8 + Tregs).
본 발명은 Foxp3를 발현하는 수지상 세포를 유효성분으로 포함하는, 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공한다. The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, including dendritic cells expressing Foxp3 as an active ingredient.
본 발명은 혈액 내 존재하는 골수유래억제세포 (myeloid-derived suppressor cell; MDSC)를 분리하는 단계; 및 상기 분리한 골수유래억제세포를 GM-CSF가 포함된 배지에서 배양하는 단계를 포함하는, Foxp3를 발현하는 수지상 세포의 제조방법을 제공한다. The present invention comprises the steps of separating the myeloid-derived suppressor cells (MDSC) present in the blood; And it provides a method for producing dendritic cells expressing Foxp3 comprising the step of culturing the isolated bone marrow-derived suppressor cells in a medium containing GM-CSF.
본 발명의 일 구현 예로서, 상기 골수유래억제세포는 단핵구성 골수유래억제세포 (Monocytic myeloid-derived suppressor cell; M-MDSC)일 수 있다. In one embodiment of the present invention, the myeloid-derived suppressor cells may be monocytic myeloid-derived suppressor cells (M-MDSC).
본 발명은 상기 약학적 조성물을 개체에 투여하는 단계를 포함하는 염증성 질환의 치료방법을 제공한다.The present invention provides a method for treating an inflammatory disease comprising administering the pharmaceutical composition to a subject.
본 발명은 염증성 질환의 치료제 제조를 위한 Foxp3를 발현하는 수지상 세포의 신규한 용도를 제공한다.The present invention provides a novel use of dendritic cells expressing Foxp3 for the preparation of a therapeutic agent for an inflammatory disease.
본 발명에 따른 조성물은 Foxp3를 발현하는 수지상 세포 (fxDC)를 유효성분으로 포함하며, 상기 fxDC는 혈액 자체의 면역 항상성에 관여하는 핵심적인 면역세포로서, 감염성 질환과 같은 면역 불균형 상태에서 그 개체수가 증가하고, CD8+ 조절 T 세포 (CD8+ Treg) 증식의 조절을 통해 혈액 내 활성 T (effector T) 세포의 증식을 강력하게 억제시킬 수 있는바, 만성 염증성 면역 질환의 예방 또는 치료를 위한 약학 조성물로 유용하게 사용될 수 있을 것으로 기대된다. 특히, CD8+ Treg 세포 감소는 면역 노화 (immune senescence)의 특징 중의 하나로, 노령층은 CD8+ Treg 세포 감소로 인해 면역 항상성이 조절이 되지 않아 만성 염증성 질환이나 자가면역질환 환자가 증가하게 되는데 아직까지 CD8+ Treg의 감소 원인을 찾지 못하였다. 그러나 본 발명에 의하면 혈중 fxDC가 CD8+ Treg의 증식을 유도하는 원인 면역세포임을 밝혀 Foxp3를 발현하는 수지상 세포를 면역 노화의 치료를 위한 약학적 조성물로 사용될 수 있음을 밝혔다.The composition according to the present invention comprises a dendritic cell (fxDC) expressing Foxp3 as an active ingredient, the fxDC is a key immune cell involved in the immune homeostasis of the blood itself, the number of individuals in an imbalanced state such as an infectious disease It is possible to strongly inhibit the proliferation of effector T cells in the blood through the regulation of CD8 + regulatory T cell (CD8 + Treg) proliferation, which is useful as a pharmaceutical composition for the prevention or treatment of chronic inflammatory immune diseases. It is expected to be able to be used. In particular, the reduction of CD8 + Treg cells is one of the characteristics of immune senescence, and in the elderly, the CD8 + Treg cells decrease the immune homeostasis and increase the number of patients with chronic inflammatory diseases or autoimmune diseases. No cause of decline was found. However, according to the present invention, it was revealed that fxDC in the blood is a causative immune cell that induces the proliferation of CD8 + Tregs. Thus, dendritic cells expressing Foxp3 can be used as a pharmaceutical composition for the treatment of immune aging.
도 1은 림프기관에 상주하는 면역세포들 중 Foxp3를 발현하는 수지상 세포 (fxDC)의 존재를 FACS로 분석한 결과이다. 1 shows the results of FACS analysis of the presence of dendritic cells (fxDC) expressing Foxp3 among immune cells residing in lymphoid organs.
도 2a는 마우스 말초혈액 단핵구세포 (mPBMC)에서, fxDC의 존재를 T 세포 마커 (CD3)로 reverse gating하여 재분석한 결과이다.Figure 2a is the result of re-analysis by reverse gating the presence of fxDC with a T cell marker (CD3) in mouse peripheral blood mononuclear cells (mPBMC).
도 2b는 plasmacytoid DC(pDC) 특이적 표면 항원 (CD11c, 및 B220)을 이용하여, fxDC가 pDC가 아님을 확인한 결과이다.Figure 2b is a result confirming that fxDC is not pDC using plasmacytoid DC (pDC) specific surface antigen (CD11c, and B220).
도 2c는 말초혈액에 존재하는 fxDC에서 Macrophage (대식세포) 특이적 표면항원 (CD11b, CD14, F4/80 및 CD64)의 발현을 조사하여 fxDC가 단핵구나 대식세포가 아님을 확인한 결과이다. Figure 2c is a result of investigating the expression of Macrophage (macrophage) specific surface antigens (CD11b, CD14, F4 / 80 and CD64) in fxDC present in peripheral blood to confirm that fxDC is not monocytes or macrophages.
도 3은 정상 상태와 급성 (LCMV armstrong) 및 만성 (LCML clone13) 바이러스 감염 마우스 혈액에서 fxDC의 존재 및 개체 수의 변화를 확인한 결과이다. Figure 3 shows the results of the change in the presence and population of fxDC in the normal and acute (LCMV armstrong) and chronic (LCML clone13) virus infected mouse blood.
도 4는 fxDC의 CCL19에 대한 이동능을 transwell 실험으로 분석한 후, FACS로 확인한 결과이다. Figure 4 shows the results confirmed by FACS after analyzing the mobility of the fxDC CCL19 by transwell experiments.
도 5는 fxDC의 전구세포를 알아보기 위해, mPBMC를 CD11c+ 세포군과 CD11c- 세포군으로 나눈 후, 각 군에서 fxDC로의 분화를 확인한 결과이다.5 is a result of dividing mPBMC into CD11c + cell group and CD11c- cell group in order to identify progenitor cells of fxDC, and confirming the differentiation into fxDC in each group.
도 6은 MDSCs 아군 (DP-MDSC, G-MDSC, M-MDSC, 및 DN cell)을 GM-CSF 존재하에서 3일 동안 배양한 후, fxDC로의 분화 여부를 FACS 분석한 결과이다. Figure 6 shows the results of FACS analysis of differentiation of fxDC after culturing MDSCs subgroups (DP-MDSC, G-MDSC, M-MDSC, and DN cells) in the presence of GM-CSF for 3 days.
도 7은 CFSF로 표지된 동형 (syngeneic) T 세포와 mDC, G-MDSC, M-MDSC, DP-MDSC, 및 fxDC를 3일간 공배양하고, T 세포의 증식 정도를 확인한 결과이다. 7 is a result of co-culture of CFSF-labeled syngeneic T cells with mDC, G-MDSC, M-MDSC, DP-MDSC, and fxDC for 3 days to confirm the proliferation of T cells.
