JP2021502796A - Nk細胞培養用組成物、及びそれを利用してnk細胞を培養する方法 - Google Patents
Nk細胞培養用組成物、及びそれを利用してnk細胞を培養する方法 Download PDFInfo
- Publication number
- JP2021502796A JP2021502796A JP2020513912A JP2020513912A JP2021502796A JP 2021502796 A JP2021502796 A JP 2021502796A JP 2020513912 A JP2020513912 A JP 2020513912A JP 2020513912 A JP2020513912 A JP 2020513912A JP 2021502796 A JP2021502796 A JP 2021502796A
- Authority
- JP
- Japan
- Prior art keywords
- cells
- cell
- culturing
- composition
- cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000000822 natural killer cell Anatomy 0.000 title claims abstract description 250
- 239000000203 mixture Substances 0.000 title claims abstract description 65
- 238000012258 culturing Methods 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 46
- 210000004027 cell Anatomy 0.000 claims abstract description 131
- 238000004113 cell culture Methods 0.000 claims abstract description 58
- 102000003812 Interleukin-15 Human genes 0.000 claims abstract description 55
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 52
- 201000011510 cancer Diseases 0.000 claims abstract description 51
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims abstract description 30
- 238000011282 treatment Methods 0.000 claims abstract description 26
- 239000003814 drug Substances 0.000 claims abstract description 7
- 230000002265 prevention Effects 0.000 claims abstract description 6
- 239000002609 medium Substances 0.000 claims description 15
- 230000035755 proliferation Effects 0.000 claims description 11
- 230000004913 activation Effects 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 claims description 5
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 claims description 5
- 230000000890 antigenic effect Effects 0.000 claims description 5
- 239000006143 cell culture medium Substances 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 229910052711 selenium Inorganic materials 0.000 claims description 3
- 239000011669 selenium Substances 0.000 claims description 3
- 210000005259 peripheral blood Anatomy 0.000 claims description 2
- 239000011886 peripheral blood Substances 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 230000006907 apoptotic process Effects 0.000 abstract description 6
- 230000002147 killing effect Effects 0.000 abstract description 6
- 210000002865 immune cell Anatomy 0.000 abstract description 5
- 229940124597 therapeutic agent Drugs 0.000 abstract description 4
- 230000006051 NK cell activation Effects 0.000 abstract 1
- 238000010586 diagram Methods 0.000 abstract 1
- 231100000135 cytotoxicity Toxicity 0.000 description 23
- 230000003013 cytotoxicity Effects 0.000 description 23
- 230000014509 gene expression Effects 0.000 description 21
- 230000000694 effects Effects 0.000 description 17
- 102000004127 Cytokines Human genes 0.000 description 14
- 108090000695 Cytokines Proteins 0.000 description 14
- 239000008280 blood Substances 0.000 description 14
- 230000001965 increasing effect Effects 0.000 description 13
- 102000003952 Caspase 3 Human genes 0.000 description 12
- 108090000397 Caspase 3 Proteins 0.000 description 12
- 210000004369 blood Anatomy 0.000 description 11
- 201000010099 disease Diseases 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 102100037850 Interferon gamma Human genes 0.000 description 10
- 108010074328 Interferon-gamma Proteins 0.000 description 10
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 206010033128 Ovarian cancer Diseases 0.000 description 8
- 206010061535 Ovarian neoplasm Diseases 0.000 description 8
- 238000012790 confirmation Methods 0.000 description 8
- 230000012010 growth Effects 0.000 description 8