도 8은 Foxp3-GFP 마우스 T 세포에 CD3 항체 및 CD28 항체를 처리하여 활성화시킨 후, mDC, G-MDSS, M-MDSC, DP-MDSC, 또는 fxDC와 공배양한 경우, 생성되는 CD4+ 조절 T 세포 (CD4+ Treg) 및 CD8+ 조절 T 세포 (CD8+ Treg)를 확인한 결과이다.FIG. 8 shows CD4 + regulatory T cells that are generated when Foxp3-GFP mouse T cells are treated with CD3 and CD28 antibodies and co-cultured with mDC, G-MDSS, M-MDSC, DP-MDSC, or fxDC. (CD4 + Tregs) and CD8 + regulatory T cells (CD8 + Tregs).
도 9는 정상 마우스 T 세포에 CD3 항체 및 CD28 항체를 처리하여 활성화시킨 후 fxDC와 접촉성 공배양과 Transwell plate (세포결합 차단)를 이용한 공배양을 통해 fxDC의 CD8+T세포의 증식억제가 사이토카인에 의한 것이 아니라 직접 접촉에 기인한 것임을 확인한 결과이다. FIG. 9 shows the inhibition of proliferation of CD8 + T cells of fxDC by fxDC and coculture with fxDC and coculture with a transwell plate (cell binding block) after activation and activation of CD3 and CD28 antibodies to normal mouse T cells. The results were confirmed to be due to direct contact and not by cain.
도 10은 Foxp3-GFP 마우스 T 세포에 CD3 항체 및 CD28 항체를 처리하여 활성화시킨 후, mDC, G-MDSS, M-MDSC, DP-MDSC, 또는 fxDC와 공배양한 경우, 생성되는 CD4+ 조절 T 세포 (CD4+ Treg) 및 CD8+ 조절 T 세포 (CD8+ Treg)를 확인한 결과이다. FIG. 10 shows CD4 + regulatory T cells generated when Foxp3-GFP mouse T cells are treated with CD3 and CD28 antibodies and then co-cultured with mDC, G-MDSS, M-MDSC, DP-MDSC, or fxDC. (CD4 + Tregs) and CD8 + regulatory T cells (CD8 + Tregs).
본 발명자들은, 림프기관 (lymphoid organ) 또는 다른 조직과 달리, 혈액에서, Foxp3+ 수지상 세포 (fxDC)의 존재를 확인하였다. 또한, 만성 감염 질환 모델에서의 fxDC의 개체수 증가를 확인하였으며, 림프절로의 이동능 분석에서, fxDC는 림프절로 이동능이 없음을 확인하였다. 또한, fxDC 전구세포를 밝히는 과정에서, 기존 cDC와는 달리 fxDC는 CD11c- 세포군에서 분화됨을 확인하였으며, 한 단계 더 나아가, 골수유래억제세포 (MDSC) 아군을 이용한 분화 연구를 통해 혈중 단핵구성 골수유래억제세포 (M-MDSC)로부터 fxDC가 분화됨을 규명하였다. 아울러, 혈액 fxDC는 다른 면역 조절 DC와 달리, CD4+ 조절 T 세포 (CD4+ Treg) 가 아닌 CD8+ 조절 T 세포 (CD8+ Treg)의 증식을 유도하여 T 세포 활성을 조절한다는 새로운 사실을 확인하고 본 발명을 완성하였다.We have confirmed the presence of Foxp3 + dendritic cells (fxDC) in the blood, unlike lymphoid organs or other tissues. In addition, the increase in the number of fxDC in the chronic infection disease model was confirmed, and in the analysis of the mobility to lymph nodes, it was confirmed that fxDC has no mobility to lymph nodes. In addition, in the process of identifying fxDC progenitor cells, unlike conventional cDC, it was confirmed that fxDC is differentiated in CD11c-cell population. Furthermore, furthermore, fxDC progenitor myeloid-derived suppression of blood mononuclear bone marrow derived through a differentiation study using bone marrow-derived suppressor cells (MDSC) The fxDCs were differentiated from the cells (M-MDSCs). In addition, blood fxDC, unlike other immunoregulatory DCs, confirms the novel fact that it induces proliferation of CD8 + regulatory T cells (CD8 + Tregs) rather than CD4 + regulatory T cells (CD4 + Tregs) to regulate T cell activity and completes the present invention It was.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 Foxp3를 발현하는 수지상 세포를 유효성분으로 포함하는, 면역 조절용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for immunomodulation comprising dendritic cells expressing Foxp3 as an active ingredient.
종래 Foxp3 단백질은 조절 T 세포 (Treg)에서만 특이적으로 발현되는 것으로 알려졌으나, 혈중 Foxp3를 발현하는 수지상 세포 (fxDC)가 면역 항상성 유지에 핵심적으로 관여하고 있다는 점에 대해서는 보고된 바가 없다. 상기 Foxp3 단백질은 서열번호 1로 기재되는 아미노산 서열을 가지는 것이 바람직하나, 이에 제한되는 것은 아니고, 상기 아미노산 서열과 70% 이상, 바람직하게는 80% 이상, 보다 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 상동성을 갖는 아미노산 서열로 표시되는 단백질을 포함할 수 있다. 또한, 본 발명에서, 수지상 세포 (Dendritic Cell, DC)는 항원 특이적인 적응 면역반응을 개시하는 중요한 항원 제시 세포 (APC) 중 하나로서, 인간 또는 마우스 유래일 수 있으며, 바람직하게는 인간 혈액으로부터 수지상 세포를 분리하거나, 혈액에서 분리된 단핵구로부터 수지상 세포로 분화시킬 수 있으나, 이에 제한되는 것은 아니다. It is known that Foxp3 protein is specifically expressed only in regulatory T cells (Treg), but it has not been reported that dendritic cells expressing Foxp3 (fxDC) in blood are critically involved in maintaining immune homeostasis. The Foxp3 protein preferably has an amino acid sequence as set forth in SEQ ID NO: 1, but is not limited thereto, and 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 70% or more of the amino acid sequence. May comprise a protein represented by an amino acid sequence having at least 95% homology. In addition, in the present invention, dendritic cells (Dendritic Cells, DC) is one of the important antigen presenting cells (APC) that initiates an antigen specific adaptive immune response, which may be derived from human or mouse, preferably dendritic from human blood Cells may be isolated or differentiated from monocytes isolated from blood into dendritic cells, but are not limited thereto.