- 108010002350 Interleukin-2 Proteins 0.000 description 7
- 230000002062 proliferating effect Effects 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 230000004663 cell proliferation Effects 0.000 description 6
- 238000002955 isolation Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 108010002586 Interleukin-7 Proteins 0.000 description 5
- 238000002659 cell therapy Methods 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 239000002033 PVDF binder Substances 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- 102000043129 MHC class I family Human genes 0.000 description 3
- 108091054437 MHC class I family Proteins 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- BQRGNLJZBFXNCZ-UHFFFAOYSA-N calcein am Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)=C(OC(C)=O)C=C1OC1=C2C=C(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(=O)C)C(OC(C)=O)=C1 BQRGNLJZBFXNCZ-UHFFFAOYSA-N 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000009169 immunotherapy Methods 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 101800004419 Cleaved form Proteins 0.000 description 2
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 description 2
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 239000006147 Glasgow's Minimal Essential Medium Substances 0.000 description 2
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 2
- 239000007760 Iscove's Modified Dulbecco's Medium Substances 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 208000002495 Uterine Neoplasms Diseases 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 238000002784 cytotoxicity assay Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000007884 disintegrant Substances 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 238000001378 electrochemiluminescence detection Methods 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 229960003130 interferon gamma Drugs 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 239000012139 lysis buffer Substances 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
- 230000035800 maturation Effects 0.000 description 2
- 210000000581 natural killer T-cell Anatomy 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000012146 running buffer Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 206010046766 uterine cancer Diseases 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 102000004556 Interleukin-15 Receptors Human genes 0.000 description 1
- 108010017535 Interleukin-15 Receptors Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 108091093105 Nuclear DNA Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 238000009175 antibody therapy Methods 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 235000019700 dicalcium phosphate Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000009429 distress Effects 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 108091008042 inhibitory receptors Proteins 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000014828 interferon-gamma production Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229940031703 low substituted hydroxypropyl cellulose Drugs 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000031942 natural killer cell mediated cytotoxicity Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 229940079832 sodium starch glycolate Drugs 0.000 description 1
- 239000008109 sodium starch glycolate Substances 0.000 description 1
- 229920003109 sodium starch glycolate Polymers 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 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
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- 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