또한, 본 발명에 따른 Foxp3를 발현하는 수지상세포 (fxDC)는 혈액 자체의 면역 항상성에 관여하는 핵심적인 면역세포로서, 정상 상태에서는 존재하지 않으며, 면역 불균형 상태에서만 새롭게 생성되어, CD8+ 조절 T 세포 (CD8+ Treg) 증식의 조절을 통해 혈액 내 활성 T (effector T) 세포의 증식을 강력하게 억제시킬 수 있는바, 염증성 질환의 예방 또는 치료를 위한 약학 조성물로 유용하게 사용될 수 있을 것으로 기대된다.In addition, dendritic cells (fxDC) expressing Foxp3 according to the present invention are essential immune cells involved in the immune homeostasis of the blood itself, and do not exist in a normal state, but are newly generated only in an imbalanced state, thereby generating CD8 + regulatory T cells ( CD8 + Tregs) can be strongly inhibited the proliferation of effector T cells in the blood through the regulation of proliferation, it is expected to be useful as a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
한편, 지금까지 체내에서 Foxp3를 발현하는 수지상세포는 보고된 적이 없지만, 이전 논문에서 정상 수지상세포에 Foxp3 유전자를 도입하여 인위적으로 Foxp3를 발현하는 수지상세포를 만들고 그 기능을 조사한 연구가 보고되었다 (Lipscomb et al., Eur J Immunol. 2010 February ; 40(2): 480493). 이들에 따르면, 인위적으로 Foxp3를 발현시킨 수지상세포는 T 세포와 공배양 시 CD4 Treg을 유도하고 모든 T 세포 활성을 억제한다고 밝히고 있으나, 본 발명자가 체내에서 발견한 Foxp3 발현 수지상세포의 경우, T 세포와 공배양시 CD8 Treg을 유도하고 CD4 Treg은 유도하지 않으며 CD8 T 세포활성만 특이적으로 억제하는 것으로 나타나는바, 기존 재조합 FoxP3 발현 수지상세포와는 특성이 다른 것으로 확인되었다. 또한, 재조합 Foxp3 발현 수지상세포는 anti-TGF-β 항체를 처리하면 면역 억제 활성이 사라지는 것으로 보아, 사이토카인에 의해 면역억제활성을 나타내지만 체내 Foxp3 발현 수지상세포나 in vitro 분화를 통해 재조한 Foxp3 발현 수지상세포는 사이토카인에 의한 간접적인 면역 억제가 아니라 직접적인 접촉에 의해 T 세포면역을 억제하는 특성을 보여 재조합 수지상세포와는 다른 것으로 확인되었다.In the meantime, there have been no reports of dendritic cells expressing Foxp3 in the body, but previous studies have reported the introduction of Foxp3 gene into normal dendritic cells to create dendritic cells expressing Foxp3 artificially and investigated their function (Lipscomb). et al., Eur J Immunol. 2010 February; 40 (2): 480493). According to these studies, dendritic cells expressing Foxp3 artificially induced CD4 Tregs and co-cultured with T cells, but inhibited all T cell activity. However, in the case of Foxp3 expressing dendritic cells found in the body, T cells In co-culture, CD8 Tregs are induced, CD4 Tregs are not induced, and CD8 T cell activity is specifically inhibited, which is different from the existing recombinant FoxP3 expressing dendritic cells. In addition, the recombinant Foxp3 expressing dendritic cells treated with the anti-TGF-β antibody disappeared the immunosuppressive activity, and showed immunosuppressive activity by cytokines, but Foxp3 expressing dendritic cells in the body or prepared through in vitro differentiation. Dendritic cells were found to be different from recombinant dendritic cells because they exhibited the property of inhibiting T cell immunity by direct contact rather than indirect immune suppression by cytokines.
또한, 본 발명에서 사용되는 용어, "면역 조절"이란, 혈액 내 면역 불균형을 해소하고 면역 항상성을 유지하는 것을 의미한다. 면역 항상성 유지는 면역을 억제시키는 메카니즘인 면역관용 (tolerance)과 면역을 증진하는 면역반응 (immunity)간 균형을 이룬 상태를 일컫는 것으로, 이러한 상태의 유지는 면역 질환 치료, 특히 자가면역 질환의 치료에 있어서 필수적인 요소이다. In addition, the term "immunoregulation" used in the present invention means to alleviate immune imbalance in the blood and maintain immune homeostasis. Maintaining immune homeostasis refers to a condition in which a balance between immune tolerance and immune response-immunity-promoting mechanisms is maintained. Is an essential element.
종래 혈액 내 이러한 항상성은 대부분 주위 림프 조직에서 담당하는 것으로 알려져 있는바, 현재, 혈액 자체의 항상성 유지 기전에 대한 연구는 전무한 실정이다. 이에, 본 발명은 혈액 내 면역 불균형이 초래되면 혈액 내 존재하는 fxDC가 증가되고, 이들이 CD8+ 조절 T 세포 (CD8+ Treg) 증식을 유도하여, 혈액 내 면역 항상성을 유지한다는 점을 처음으로 밝힌 점에 기술적 특징이 있다.It is known that such homeostasis in the blood is mostly responsible for surrounding lymphoid tissues. Currently, there is no research on the mechanism of maintaining the homeostasis of the blood itself. Thus, the present invention is the first to reveal that when the imbalance in the blood caused fxDC present in the blood increases, they induce CD8 + regulatory T cells (CD8 + Tregs) proliferation, maintaining immune homeostasis in the blood There is a characteristic.
또한, 본 발명은 Foxp3를 발현하는 수지상 세포를 유효성분으로 포함하는, 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, including dendritic cells expressing Foxp3 as an active ingredient.
본 발명의 예방 또는 치료 대상 질병인 "염증성 질환"은 염증을 주 병변으로 하는 질병을 총칭하는 것으로서, 부종, 알레르기, 천식, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 위염, 크론병, 대장염, 치질, 통풍, 강직성 척추염, 류마티스성 열, 루푸스, 섬유근통 (fibromyalgia), 건선 관절염, 골관절염, 류마티스성 관절염, 견관절주위염, 건염, 건초염, 근육염, 간염, 방광염, 신장염, 쇼그렌 증후군 (sjogren's syndrome) 및 다발성 경화증으로 구성된 군으로부터 선택되는 어느 하나에 해당할 수 있으나, 이에 제한되는 것은 아니다."Inflammatory disease", which is a disease to be prevented or treated according to the present invention, refers to a disease that is mainly a inflammation, swelling, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome ( sjogren's syndrome) and multiple sclerosis, but may be any one selected from, but is not limited thereto.
본 발명에서 사용되는 용어, "예방"은 본 발명에 따른 약학적 조성물의 투여에 의해 염증성 질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" means any action that inhibits or delays the development of an inflammatory disease by administration of a pharmaceutical composition according to the present invention.
본 발명에서 사용되는 용어, "치료"는 본 발명에 따른 약학적 조성물의 투여에 의해 염증성 질환에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "treatment" means any action in which symptoms for inflammatory diseases are improved or beneficially altered by administration of the pharmaceutical composition according to the invention.
본 발명의 일 실시예에서는 마우스 체내 여러 림프기관 (Spleen, inguinal lymph node, mesenteric lymph node, peyer's patch and thymus, Lung, Bone marrow) 및 혈액에서 CD11c+ Foxp3+ 수지상 세포 (fxDC)의 존재 여부를 확인한 결과, Foxp3는 대부분 림프기관이나 조직의 조절 T 세포 (Treg cell)에서 높게 발현된 반면, 림프기관들에서 분리된 면역세포에서는 Foxp3를 발현하지 않음을 확인하였다 (실시예 1 참조). In an embodiment of the present invention, as a result of confirming the presence of CD11c + Foxp3 + dendritic cells (fxDC) in various lymphoid organs (Spleen, inguinal lymph node, mesenteric lymph node, peyer's patch and thymus, Lung, Bone marrow) and blood in the mouse body, Foxp3 was highly expressed in regulatory T cells (Treg cells) of lymphoid organs or tissues, whereas Foxp3 was not expressed in immune cells isolated from lymphoid organs (see Example 1).
또한, 본 발명의 다른 실시예에서는 fxDC가 pDC로부터 유래한 것인지 여부를 확인한 결과, B220+로 gating 한 세포는 CD11c 발현 유무와 무관하게 어떤 경우에도 GFP (Foxp3)를 발현하지 않았으며, CD11c로 gating한 경우에도 B220+ 세포는 GFP (Foxp3)를 발현하지 않음을 확인하였으며, 나아가 CD11b를 발현하는 Foxp3+DC를 gating하여 F4/80 및 CD64 발현을 확인한 결과, 본 발명의 Foxp3+DC는 대식세포와의 오염에 의해 나타나는 것이 아닌, 독자적인 수지상세포임을 확인하였다 (실시예 2 참조).Further, in another embodiment of the present invention, as a result of confirming whether fxDC is derived from pDC, cells gated with B220 + did not express GFP (Foxp3) in any case regardless of CD11c expression, and gating with CD11c. In the case of B220 + cells did not express GFP (Foxp3), and further confirmed that the expression of F4 / 80 and CD64 by gating Foxp3 + DC expressing CD11b, Foxp3 + DC of the present invention is contaminated with macrophages It was confirmed that the cells were independent dendritic cells, not shown by (see Example 2).