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- 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
- A61K39/4613—Natural-killer cells [NK or NK-T]
-
- 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
- A61K39/4644—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
- A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2315—Interleukin-15 (IL-15)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2318—Interleukin-18 (IL-18)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2327—Interleukin-27 (IL-27)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Epidemiology (AREA)
- Hematology (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
他の態様は、前記NK細胞を培養する方法によって製造されたNK細胞を提供する。
他の態様は、前記NK細胞を培養する方法によって製造されたNK細胞を含む癌予防用又は癌治療用の組成物を提供する。
(1.1)研究対象選定、血液及びPBMCの分離、NK細胞分離
満20歳ないし65歳の健康な男性及び女性であった、本研究のための採血に同意した対象者を対象にし、問診、体重、バイタルサインを測定し、研究対象適合者を選定した。適合者は、下記のような基準で選定した。
1)問診を介して、以下の除外条件がない者
−高血圧などの心血管系疾患、腎臓疾患、糖尿病、癌診断を受けた過去歴がある者
−宗教的な理由などで輸血ができない者
−妊婦
2)体重は、男性50kg以上、及び女性45kg以上である者
3)バイタルサインは、下記の条件を満足する者
−血圧(mmHg):収縮期90〜179、弛緩期100未満
−体温(℃):37.5℃以下
−脈拍(回/分):50〜100
(1)で得られたNK細胞を、1x105細胞/ml濃度で、12又は24−ウェルのティッシュ培養プレート(tissue culture plate)の各ウェルに入れた後、CellGro(R)無血清培地(serum-free medium)(CellGenix、米国)、10%ヒト血清(human serum)(Sigma Aldrich、米国)、10,000U/mLペニシリン/ストレプトマイシン(Pen/Strep)(Gibco/Life Technologies,Carlsbad,CA)、サイトカイン(IL−15、IL−27、1〜100ng/ml;Peperotech,Inc.NJ、米国;IL−18、1〜100ng/ml;R&D Systems Inc.,MN、米国)、ITS(insulin−transferrin−selenium−G Supplement 100X,GibcoTM)を添加し、37℃、5%のCO2恒温器で21日間培養した。
(2.1)NK細胞培養用組成物を添加し、NK細胞培養後のNK細胞の増殖能確認
(1.2)のように培養している間、増殖されたNK細胞の数は、初期6−ウェル組織培養プレートに、1x105〜1x106/mlのNK細胞培養を始まりに、2〜3日間隔で、T25、T75、そして最終的にBAG(NIPRO cell culture bags,A−1000NL,A−350NL,Funakoshi Co.,Ltd.)を利用して大量培養した。生存率は、増殖されたNK細胞数をトリパンブルー(trypan blue)染色剤(Thermo Fisher Scientific、米国)を利用して染色した後、血球計算板(hemocytometer)を利用して測定した。
(1.2)のように培養している間、NK細胞の活性度(activity)と受容体(receptor)の発現程度との差を確認した。
NK細胞を培養している間、ITSを添加し、NK細胞の増殖効果が増大するか否かということを確認した。IL−2、IL−15及びIL−18、並びにIL−15、IL−18及びIL−27をそれぞれ添加した3個の培養液に、ITS(insulin-transferrin-selenium−G Supplement 100X、GibcoTM)を追加したり、追加しなかったりして、21日間培養した。その培養結果を図5に示した。
従って、ITSを追加して添加する場合、本発明による培養用組成物において、NK細胞増殖効果をさらに向上させることができるということを確認した。
前述のところで確認したNK細胞の培養方法を、NK細胞の大量生産に適用することができるか否かということを確認した。具体的には、同一対象者からのNK細胞を利用し、初期0〜7日に、IL−15/18/27を利用してNK細胞を活性化させた後、既存NK細胞培養液にITSを添加し、T25プレートで培養した。その後、7〜12日に、いくつかのT25プレートに細胞を移して培養した。培養12〜14日に、培養バッグに細胞を移し、21日まで培養した。
(3.1)NK細胞培養用組成物を添加して培養されたNK細胞の癌細胞に対する細胞毒性の確認1
(3.1.1)細胞毒性分析(cytotoxicity assay)
細胞毒性は、NK細胞に対する感受性が高く、NK細胞の活性測定に主に使用されているK562細胞(ヒト慢性骨髄性白血病細胞系統;human chronic myelogenous leukemia cell line)を対象に、PromegaのCytotTox−GloTM細胞毒性分析キットを利用し、細胞毒性分析を行った。それは、損傷された細胞膜から遊離された酵素を測定する方法であり、ルミノジェニックペプチド基質(luminogenic peptide substrate)(アラニル−アラニル−フェニルアラニル−アミノルシフェリン;alanyl-alanyl-phenylalanyl-aminoluciferin,AAF−Glo substrate)を測定することにより、死んだ細胞の酵素反応を測定することができる方法である。
ポリ−D−リシンがコーティングされた6−ウェルプレートの各ウェルに、Calcein AM(Thermo Fisher Scientific)で染色されたK562細胞を1x105細胞で分注し、細胞毒性を測定するNK92とNK細胞とを、0:1、1.25:1、2.5:1、5:1、10:1のE:T比率で入れ、21日間共培養した。その後、Calcein AM放出分析(release assay)を介して、K562細胞がNK細胞によって溶解(lysis)される程度を、蛍光顕微鏡(zeiss microscope)を利用して観察した。
(3.3.1)カスパーゼ−3免疫ブロット
本発明のNK細胞培養用組成物を添加して培養されたNK細胞のインビトロ毒性を評価するために、卵巣癌細胞とNK細胞とを培養した後、卵巣癌細胞でのカスパーゼ−3(caspase−3)の活性レベル及び発現レベルを確認した。
次に、癌細胞内アポトーシス進行いかんを確認した。
Claims (12)
- IL−15、IL−18、IL−27、又はそれらの組み合わせを含むNK(ナチュラルキラー)細胞培養用組成物。
- ITS(インスリン−トランスフェリン−セレニウム)をさらに含むことを特徴とする請求項1に記載の組成物。
- 前記培養は、NK細胞の増殖又は活性化のためであることを特徴とする請求項1に記載の組成物。
- 細胞培養培地において、前記IL−15の濃度は、0.1ng/mlないし1,000ng/ml、IL−18の濃度は、0.25ng/mlないし2,500ng/ml、及びIL−27の濃度は、0.20ng/mlないし2,000ng/mlであることを特徴とする請求項1に記載の組成物。
- 前記NK細胞は、CD3−及びCD56+の表面抗原特性を有することを特徴とする請求項1に記載の組成物。
- NK(ナチュラルキラー)細胞を、IL−15、IL−18及びIL−27を含むNK細胞培養用組成物を含む培地で培養する工程を含むNK細胞を培養する方法。
- 前記培養する工程前に、
末梢血から末梢血単核細胞(PBMC)を得る工程と、
得られたPBMCからNK細胞を分離する工程と、
をさらに含むことを特徴とする請求項6に記載の方法。 - 前記培養は、7日間ないし30日間行われることを特徴とする請求項6に記載の方法。
- 請求項6に記載の方法によって製造されたNK細胞。
- 請求項6に記載の方法によって製造されたNK細胞を含む癌予防用又は癌治療用の組成物。
- 癌予防用又は癌治療用の医薬製造に使用するための請求項6に記載の方法によって製造されたNK細胞の用途。
- 有効量の、請求項6に記載の方法によって製造されたNK細胞を投与する工程を含む、癌を予防又は治療する方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2017-0158577 | 2017-11-24 | ||
KR1020170158577A KR102265437B1 (ko) | 2017-11-24 | 2017-11-24 | Nk 배양용 조성물 및 이를 이용하여 nk 세포를 배양하는 방법 |
PCT/KR2018/014294 WO2019103436A2 (ko) | 2017-11-24 | 2018-11-20 | Nk 세포 배양용 조성물 및 이를 이용하여 nk 세포를 배양하는 방법 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2021502796A true JP2021502796A (ja) | 2021-02-04 |
JP7119076B2 JP7119076B2 (ja) | 2022-08-16 |
Family
ID=66630757
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2020513912A Active JP7119076B2 (ja) | 2017-11-24 | 2018-11-20 | Nk細胞培養用組成物、及びそれを利用してnk細胞を培養する方法 |
Country Status (4)
Country | Link |
---|---|
US (2) | US11746327B2 (ja) |
JP (1) | JP7119076B2 (ja) |
KR (1) | KR102265437B1 (ja) |
WO (1) | WO2019103436A2 (ja) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR102236011B1 (ko) * | 2020-11-11 | 2021-04-05 | 한바이오 주식회사 | Nk 세포의 대량증식 배양방법 |
WO2024014643A1 (ko) * | 2022-07-11 | 2024-01-18 | 주식회사 노보셀바이오 | 세포 독성이 향상된 면역세포 제조방법 |
KR20240069648A (ko) | 2022-11-10 | 2024-05-20 | 의료법인 성광의료재단 | 전분화능 줄기세포에서 자연살해세포로의 분화 유도용 배지 조성물 및 이를 이용한 분화 방법 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006514057A (ja) * | 2002-12-31 | 2006-04-27 | シェーリング コーポレイション | 哺乳類のサイトカイン;関連試薬の使用 |
JP2007538108A (ja) * | 2004-05-20 | 2007-12-27 | ザイモジェネティクス, インコーポレイテッド | Il−21およびモノクローナル抗体治療を用いる癌を処置する方法 |
JP2017012010A (ja) * | 2015-06-26 | 2017-01-19 | チャ バイオテック カンパニー リミテッド | 自然殺害細胞増殖方法、及び自然殺害細胞増殖用の組成物 |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100254963B1 (ko) | 1997-11-28 | 2000-05-01 | 조생현 | 세포 배양용 무혈청 배지 |
EP2411507B1 (en) | 2009-03-26 | 2019-09-25 | CellProtect Nordic Pharmaceuticals AB | Expansion of nk cells |
CA2830080C (en) * | 2011-03-18 | 2021-10-26 | Jan Spanholtz | Generation of nk cells and nk-cell progenitors |
US9834753B2 (en) | 2011-12-22 | 2017-12-05 | Green Cross Labcell | Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same |
WO2016122014A1 (ko) | 2015-01-27 | 2016-08-04 | 한국생명공학연구원 | 자연살해세포의 대량생산 방법 및 상기 방법으로 수득된 자연살해세포의 항암제로서의 용도 |
KR101909879B1 (ko) | 2015-06-24 | 2018-10-19 | 주식회사 차바이오텍 | 자연살해세포의 증식 방법 및 자연살해세포 증식용 조성물 |
WO2016209021A1 (ko) | 2015-06-24 | 2016-12-29 | 주식회사 차바이오텍 | 자연살해세포의 증식 방법 및 자연살해세포 증식용 조성물 |
-
2017
- 2017-11-24 KR KR1020170158577A patent/KR102265437B1/ko active IP Right Grant
-
2018
- 2018-11-20 US US16/645,215 patent/US11746327B2/en active Active
- 2018-11-20 JP JP2020513912A patent/JP7119076B2/ja active Active
- 2018-11-20 WO PCT/KR2018/014294 patent/WO2019103436A2/ko active Application Filing
-
2023
- 2023-07-17 US US18/353,182 patent/US11981924B2/en active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006514057A (ja) * | 2002-12-31 | 2006-04-27 | シェーリング コーポレイション | 哺乳類のサイトカイン;関連試薬の使用 |
JP2007538108A (ja) * | 2004-05-20 | 2007-12-27 | ザイモジェネティクス, インコーポレイテッド | Il−21およびモノクローナル抗体治療を用いる癌を処置する方法 |
JP2017012010A (ja) * | 2015-06-26 | 2017-01-19 | チャ バイオテック カンパニー リミテッド | 自然殺害細胞増殖方法、及び自然殺害細胞増殖用の組成物 |
Non-Patent Citations (3)
Title |
---|
FRIAS A. M. ET AL., EXPERIMENTAL HEMATOLOGY, vol. 36 (2008), JPN6021015639, pages 61 - 68, ISSN: 0004664474 * |
MARTINEZ A. P. ET AL.: "Clinical grade activated natural killer products for adoptive immunotherapy against high-risk malign", HAEMATOLOGICA, vol. Vol.100 Suppl.1 (2015), JPN6021015636, pages 289 - 290, ISSN: 0004664475 * |