또한, 본 발명의 또 다른 실시예에서는 만성 감염 질환 모델에서 fxDC 개체수가 크게 증가하는 것을 확인하였고 (실시예 3 참조), fxDC가 림프절로 이동하는지 여부를 확인한 결과, fxDC는 림프절로 이동하지 않음을 확인하였다 (실시예 4 참조).In another embodiment of the present invention, it was confirmed that the fxDC population increased significantly in the chronic infection disease model (see Example 3). As a result of confirming whether fxDC moves to the lymph node, fxDC did not move to the lymph node. It was confirmed (see Example 4).
또한, 본 발명의 또 다른 실시예에서는 종양 조직 내에서의 분화 기전에 대해서 조사한 결과, 단핵구성 골수유래억제세포 (M-MDSC)가 종양 조직으로 이동한 후, fxDC로 분화됨을 확인하였으며 (실시예 5 참조), fxDC를 활성화되어 빨리 증식하는 T 세포와 공배양시, T 세포 증식이 강력하게 억제됨을 확인하였다. 이러한 효과는 fxDC에 의한 CD8+ 조절 T 세포 (CD8+ Treg)의 증식에 기인하는바, 상기 Foxp3를 발현하는 수지상 세포는 세포 면역 조절용 및/또는 염증성 질환 예방 또는 치료용 약학적 조성물의 유효성분으로 이용될 수 있음을 시사한다 (실시예 6 참조).In another embodiment of the present invention, as a result of investigating differentiation mechanisms in tumor tissues, it was confirmed that monocyte-derived myeloid-derived suppressor cells (M-MDSCs) migrated to tumor tissues and then differentiated into fxDC (Example 5), it was confirmed that when co-culture with fxDC activated and rapidly proliferating T cells, T cell proliferation was strongly inhibited. This effect is due to the proliferation of CD8 + regulatory T cells (CD8 + Tregs) by fxDC. The dendritic cells expressing Foxp3 may be used as an active ingredient of a pharmaceutical composition for regulating cellular immune and / or preventing or treating inflammatory diseases. Suggests that it can (see Example 6).
본 발명의 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 또한, 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. It may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions according to conventional methods.
상기 조성물에 포함될 수 있는 담체, 부형제 및 희석제는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등이 있다. 상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.Carriers, excipients and diluents which may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. In formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
본 발명에 따른 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 따른 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
구체적으로, 본 발명에 따른 약학적 조성물의 유효량은 환자의 연령, 성별, 상태, 체중, 체내에서 활성 성분의 흡수도, 불활성율 및 배설속도, 질병 종류, 병용되는 약물에 따라 달라질 수 있으며, 일반적으로는 1일 0.1 mg/kg 내지 100 mg/kg으로, 바람직하게는 1 내지 30 mg/kg의 양으로 투여할 수 있으며, 하루에 한번 또는 수 회 나누어 투여할 수도 있다.Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general It may be administered in an amount of 0.1 mg / kg to 100 mg / kg, preferably in an amount of 1 to 30 mg / kg per day, it may be administered once or several times a day.
본 발명의 약학적 조성물은 개체에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 피하, 정맥, 근육 또는 자궁 내 경막 또는 뇌혈관 내 주사에 의해 투여될 수 있다. 본 발명의 약학적 조성물은 치료할 질환, 투여 경로, 환자의 연령, 성별 및 체중 및 질환의 중등도 등의 여러 관련 인자와 함께, 활성성분인 약물의 종류에 따라 결정된다. The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, by subcutaneous, intravenous, intramuscular or intrauterine dural or cerebrovascular injections. The pharmaceutical composition of the present invention is determined according to the type of drug that is the active ingredient, along with various related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient and the severity of the disease.
본 발명의 다른 양태로서, 본 발명은 상기 약학적 조성물을 개체에 투여하는 단계를 포함하는 염증성 질환의 치료방법을 제공한다. 본 발명에서 "개체"란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는 인간 또는 비-인간인 영장류, 생쥐 (mouse), 쥐 (rat), 개, 고양이, 말 및 소 등의 포유류를 의미한다.In another aspect of the invention, the invention provides a method of treating an inflammatory disease comprising administering the pharmaceutical composition to a subject. As used herein, "individual" means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses and cattle, etc. Mean mammal.
본 발명의 또 다른 양태로서, 본 발명은 혈액 내 존재하는 골수유래억제세포 (myeloid-derived suppressor cell; MDSC)를 분리하는 단계; 및 상기 분리한 골수유래억제세포를 과립구-대식세포 콜로니 자극인자 (Granulocyte-macrophage colony-stimulating factor; GM-CSF)가 포함된 배지에서 배양하는 단계를 포함하는, Foxp3를 발현하는 수지상 세포 제조방법을 제공한다. As another aspect of the invention, the present invention comprises the steps of separating the myeloid-derived suppressor cells (MDSC) present in the blood; And culturing the isolated bone marrow-derived suppressor cells in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF). to provide.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
실시예 1. 다양한 세포에서의 Foxp3 발현 여부 확인Example 1 Confirmation of Foxp3 Expression in Various Cells
1-1. 림프기관에 상주하는 면역세포들의 Foxp3 발현 1-1. Foxp3 Expression in Immune Cells Residing in Lymphoid Organs
마우스 체내 여러 림프기관 및 점막 조직 (Spleen, inguinal lymph node, mesenteric lymph node, peyer's patch, thymus, lung, bone marrow)에서, CD11c+ Foxp3+ 수지상 세포의 존재 여부를 FACS (Fluorescence activated cell sorter)를 통하여 분석하였다. The presence of CD11c + Foxp3 + dendritic cells in various lymphoid organs and mucosal tissues (Spleen, inguinal lymph node, mesenteric lymph node, peyer's patch, thymus, lung, bone marrow) in the mouse were analyzed by FACS (Fluorescence activated cell sorter). .
그 결과, 도 1에 나타낸 바와 같이, Foxp3는 대부분 림프기관이나 조직의 조절 T 세포 (Treg cell)에서 높게 발현된 반면, 림프기관들에서 분리된 면역세포에서는 Foxp3를 발현하지 않았다. As a result, as shown in FIG. 1, Foxp3 was highly expressed in regulatory T cells of lymphoid organs or tissues, whereas Foxp3 was not expressed in immune cells isolated from lymphoid organs.
실시예 2. Foxp3를 발현하는 수지상세포의 확인Example 2. Identification of Dendritic Cells Expressing Foxp3
가장 먼저, Foxp3는 CD4+ 조절 T세포에서만 발현된다고 보고된 바, CD3 reverse gating으로 재분석하여도 Foxp3+ 발현 수지상세포가 Foxp3+ 조절 T세포의 오염에 의한 것이 아님을 확인하였다 (도 2a). First, Foxp3 was reported to be expressed only in CD4 + regulatory T cells, and reanalysis by CD3 reverse gating confirmed that Foxp3 + expressing dendritic cells were not caused by contamination of Foxp3 + regulatory T cells (FIG. 2A).
한편, 혈액에 존재하는 수지상세포는 common DC precursor (CDP)에서 바로 분화된 plasmacytoid DC (pDC)와 전구세포 형태를 띤 pre-conventional DC (pre-cDC)로 구성되며, 이들 중 pre-cDC는 다시 림프 조직이나 장기로 이동하여 CD4+ DC, CD8a+ DC, CD4-CD8- double negative DC (DN-DC), 또는 CD103+ DC, CD11b+ DC 등으로 분화된 뒤, 조직이나 장기에서 면역반응을 담당하는 것으로 알려져 왔다. 따라서 혈액 내 수지상 세포는 CDP에서 바로 분화된 pDC, 또는 분화 중간단계의 pre-DC 중 하나일 수 있다. 이에, 본 발명자들은 상기 fxDC가 pDC로부터 유래한 것인지 여부를 알아보기 위해 pDC 특이적 표면 항원 발현을 조사하였다. Dendritic cells present in the blood are composed of plasmacytoid DC (pDC) directly differentiated from common DC precursor (CDP) and pre-conventional DC (pre-cDC) in the form of progenitor cells. Has been known to be responsible for immune responses in tissues and organs after migration to lymphoid tissues or organs and differentiated into CD4 + DC, CD8a + DC, CD4-CD8 double negative DC (DN-DC), or CD103 + DC, CD11b + DC. . Thus, dendritic cells in the blood may be either pDCs differentiated directly from CDP, or pre-DCs of differentiation stages. Thus, the present inventors examined pDC specific surface antigen expression to determine whether the fxDC is derived from pDC.
그 결과, 도 2b에 나타낸 바와 같이, B220+로 gating 한 세포는 CD11c 발현 유무와 무관하게 어떤 경우에도 GFP (Foxp3)를 발현하지 않았으며, CD11c로 gating한 경우에도 B220+ 세포는 GFP (Foxp3)를 발현하지 않음을 확인하였다.As a result, as shown in FIG. 2B, the cells gated with B220 + did not express GFP (Foxp3) in any case irrespective of the expression of CD11c, and even when gated with CD11c, B220 + cells expressed GFP (Foxp3). It was confirmed not to.
이러한 결과는, 본 발명의 fxDC가 ppDC로부터 유래된 것이 아님을 의미한다. This result means that the fxDC of the present invention is not derived from ppDC.
또한, 대식세포는 수지상세포와 동일한 항원제시세포로 면역을 유도 또는 억제하는 M1, M2 및 TAM (Tumor associated macrophage)이 존재한다. 이에 Foxp3+DC가 대식세포와의 오염으로 인해 나타나는 것인지 여부를 확인하기 위하여, 대식세포 특이적 표면항원 (CD11b+CD14+F4/80+CD64+)을 이용하여 실험하였다.In addition, macrophages have M1, M2, and TAM (Tumor associated macrophage) that induce or inhibit immunity to the same antigen presenting cells as dendritic cells. In order to determine whether Foxp3 + DC appears due to contamination with macrophages, experiments were performed using macrophage specific surface antigens (CD11b + CD14 + F4 / 80 + CD64 +).
그 결과, 도 2c에 나타낸 바와 같이, Foxp3+DC로 gating한 세포는 대부분이 CD11b를 높게 발현하고 있는 반면, CD14는 전혀 발현하지 않음을 확인하였으며, 나아가 CD11b를 발현하는 Foxp3+DC를 gating하여 F4/80 및 CD64 발현을 확인한 결과, 본 발명의 Foxp3+DC는 대식세포와의 오염에 의해 나타나는 것이 아닌, 독자적인 수지상세포임을 확인하였다.As a result, as shown in Figure 2c, the cells gating with Foxp3 + DC was found that most of them express high CD11b, while not expressing CD14 at all, and further, by gating Foxp3 + DC expressing CD11b F4 As a result of confirming / 80 and CD64 expression, it was confirmed that Foxp3 + DC of the present invention was independent dendritic cells, not caused by contamination with macrophages.
이에, 이하 실시예에서는 Foxp3를 발현하는 수지상세포 (Foxp3+ 수지상세포)를 fxDC로 명명하였다.Thus, in the following examples, dendritic cells (Foxp3 + dendritic cells) expressing Foxp3 were named fxDC.
실시예 3. 만성 감염 질환 모델에서의 Foxp3가 발현된 수지상 세포 (fxDC) 개체수의 증가 확인Example 3 Confirmation of Increase of Foxp3 Expressed Dendritic Cell (fxDC) Population in Chronic Infectious Disease Model
혈중 cDC는 침입한 병원체 또는 외부 항원을 포획 및 분해하면서 활성화되며, 비장 또는 국부 림프절로 이동하여 T 세포에 항원을 전달함으로써, 후천적 면역을 활성화시킨다. 본 발명자들은 fxDC가 난치성 면역질환에 있어서, 중요한 역할을 담당할 것으로 예상하고, 이에, lymphocytic chriomeningitis virus (LCMV)를 이용한 급성 감염모델 (LCMV Arm), 및 만성 감염모델 (LCMV C13)의 혈액에서 fxDC 개체수 변화를 조사하였다.Blood cDCs are activated by capturing and breaking down invading pathogens or foreign antigens, and migrate to the spleen or local lymph nodes to deliver antigens to T cells, thereby activating acquired immunity. The present inventors expect fxDC to play an important role in intractable immune disease, and therefore, fxDC in the blood of acute infection model (LCMV Arm), and chronic infection model (LCMV C13) using lymphocytic chriomeningitis virus (LCMV). Population changes were investigated.
그 결과, 도 3에 나타낸 바와 같이, 감염 1주일 후 급성 감염모델에서, fxDC는 WT에 비해 그 개체수가 현저하게 감소한 반면, Foxp3를 발현하지 않는 DC의 개체수가 9 배 증가하였다. 그러나 만성 감염모델에서는, 감염 1달 후 fxDC의 개체수가 현저히 (약 15 배) 증가하였고, Foxp3를 발현하지 않는 DC의 개체수는 상대적으로 크게 증가하지 않았다 (약 4배). 상기 결과는 만성감염 모델에서, 장기적 면역 불균형을 해소하기 위해 fxDC 개체수가 크게 증가되었음을 의미한다. As a result, as shown in Figure 3, in the acute infection model 1 week after infection, fxDC significantly decreased compared to WT, whereas the number of DCs that do not express Foxp3 increased 9 times. In the chronic infection model, however, the number of fxDCs increased significantly (about 15-fold) after one month of infection, and that of DCs that did not express Foxp3 did not increase significantly (about 4-fold). The results indicate that in chronic infection models, the fxDC population increased significantly to resolve long-term immune imbalances.
실시예 4. Foxp3를 발현하는 수지상 세포 (fxDC)의 이동능 확인Example 4 Confirmation of Mobility of Dendritic Cells (fxDC) Expressing Foxp3
4-1. fxDC의 림프절로의 이동능 4-1. Mobility of fxDC into lymph nodes
Foxp3+ 수지상 세포 (fxDC)가 림프절로 이동하는지 확인하기 위해 transwell plate를 이용한 in vitro chemotaxis assay을 실시하였다. 즉, 말초혈액 단핵구세포(PBMC)에서 CD11c bead로 순수 분리한 수지상 세포를 24-well transwell plate (8 μM pore size polycarbonate filter, Corning Costar, Cambridge, MA)의 upper chambers에 채우고, lower chambers에 0.6 ml의 serum-free RPMI 1640 배지 및 희석된 CCL19 (300 ng/ml)를 채운 후, trans well plate를 37℃에서 3시간 동안 CO2 incubator에 방치하였다. upper chamber와 lower chamber로 이동한 Foxp3+ 세포 (Foxp3-GFP)를 FACS로 분석하였다. In vitro chemotaxis assay was performed using a transwell plate to confirm that Foxp3 + dendritic cells (fxDC) migrated to lymph nodes. In other words, dendritic cells purely isolated from peripheral blood mononuclear cells (PBMC) with CD11c bead were filled in the upper chambers of a 24-well transwell plate (8 μM pore size polycarbonate filter, Corning Costar, Cambridge, Mass.) And 0.6 ml in the lower chambers. After filling with serum-free RPMI 1640 medium and diluted CCL19 (300 ng / ml), the trans well plate was left in a CO 2 incubator at 37 ° C. for 3 hours. Foxp3 + cells (Foxp3-GFP) migrated to the upper chamber and the lower chamber were analyzed by FACS.
그 결과, 도 4에 나타낸 바와 같이, fxDC는 lower chamber (CCL19)로 전혀 이동하지 않았다. 이에, 상기 실시예 1에서, Foxp3+ 수지상 세포가 종양 조직에 상당히 존재하는 점, 및 본 실시예에서, Foxp3+ 수지상 세포가 림프절로 이동하지 않는 점을 미루어 보아, Foxp3+ 수지상 세포가 혈액에서 종양 조직으로 이동할 가능성이 있는지 여부를 조사하였다. As a result, as shown in FIG. 4, fxDC did not move to the lower chamber (CCL19) at all. Thus, in Example 1 above, Foxp3 + dendritic cells are significantly present in the tumor tissue, and in the present Example, Foxp3 + dendritic cells do not migrate to the lymph nodes, thereby allowing Foxp3 + dendritic cells to migrate from blood to tumor tissue. We investigated whether there is a possibility.
실시예 5. 종양 조직에서의 fxDC 분화 확인Example 5. Confirmation of fxDC Differentiation in Tumor Tissue
5-1. pre-conventional DC (pre-cDC)의 fxDC 전구세포 가능성 조사5-1. Investigation of the fxDC progenitor potential of pre-conventional DC (pre-cDC)
상기 실시예 2에서 본 발명의 fxDC는 plasmacytoid DC (pDC)로부터 유래된 것이 아님을 확인하였는바, CD11c+ pre-conventional DC (pre-cDC)로부터 유래한 것인지 여부를 확인하고자 하였다. 구체적으로, 마우스 혈액 PBMC를 CD11c+ 세포와 CD11c- 세포로 나눈 후, GM-CSF로 하루 동안 추가 분화시키고, 각각의 Foxp3 (GFP) 발현을 조사하였다.In Example 2, it was confirmed that the fxDC of the present invention was not derived from plasmacytoid DC (pDC), and it was intended to confirm whether it is derived from CD11c + pre-conventional DC (pre-cDC). Specifically, mouse blood PBMCs were divided into CD11c + cells and CD11c− cells, followed by further differentiation with GM-CSF for one day, and the expression of each Foxp3 (GFP) was examined.
그 결과, 도 5에 나타낸 바와 같이, CD11c+ 세포군에서는 Foxp3 (GFP) 발현 세포군은 5.36에서 5.60으로 큰 차이가 없었던 반면, CD11c(-) 세포군 (Foxp3+ 세포는 모두 Treg임.) 배양 후 CD11c+ 세포를 gating한 곳에서는 4.70% 가량의 fxDC 세포군이 새로 생성되었다. 이러한 결과를 통해, 본 발명의 fxDC는 pre-cDC, 즉 DC precursor 세포로 부터 유래한 것이 아니라, CD11c(-) 인 전구세포에서 CD11c+ fxDC로 분화된 것임을 확인하였다. 즉, fxDC의 전구세포는 CD11c- 세포군에 존재함을 의미한다.As a result, as shown in Figure 5, CD11c + cell population in the Foxp3 (GFP) expression cell population while there was no significant difference in the 5.36 to 5.60, CD11c (-) cell population (. Foxp3 + cells being all Treg) CD11c + cells after culture In the gating site, approximately 4.70% of fxDC cells were newly generated. Through these results, it was confirmed that the fxDC of the present invention is not derived from pre-cDC, that is, DC precursor cells, but differentiated into CD11c + fxDC in CD11c (-) precursor cells. In other words, fxDC progenitor cells are present in the CD11c- cell population.
5-2. fxDC의 전구세포 탐색 및 분화5-2. Progenitor Cell Exploration and Differentiation of fxDC
본 발명자들은 기존의 종양모델의 혈액에서 fxDC 개체수가 현저히 증가하며, fxDC는 기존 수지상 세포 전구세포 (pre-cDC)로부터 유래하지 않음을 상기 실험을 통해 확인하였다. 한편, 골수유래억제세포 (Myeloid derived suppressor cells, MDSC)는 두 가지 주요한 아형 즉, 과립구성 골수유래억제세포(CD11b+Ly6G+Ly6Clow granulocytic MDSCs, G-MDSCs), 및 단핵구성 골수유래억제세포 (CD11b+Ly6G-Ly6Chigh monocytic MDSCs, M-MDSCs)로 나뉘어지며, 이들 중 M-MDSC는 GM-CSF에 의해 tumor-associated macrophage (TAM), 또는 면역에 관여하는 수지상 세포로 분화되는 것으로 알려져 왔다 (Nat Rev Immunol 9:162-174, 2009). 이에 기초하여 본 발명자는 M-MDSC가 fxDC의 전구세포일 가능성을 조사하였다.The present inventors confirmed that the fxDC population increased significantly in the blood of the existing tumor model, and fxDC was not derived from the existing dendritic cell progenitor cells (pre-cDC) through the above experiments. Myeloid derived suppressor cells (MDSCs), on the other hand, have two main subtypes: CD11b + Ly6G + Ly6C low granulocytic MDSCs (G-MDSCs), and mononuclear myeloid suppressor cells (MDSCs). CD11b + Ly6G - Ly6C high monocytic MDSCs (M-MDSCs), of which M-MDSC has been known to differentiate into tumor-associated macrophage (TAM), or dendritic cells involved in immunity by GM-CSF ( Nat Rev Immunol 9: 162-174, 2009). Based on this, we investigated the possibility that M-MDSC is a precursor of fxDC.
GM-CSF 처리는 혈중 전구세포에서 DC로의 분화에 필수적인 요소이므로 혈액에서 분리한 MDSCs 아군들 (DP-MDSC, G-MDSC, M-MSDC, DN cell)을 3일 동안 GM-CSF 배지에서 수지상 세포로 분화시킨 후, 아군별 분화된 세포에서 Foxp3 (GFP) 발현 여부를 조사하였다. 또한, 이 배양과정 전에 MDSCs 아군들을 CFSF로 표지하여, 분화 과정에서 세포 증식 여부를 FACS로 확인하였다. Since GM-CSF treatment is an essential factor in the differentiation of DCs from blood progenitor cells, dendritic cells in GM-CSF medium for 3 days were treated with MDSCs subgroups (DP-MDSC, G-MDSC, M-MSDC, DN cells) isolated from blood. After differentiation, the expression of Foxp3 (GFP) in subdivision differentiated cells was examined. In addition, MDSCs subgroups were labeled with CFSF prior to the incubation process, and FACS was confirmed for cell proliferation during differentiation.
그 결과, 도 6에 나타낸 바와 같이, 다양한 MDSC 아군 중에서, Double positve (DP)-MDSC 및 M-MDSC 중 일부가 fxDC로 분화됨을 확인하였으며, 특히, (DP)-MDSC와 비교하여, M-MDSC가 더욱 높은 비율로 fxDC로 분화되었다. As a result, as shown in Figure 6, among the various MDSC subgroups, it was confirmed that some of the double positve (DP) -MDSC and M-MDSC is differentiated into fxDC, in particular, compared to the (DP) -MDSC, M-MDSC Was differentiated into fxDC at a higher rate.
상기 내용을 종합한 결과, 본 발명의 fxDC는 혈액 내 다량으로 존재하는 M-MDSC의 일부가 염증성 환경에서 분비되는 GM-CSF에 의해 fxDC로 분화되었음을 규명하였다. As a result of the above, it was found that fxDC of the present invention was differentiated into fxDC by GM-CSF secreted in an inflammatory environment, a part of M-MDSC present in a large amount in blood.
실시예 6. Foxp3가 발현된 수지상 세포 (fxDC)의 T 세포 활성 조절능 확인Example 6. Confirmation of T cell activity regulation ability of Foxp3 expressing dendritic cells (fxDC)
fxDC의 T 세포 활성 조절능을 조사하기 위해 T 세포와 M-MDSC에서 분화시킨 fxDC를 공배양하여 T 세포 증식 여부를 확인하였다. 또한, T 세포 면역 조절 기전을 구체적으로 조사하기 위해 fxDC 처리에 따른 CD4+ 조절 T 세포 (CD4+ Treg) 및 CD8+ 조절 T 세포 (CD8+ Treg)의 증식 여부를 조사하였다. In order to investigate the ability of fxDC to regulate T cell activity, T cell proliferation was confirmed by co-culture of fxDC differentiated from T cells and M-MDSC. In addition, to investigate the mechanism of T cell immune regulation specifically, the proliferation of CD4 + regulatory T cells (CD4 + Tregs) and CD8 + regulatory T cells (CD8 + Tregs) following fxDC treatment was investigated.
그 결과, 도 7 내지 10에 나타낸 바와 같이, G-MDSC나 fxDC의 전구세포인 M-MDSC는 mature BMDC (mDC)와 비슷한 수준으로 항원 특이적인 T 세포 증식을 유도하는 반면, DP-MDSC 또는 fxDC는 T 세포 증식을 거의 유도하지 못함을 확인하였다 (도 7). 또한, CD3/CD28에 의해 활성화/증식 중인 T세포를 fxDC와 공배양한 경우, CD4+T세포 증식은 전혀 억제하지 못한 반면, CD8+T세포만 선택적으로 증식을 억제하였으며, Foxp3/DTR 마우스를 사용하여 blood DC에서 fxDC만 제거한 경우, CD8+T세포의 증식이 회복되는 것을 확인하였다 (도 8). 이는 사이토카인에 의한 T세포 억제기전을 가진 재조합 Foxp3발현 수지상세포와는 달리 fxDC가 직접 T 세포와 접촉하여 T 세포면역을 조절함을 확인하였다 (도 9). 또한, MDSC 아군들은 CD4+ 조절 T 세포(CD4+ Treg) 증식을 유도하여 T 세포 활성을 조절하지만, 이와는 달리 fxDC는 CD8+ 조절 T 세포 (CD8+ Treg) 개체수를 크게 증가시키며 CD4+ 조절 T (CD4+ Treg)은 전혀 조절하지 않음을 확인하였다 (도 10).As a result, as shown in Figures 7 to 10, M-MDSC, a precursor of G-MDSC or fxDC, induces antigen-specific T cell proliferation to a level similar to mature BMDC (mDC), while DP-MDSC or fxDC Confirmed little induction of T cell proliferation (FIG. 7). In addition, when T cells activated / proliferated by CD3 / CD28 were co-cultured with fxDC, CD4 + T cell proliferation was not inhibited at all, whereas only CD8 + T cells selectively inhibited proliferation, and Foxp3 / DTR mice were suppressed. When only fxDC was removed from blood DC, it was confirmed that the proliferation of CD8 + T cells was recovered (FIG. 8). It was confirmed that unlike recombinant Foxp3 expressing dendritic cells with cytokine-induced T cell suppression mechanism, fxDC directly contacts T cells to regulate T cell immunity (FIG. 9). In addition, MDSC subgroups induce CD4 + regulatory T cell (CD4 + Tregs) proliferation to regulate T cell activity, whereas fxDC significantly increases CD8 + regulatory T cell (CD8 + Tregs) population and CD4 + regulatory T (CD4 + Tregs) at all. It was confirmed that no adjustment was made (FIG. 10).
이러한 결과는, fxDC가 CD8+ Treg 의 증식을 통해 항원 특이적으로 CD8+ effector T 세포 (CTL)의 증식활성을 강력하게 조절함을 시사한다. These results suggest that fxDC strongly regulates the proliferative activity of CD8 + effector T cells (CTL) antigen-specifically through the proliferation of CD8 + Tregs.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
본 발명에 따른 조성물은 Foxp3를 발현하는 수지상 세포 (fxDC)를 유효성분으로 포함하며, 상기 fxDC는 혈액 자체의 면역 항상성에 관여하는 핵심적인 면역세포로서, 감염성 질환과 같은 면역 불균형 상태에서 그 개체수가 증가하고, CD8+ 조절 T 세포 (CD8+ Treg) 증식의 조절을 통해 혈액 내 활성 T (effector T) 세포의 증식을 강력하게 억제시킬 수 있는바, 만성 염증성 면역 질환의 예방 또는 치료를 위한 약학 조성물로 유용하게 사용될 수 있을 것으로 기대된다. 특히, CD8+ Treg 세포 감소는 면역 노화 (immune senescence)의 특징 중의 하나로, 노령층은 CD8+ Treg 세포 감소로 인해 면역 항상성이 조절이 되지 않아 만성 염증성 질환이나 자가면역질환 환자가 증가하게 되는데 아직까지 CD8+ Treg의 감소 원인을 찾지 못하였다. 그러나 본 발명에 의하면 혈중 fxDC가 CD8+ Treg의 증식을 유도하는 원인 면역세포임을 밝혀 Foxp3를 발현하는 수지상 세포를 면역 노화의 치료를 위한 약학적 조성물로 사용될 수 있음을 밝혔다.The composition according to the present invention comprises a dendritic cell (fxDC) expressing Foxp3 as an active ingredient, the fxDC is a key immune cell involved in the immune homeostasis of the blood itself, the number of individuals in an imbalanced state such as an infectious disease It is possible to strongly inhibit the proliferation of effector T cells in the blood through the regulation of CD8 + regulatory T cell (CD8 + Treg) proliferation, which is useful as a pharmaceutical composition for the prevention or treatment of chronic inflammatory immune diseases. It is expected to be able to be used. In particular, the reduction of CD8 + Treg cells is one of the characteristics of immune senescence, and in the elderly, the CD8 + Treg cells decrease the immune homeostasis and increase the number of patients with chronic inflammatory diseases or autoimmune diseases. No cause of decline was found. However, according to the present invention, it was revealed that fxDC in the blood is a causative immune cell that induces the proliferation of CD8 + Tregs. Thus, dendritic cells expressing Foxp3 can be used as a pharmaceutical composition for the treatment of immune aging.
Claims (10)
- Foxp3를 발현하는 수지상 세포를 유효성분으로 포함하는, 면역 조절용 약학적 조성물. Immune regulating pharmaceutical composition comprising a dendritic cell expressing Foxp3 as an active ingredient.
- 제1항에 있어서,The method of claim 1,상기 Foxp3는 서열번호 1로 기재된 아미노산 서열로 이루어지는 것을 특징으로 하는, 약학적 조성물. Foxp3 is characterized in that consisting of the amino acid sequence shown in SEQ ID NO: 1.
- 제1항에 있어서,The method of claim 1,상기 수지상 세포는 혈액에서 분리된 것임을 특징으로 하는, 약학적 조성물. The dendritic cell is characterized in that isolated from the blood, pharmaceutical composition.
- 제1항에 있어서,The method of claim 1,상기 조성물은 혈액 내 면역 항상성을 조절하는 것을 특징으로 하는, 약학적 조성물. The composition is characterized in that to regulate immune homeostasis in the blood, pharmaceutical composition.
- 제1항에 있어서, The method of claim 1,상기 조성물은 CD8+ 조절 T 세포 (CD8+ Treg)를 증식시키는 것을 특징으로 하는, 약학적 조성물. The composition is characterized by propagating CD8 + regulatory T cells (CD8 + Tregs).
- Foxp3를 발현하는 수지상 세포를 유효성분으로 포함하는, 염증성 질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating inflammatory diseases, comprising dendritic cells expressing Foxp3 as an active ingredient.
- 혈액 내 존재하는 골수유래억제세포 (myeloid-derived suppressor cell; MDSC)를 분리하는 단계; 및 상기 분리한 골수유래억제세포를 과립구-대식세포 콜로니 자극인자 (Granulocyte-macrophage colony-stimulating factor; GM-CSF)가 포함된 배지에서 배양하는 단계를 포함하는, Foxp3를 발현하는 수지상 세포의 제조방법.Separating the myeloid-derived suppressor cells (MDSC) present in the blood; And culturing the isolated bone marrow-derived cells in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF). .
- 제7항에 있어서, The method of claim 7, wherein상기 골수유래억제세포는 단핵구성 골수유래억제세포 (Monocytic myeloid-derived suppressor cell; M-MDSC)인 것을 특징으로 하는, Foxp3를 발현하는 수지상 세포의 제조방법.The bone marrow-derived suppressor cells are monocytic myeloid-derived suppressor cells (M-MDSC), characterized in that the production method of dendritic cells expressing Foxp3.
- 제6항의 조성물을 개체에 투여하는 단계를 포함하는 염증성 질환의 치료방법.A method of treating an inflammatory disease comprising administering to a subject a composition of claim 6.
- 염증성 질환의 치료제 제조를 위한 Foxp3를 발현하는 수지상 세포의 용도.Use of dendritic cells expressing Foxp3 for the preparation of a therapeutic agent for an inflammatory disease.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150150688A KR101734697B1 (en) | 2015-10-29 | 2015-10-29 | Pharmaceutical composition for immune regulation comprising dendritic cells expressed Foxp3 |
| KR10-2015-0150688 | 2015-10-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2017074118A1 true WO2017074118A1 (en) | 2017-05-04 |
Family
ID=58630639
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2016/012289 WO2017074118A1 (en) | 2015-10-29 | 2016-10-28 | Pharmaceutical composition containing dendritic cell expressing foxp3 for regulating immunity |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR101734697B1 (en) |
| WO (1) | WO2017074118A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3690030A4 (en) * | 2017-09-28 | 2021-06-23 | Industry-Academic Cooperation Foundation, Yonsei University | Method for producing myeloid-derived suppressor cells, myeloid-derived suppressor cells produced thereby, and uses thereof |
| KR101944723B1 (en) * | 2017-09-28 | 2019-02-07 | 연세대학교 산학협력단 | Method for preparing myeloid-derived suppressor cells using type 1 interferon, myeloid-derived suppressor cells prepared thereby, and use thereof |
| KR102118116B1 (en) * | 2019-03-20 | 2020-06-02 | 충남대학교 산학협력단 | Nanoparticle comprising Foxp3 or a gene encoding of the same and uses thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150116614A (en) * | 2014-04-08 | 2015-10-16 | 성균관대학교산학협력단 | Composition for preventing or treating cancers comprising dendritic cells with Dab2 gene silenced |
-
2015
- 2015-10-29 KR KR1020150150688A patent/KR101734697B1/en active IP Right Grant
-
2016
- 2016-10-28 WO PCT/KR2016/012289 patent/WO2017074118A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150116614A (en) * | 2014-04-08 | 2015-10-16 | 성균관대학교산학협력단 | Composition for preventing or treating cancers comprising dendritic cells with Dab2 gene silenced |
Non-Patent Citations (4)
| Title |
|---|
| DATABASE protein [O] 4 June 2017 (2017-06-04), "forkhead box protein P3 [Mus musculus]", XP055380219, retrieved from NCBI Database accession no. np_473380.1 * |
| GONG, Y. -B. ET AL.: "Effects of Foxp3 Gene Modified Dendritic Cells on Mouse Corneal Allograft Rejection", INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE, vol. 8, no. 3, March 2015 (2015-03-01), pages 3965 - 3973, XP055380205 * |
| LIPSCOMB, M. W. ET AL.: "DC Expressing Transgene Foxp3 are Regulatory APC", EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 40, 2010, pages 480 - 493, XP055380202 * |
| VAN DE LAAR, L. ET AL.: "Regulation of Dendritic Cell Development by GM-CSF: Molecular Control and Implications for Immune Homeostasis and Therapy", BLOOD, vol. 119, no. 15, 2012, pages 3383 - 3393, XP055380210 * |
Also Published As
| Publication number | Publication date |
|---|---|
| KR101734697B1 (en) | 2017-05-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2017142187A1 (en) | Medium addition kit for culturing nk cells and nk cell culturing method using kit | |
| WO2010013947A2 (en) | Growth method for natural killer cells | |
| WO2017074118A1 (en) | Pharmaceutical composition containing dendritic cell expressing foxp3 for regulating immunity | |
| WO2019117633A1 (en) | Cosmetic composition and pharmaceutical composition for alleviating atopic dermatitis, hair loss, and wounds or reducing skin wrinkles | |
| JP2000509604A (en) | Use of interleukin-10 to generate suppressor cell populations | |
| Bissonnette et al. | Interferons differentially regulate histamine and TNF-alpha in rat intestinal mucosal mast cells. | |
| WO2022102887A1 (en) | Mass proliferation culture method of nk cells | |
| WO2019198995A1 (en) | Exosome-based conversion method for immune cells | |
| WO2019124666A2 (en) | Pharmaceutical composition comprising interleukin-17 inhibitor and tumor necrosis factor-alpha inhibitor as effective ingredient for preventing or treating neutrophilic lung inflammation disease | |
| Tamesis et al. | The role of natural killer cells in the development of herpes simplex virus type 1 induced stromal keratitis in mice | |
| WO2021150078A1 (en) | Off-the-shelf stem cells and immune cells, and pharmaceutical composition comprising same | |
| WO2015199402A1 (en) | Method for preparing dendritic cells with increased specific gene expression, and composition for treating or preventing autoimmune diseases, containing dendritic cells prepared using same | |
| WO2017069512A1 (en) | Method for inducing and proliferating virus antigen-specific t cells | |
| US8105601B2 (en) | Compositions and methods for modulating an immune response | |
| WO2019103436A9 (en) | Composition for culturing nk cells and method for culturing nk cells using same | |
| WO2012050269A1 (en) | Composition comprising mycobacterium tuberculosis rv0351 protein for promoting the maturation of dendritic cells | |
| WO2019168222A1 (en) | Culture medium composition for culturing of anti-cancer activated lymphocytes derived from peripheral blood mononuclear cells and method for culturing anti-cancer activated lymphocytes using same | |
| WO2015023147A1 (en) | Mtor/stat3 signal inhibitor-treated mesenchymal stem cell having immunomodulatory activity, and cell therapy composition comprising same, for preventing or treating immune disorders | |
| WO2021177599A1 (en) | METHOD FOR PRODUCING MEMORY-LIKE NK CELLS WITH ABILITY TO EXPRESS HIGHER LEVELS OF NCRS, CYTOTOXICITY, AND IFN-γ THAN NK CELLS IN HUMAN PERIPHERAL BLOOD | |
| WO2022108165A1 (en) | Method for producing exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells, and use thereof | |
| KR20170051376A (en) | Pharmaceutical composition for immune regulation comprising dendritic cells expressed Foxp3 | |
| WO2021006420A1 (en) | Method for controlling apoptosis of t cell, by treating phospholipase a2 | |
| WO2023204615A1 (en) | Method for producing memory-like natural killer cells and anticancer use of memory-like natural killer cells produced thereby | |
| WO2023096352A1 (en) | Feeder cell line genetically engineered to express hla-e and use thereof | |
| WO2021085853A1 (en) | Composition for enhancing immune activity and method therefor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 16860301 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 16860301 Country of ref document: EP Kind code of ref document: A1 |