ZIBLAT A. ET AL., EUR. J. IMMUNOL., vol. 45 (2015), JPN6021015641, pages 192 - 202, ISSN: 0004664476 * |
Also Published As
Publication number | Publication date |
---|---|
US11981924B2 (en) | 2024-05-14 |
WO2019103436A2 (ko) | 2019-05-31 |
JP7119076B2 (ja) | 2022-08-16 |
KR102265437B1 (ko) | 2021-06-15 |
US20230365932A1 (en) | 2023-11-16 |
WO2019103436A9 (ko) | 2019-08-22 |
US11746327B2 (en) | 2023-09-05 |
WO2019103436A3 (ko) | 2019-07-18 |
KR20190060412A (ko) | 2019-06-03 |
US20210095249A1 (en) | 2021-04-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Van Acker et al. | Interleukin-15 enhances the proliferation, stimulatory phenotype, and antitumor effector functions of human gamma delta T cells | |
Chen et al. | IFN-β induces the proliferation of CD4+ CD25+ Foxp3+ regulatory T cells through upregulation of GITRL on dendritic cells in the treatment of multiple sclerosis | |
US11981924B2 (en) | Composition for culturing NK cells and method for culturing NK cells using same | |
JP7485385B2 (ja) | 適応型の表現型を示すnk細胞ならびにその製造方法および使用方法 | |
AU2008201685A1 (en) | CD4+CD25+ regulatory T cells from human blood | |
Alici et al. | Anti-myeloma activity of endogenous and adoptively transferred activated natural killer cells in experimental multiple myeloma model | |
Khammari et al. | Adoptive T cell therapy combined with intralesional administrations of TG1042 (adenovirus expressing interferon-γ) in metastatic melanoma patients | |
CZ347798A3 (cs) | Použití interleukinu-10 k produkci populace supresorových buněk | |
US11096967B2 (en) | Pharmaceutical composition for preventing or treating regulatory T cell-mediated diseases | |
Majumder et al. | Antigen-pulsed CpG-ODN-activated dendritic cells induce host-protective immune response by regulating the T regulatory cell functioning in Leishmania donovani-infected mice: critical role of CXCL10 | |
JP3904374B2 (ja) | キラー活性を増強したリンパ球 | |
KR101145391B1 (ko) | 말초혈액으로부터 자연 살해세포의 증식 방법 | |
KR20170047174A (ko) | 바이러스 항원 특이적인 t 세포의 유도 및 증식 방법 | |
Sugita et al. | Suppression of interleukin-17-producing T-helper 17 cells by retinal pigment epithelial cells | |
KR20200118449A (ko) | 형질전환 성장 인자 베타-내성 자연 살해 세포 | |
JP2022023136A (ja) | T細胞の拡張及び活性化の方法 | |
KR102032384B1 (ko) | 제대혈 단핵세포에서의 자연살해세포의 제조 방법 | |
KR20200073810A (ko) | 역분화 줄기세포 유래 중간엽 줄기세포를 포함하는 염증성 질환의 예방 또는 치료용 조성물 | |
WO2020246535A1 (ja) | Htlv-i特異的ctl活性化剤 | |
WO2023235511A1 (en) | Targeted elimination of senescent cells by gamma-delta t cells | |
Bloom | c12) United States Patent | |
KR20130131268A (ko) | 폴리감마글루탐산을 포함하는 Th17 매개성 질환 예방 또는 치료용 조성물 | |
Wang et al. | Prolongation of rat renal allograft survival by CD4+ CD25‑T cells induced by recipient dendritic cells transfected with IKK2dn | |
Fujiwara et al. | Graft-Versus-leukemia Tissue-restricted T cell alloresponses across HLA barriers: selection and identification of leukemia-restricted CTL in HLA-mismatched stimulator–responder pairs | |
Taams et al. | Type 1 IFN Maintains the Survival of Anergic |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20200514 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20210511 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20210811 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20211216 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20220314 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220614 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20220705 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20220803 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 7119076 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |