JP2010539992A - トランスフェクション方法及びそのための組成物 - Google Patents
トランスフェクション方法及びそのための組成物 Download PDFInfo
- Publication number
- JP2010539992A JP2010539992A JP2010528245A JP2010528245A JP2010539992A JP 2010539992 A JP2010539992 A JP 2010539992A JP 2010528245 A JP2010528245 A JP 2010528245A JP 2010528245 A JP2010528245 A JP 2010528245A JP 2010539992 A JP2010539992 A JP 2010539992A
- Authority
- JP
- Japan
- Prior art keywords
- tlr
- compound
- dna
- cells
- nucleic acid
- 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
- 238000003151 transfection method Methods 0.000 title claims description 5
- 239000000203 mixture Substances 0.000 title description 12
- 150000001875 compounds Chemical class 0.000 claims abstract description 87
- 239000003446 ligand Substances 0.000 claims abstract description 41
- 238000001890 transfection Methods 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 30
- 230000014509 gene expression Effects 0.000 claims abstract description 26
- 108020004414 DNA Proteins 0.000 claims description 103
- 102000039446 nucleic acids Human genes 0.000 claims description 78
- 108020004707 nucleic acids Proteins 0.000 claims description 78
- 150000007523 nucleic acids Chemical class 0.000 claims description 78
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 29
- 239000000427 antigen Substances 0.000 claims description 24
- 102000036639 antigens Human genes 0.000 claims description 24
- 108091007433 antigens Proteins 0.000 claims description 24
- 230000028993 immune response Effects 0.000 claims description 22
- 108090000623 proteins and genes Proteins 0.000 claims description 22
- 238000011282 treatment Methods 0.000 claims description 18
- 230000009881 electrostatic interaction Effects 0.000 claims description 17
- 238000004519 manufacturing process Methods 0.000 claims description 16
- 239000004055 small Interfering RNA Substances 0.000 claims description 15
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 10
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 9
- 108020004459 Small interfering RNA Proteins 0.000 claims description 9
- 229960005486 vaccine Drugs 0.000 claims description 9
- 239000002158 endotoxin Substances 0.000 claims description 7
- 229920006008 lipopolysaccharide Polymers 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 102000004895 Lipoproteins Human genes 0.000 claims description 5
- 108090001030 Lipoproteins Proteins 0.000 claims description 5
- 230000006698 induction Effects 0.000 claims description 5
- 150000002632 lipids Chemical class 0.000 claims description 5
- 230000002159 abnormal effect Effects 0.000 claims description 4
- 230000005856 abnormality Effects 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 4
- 230000002068 genetic effect Effects 0.000 claims description 4
- 125000003696 stearoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 4
- 125000003074 decanoyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(*)=O 0.000 claims description 2
- 230000009395 genetic defect Effects 0.000 claims description 2
- 125000000400 lauroyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 125000001419 myristoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 125000002801 octanoyl group Chemical group C(CCCCCCC)(=O)* 0.000 claims description 2
- 125000001312 palmitoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims 2
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 claims 1
- 108010013639 Peptidoglycan Proteins 0.000 claims 1
- 102000002689 Toll-like receptor Human genes 0.000 abstract description 78
- 108020000411 Toll-like receptor Proteins 0.000 abstract description 78
- 238000011238 DNA vaccination Methods 0.000 abstract description 4
- 238000013459 approach Methods 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract description 3
- 238000001727 in vivo Methods 0.000 abstract description 2
- 230000001629 suppression Effects 0.000 abstract description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N Arginine Chemical compound OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 159
- 210000004027 cell Anatomy 0.000 description 121
- 102000053602 DNA Human genes 0.000 description 99
- 239000004475 Arginine Substances 0.000 description 82
- HIGSLXSBYYMVKI-UHFFFAOYSA-N pralidoxime chloride Chemical compound [Cl-].C[N+]1=CC=CC=C1\C=N\O HIGSLXSBYYMVKI-UHFFFAOYSA-N 0.000 description 32
- 239000013612 plasmid Substances 0.000 description 30
- 210000004443 dendritic cell Anatomy 0.000 description 28
- 241000699670 Mus sp. Species 0.000 description 26
- 208000024891 symptom Diseases 0.000 description 13
- 210000001744 T-lymphocyte Anatomy 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 239000004472 Lysine Substances 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 10
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 10
- 102000043131 MHC class II family Human genes 0.000 description 10
- 108091054438 MHC class II family Proteins 0.000 description 10
- 239000005090 green fluorescent protein Substances 0.000 description 10
- 235000018102 proteins Nutrition 0.000 description 10
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 9
- 201000010099 disease Diseases 0.000 description 9
- 206010022000 influenza Diseases 0.000 description 9
- 102000004196 processed proteins & peptides Human genes 0.000 description 9
- 108090000695 Cytokines Proteins 0.000 description 8
- 102000004127 Cytokines Human genes 0.000 description 8
- 206010061218 Inflammation Diseases 0.000 description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- 210000000612 antigen-presenting cell Anatomy 0.000 description 8
- 230000004054 inflammatory process Effects 0.000 description 8
- 210000001165 lymph node Anatomy 0.000 description 8
- 239000012096 transfection reagent Substances 0.000 description 8
- 208000023275 Autoimmune disease Diseases 0.000 description 7
- 208000035473 Communicable disease Diseases 0.000 description 7
- 238000005917 acylation reaction Methods 0.000 description 7
- 125000002091 cationic group Chemical group 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 6
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 230000010933 acylation Effects 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 230000019491 signal transduction Effects 0.000 description 6
- 210000000952 spleen Anatomy 0.000 description 6
- 230000008685 targeting Effects 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 5
- 102100037850 Interferon gamma Human genes 0.000 description 5
- 108010074328 Interferon-gamma Proteins 0.000 description 5
- 108010028921 Lipopeptides Proteins 0.000 description 5
- 241000124008 Mammalia Species 0.000 description 5
- 102000011931 Nucleoproteins Human genes 0.000 description 5
- 108010061100 Nucleoproteins Proteins 0.000 description 5
- 102100039360 Toll-like receptor 4 Human genes 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 244000052769 pathogen Species 0.000 description 5
- 239000011347 resin Substances 0.000 description 5
- 229920005989 resin Polymers 0.000 description 5
- 229920002477 rna polymer Polymers 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 210000001132 alveolar macrophage Anatomy 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 210000002919 epithelial cell Anatomy 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 125000003473 lipid group Chemical group 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 230000035800 maturation Effects 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 230000005012 migration Effects 0.000 description 4
- 238000013508 migration Methods 0.000 description 4
- 210000001616 monocyte Anatomy 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 3
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- -1 DC maturation Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 108010060804 Toll-Like Receptor 4 Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000011481 absorbance measurement Methods 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000015788 innate immune response Effects 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 125000006239 protecting group Chemical group 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 230000001603 reducing effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- DMWMUMWKGKGSNW-OPMCLZTFSA-N (2S)-6-amino-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-4-amino-2-[[2-[[(2R)-2-amino-3-[(2R)-2,3-di(hexadecanoyloxy)propyl]sulfanylpropanoyl]amino]acetyl]amino]-4-oxobutanoyl]amino]-4-oxobutanoyl]amino]-3-carboxypropanoyl]amino]-4-carboxybutanoyl]amino]-3-hydroxypropanoyl]amino]-4-oxobutanoyl]amino]-3-methylpentanoyl]amino]-3-hydroxypropanoyl]amino]-3-phenylpropanoyl]amino]hexanoyl]amino]-4-carboxybutanoyl]amino]hexanoic acid Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](CSC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(O)=O)OC(=O)CCCCCCCCCCCCCCC DMWMUMWKGKGSNW-OPMCLZTFSA-N 0.000 description 2
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 2
- 108020000946 Bacterial DNA Proteins 0.000 description 2
- 0 CN**(N(*)OOC)OOC Chemical compound CN**(N(*)OOC)OOC 0.000 description 2
- 108010041986 DNA Vaccines Proteins 0.000 description 2
- 229940021995 DNA vaccine Drugs 0.000 description 2
- 108010040721 Flagellin Proteins 0.000 description 2
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 2
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 2
- 101710154606 Hemagglutinin Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- 101100481584 Mus musculus Tlr1 gene Proteins 0.000 description 2
- 241000202952 Mycoplasma fermentans Species 0.000 description 2
- 102000010168 Myeloid Differentiation Factor 88 Human genes 0.000 description 2
- 108010077432 Myeloid Differentiation Factor 88 Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 2
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 101710176177 Protein A56 Proteins 0.000 description 2
- 230000024932 T cell mediated immunity Effects 0.000 description 2
- 102000008228 Toll-like receptor 2 Human genes 0.000 description 2
- 108010060888 Toll-like receptor 2 Proteins 0.000 description 2
- 102100039390 Toll-like receptor 7 Human genes 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- 230000004721 adaptive immunity Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 239000012298 atmosphere Substances 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 230000009918 complex formation Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 210000002443 helper t lymphocyte Anatomy 0.000 description 2
- 239000000185 hemagglutinin Substances 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 230000028996 humoral immune response Effects 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 108010051618 macrophage stimulatory lipopeptide 2 Proteins 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 102000007863 pattern recognition receptors Human genes 0.000 description 2
- 108010089193 pattern recognition receptors Proteins 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 241000712461 unidentified influenza virus Species 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- AKIIJJAKZRNOLW-HTIIIDOHSA-N (2r)-2-[di(hexadecanoyl)amino]-3-(2,3-dihydroxypropylsulfanyl)propanoic acid Chemical compound CCCCCCCCCCCCCCCC(=O)N([C@@H](CSCC(O)CO)C(O)=O)C(=O)CCCCCCCCCCCCCCC AKIIJJAKZRNOLW-HTIIIDOHSA-N 0.000 description 1
- ZABOXPZKDZOMHY-FUBQLUNQSA-N (2r)-3-(2,3-dihydroxypropylsulfanyl)-2-(9h-fluoren-1-ylmethoxycarbonylamino)propanoic acid Chemical compound C1C2=CC=CC=C2C2=C1C(COC(=O)N[C@@H](CSCC(O)CO)C(O)=O)=CC=C2 ZABOXPZKDZOMHY-FUBQLUNQSA-N 0.000 description 1
- SCZNXLWKYFICFV-UHFFFAOYSA-N 1,2,3,4,5,7,8,9-octahydropyrido[1,2-b]diazepine Chemical compound C1CCCNN2CCCC=C21 SCZNXLWKYFICFV-UHFFFAOYSA-N 0.000 description 1
- UPAQRWMRKQCLSD-HTIIIDOHSA-N 2,3-dipalmitoyl-S-glycerylcysteine Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(CSC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCC UPAQRWMRKQCLSD-HTIIIDOHSA-N 0.000 description 1
- NHJVRSWLHSJWIN-UHFFFAOYSA-N 2,4,6-trinitrobenzenesulfonic acid Chemical compound OS(=O)(=O)C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O NHJVRSWLHSJWIN-UHFFFAOYSA-N 0.000 description 1
- 101150033839 4 gene Proteins 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- 239000000592 Artificial Cell Substances 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 1
- 102000006303 Chaperonin 60 Human genes 0.000 description 1
- 108010058432 Chaperonin 60 Proteins 0.000 description 1
- 241000938605 Crocodylia Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 1
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 1
- 101000669402 Homo sapiens Toll-like receptor 7 Proteins 0.000 description 1
- 241000588655 Moraxella catarrhalis Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 101710149951 Protein Tat Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108010038122 S-(2,3-bis(palmitoyloxy)propyl)cysteine Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 101150082427 Tlr4 gene Proteins 0.000 description 1
- 108010060825 Toll-Like Receptor 7 Proteins 0.000 description 1
- 108010060752 Toll-Like Receptor 8 Proteins 0.000 description 1
- 102000008235 Toll-Like Receptor 9 Human genes 0.000 description 1
- 108010060818 Toll-Like Receptor 9 Proteins 0.000 description 1
- 102000008229 Toll-like receptor 1 Human genes 0.000 description 1
- 108010060889 Toll-like receptor 1 Proteins 0.000 description 1
- 102100033110 Toll-like receptor 8 Human genes 0.000 description 1
- 241000223997 Toxoplasma gondii Species 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000012435 analytical chromatography Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 210000001557 animal structure Anatomy 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001767 cationic compounds Chemical class 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002079 cooperative effect Effects 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002121 endocytic effect Effects 0.000 description 1
- 239000006274 endogenous ligand Substances 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000005021 gait Effects 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-N guanidine group Chemical group NC(=N)N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 230000008348 humoral response Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 210000005007 innate immune system Anatomy 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 230000006525 intracellular process Effects 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 210000001821 langerhans cell Anatomy 0.000 description 1
- 230000029226 lipidation Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 238000003909 pattern recognition Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108010043655 penetratin Proteins 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 210000005134 plasmacytoid dendritic cell Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000034190 positive regulation of NF-kappaB transcription factor activity Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 108091069025 single-strand RNA Proteins 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013595 supernatant sample Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- 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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6018—Lipids, e.g. in lipopeptides
-
- 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
- A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
- A61K2039/622—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier non-covalent binding
-
- 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
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
- C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
- C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
- C12N2810/855—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from receptors; from cell surface antigens; from cell surface determinants
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Transplantation (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
【選択図】なし
Description
本発明は、一般的に、細胞生物学分野に関する。より具体的には、本発明は、細胞、特に、樹状細胞をトランスフェクションする方法、及び免疫応答を誘発する方法に向けられる。この目的を達成するために、本発明は、Toll様受容体(TLR)ファミリーの1つ又は複数のメンバーと相互作用する能力がある部分に連結した、核酸を結合する能力がある少なくとも1個の正荷電基を含む化合物を提供する。
本明細書に用いられる場合、用語「由来する」は、特定された完全体(integer)が、特定の供給源から得ることができるが、ただし、必ずしもその供給源から直接というわけではないことを示すものと解釈されるべきである。
1.Sambrook、Fritsch、及びManiatis、Molecular Cloning:A Laboratory Manual、Cold Spring Harbor Laboratories、New York、第2版(1989)、I巻、II巻、及びIII巻の全体、
2.DNA Cloning:A Practical Approach、I巻及びII巻(D.N.Glover編、1985)、IRL Press、Oxford、テキストの全体、
3.Oligonucletoide Synthesis:A Practical Approach(M.J.Gait編、1984)IRL Press、Oxford、テキストの全体、並びに特に、Gait、1〜22ページ;Atkinsonら、35〜81ページ;Sproatら、83〜115ページ;及びWuら、135〜151ページによるその中の論文
に記載されている。
本発明に繋がる研究において、本発明者らは、細胞に核酸をトランスフェクションする効率的な手段を生み出そうとした。それはまた、対象において、抗原提示細胞、特に樹状細胞をターゲットにし、体液性免疫応答及び細胞性免疫応答の両方を誘発する、DNAワクチン候補を作製しようとすることでもあった。
カチオン性ペプチドの合成
分岐ペプチド構築物については、リシン残基を用いて足場鋳型の分岐点を供給することによって合成した。足場内に存在する分岐点の数に依存して、ジカチオン性又はテトラカチオン性構造が生じた(図1)。これらの分岐構築物を、PEG−S RAM樹脂(Rapp Polymere、Tubingen、Germany;置換係数(substitution factor)0.27mmol/g)上で合成した。まず、等モル量のO−ベンゾトリアゾール−N,N,N,N’,N’−テトラメチル−ウロニウム−ヘキサフルオロホスフェート(HBTU;Novabiochem、Darmstadt、Germany)、1−ヒドロキシベンゾトリアゾール(HOBt)、及び1.5倍モル過剰のジイソプロピルエチルアミン(DIPEA;Sigma、Castle Hill、Australia)と共に、Fmoc−リシン(Mtt)−OH(Novabiochem、Laufelfingen、Switzerland)を4倍過剰で、樹脂に結合させた。アシル化を40分間行い、完全な反応をトリニトロベンジルスルホン酸(TNBSA)試験によって確認した(21)。α−アミノ基上のFmoc保護基の除去を2.5%ジアザビシクロ[5.4.0]ウンデカ−7−エン(Sigma、Steinheim、Germany)で達成し、Fmoc−リシン(Fmoc)−OH(Auspep、Melbourne、Australia)を、Fmoc基の除去後、2つの一級アミノ基を露出させて分岐点として働くように、結合させた。ジメチルホルムアミド(DMF;Auspep、Melbourne、Australia)を用いて、それぞれのアシル化段階と脱保護段階の間に樹脂を洗浄した。後続のアミノ酸を4倍過剰で結合させ、アシル化を60分間行った。四価の構築物の組み立てについて、アシル化のさらなるラウンドを、Fmoc−リシン(Fmoc)−OHを用いて60分間行い、4つの分岐点を生じさせた。アミノ酸を含む基の四価アルギニン構築物(R4)への化学選択的ライゲーションを可能にするために、システインを構築物のC末端に挿入し、R4−Cysを生じさせた。分岐ペプチド構築物上へのカルボキシフルオレセイン結合について、最後のアシル化反応後、N末端Fmoc基を除去しなかった。その代わり、C末端リシンのε−アミノ基上に存在するMtt保護基をジクロロメタン中1%TFA(Ajax Finechem、Seven Hills、Australia)での処理によって除去した。その後、等モル量のHOBt、HBTU、及び1.5倍モル過剰のDIPEAの存在下で、5(6)−カルボキシフルオレセイン(Fluka BioChemika、Switzerland)を4倍過剰用いて、暗闇中18時間、露出したε−アミノ基に結合させた。アシル化後、樹脂をDMFで洗浄し、N末端Fmoc基を除去した。全てのペプチドを同時に、固相支持体及び側鎖保護基から切断した。ウォーターズ(Waters)3 HPLCクロマトグラフィーシステムに設置されたバイダック(Vydac)C4カラム(4.6mm×250mm)又はC8カラム(4.6mm×250mm)を用いて逆相分析クロマトグラフィーによってペプチドの純度を評価した。限界溶媒としてH2O中0.1%TFA及びアセトニトリル中0.1%TFAを用いる1ml/分の流速を用いて、クロマトグラムを展開した。必要な場合、セミ分取用ウォーターズ又はGBC HPLCシステム及びセミ分取用バイダックC4カラム(10mm×300mm)を用いて2.5ml/分の流速でペプチドを精製した。ペプチド含有量の推定を、蛍光ペプチドの吸光度を496nmで測定するUV分光光度法によって測定し、83,000M−1cm−1のモル吸光係数を用いて濃度を計算した。合成された蛍光分岐ペプチド構築物の模式図は、図1に示されている。
R4によるDNA移動の遅延
分岐ペプチドR4とDNAの間の会合を調べるために、緑色蛍光タンパク質及びインフルエンザHAタンパク質をそれぞれコードするDNAプラスミドpEGFP−N1又はpCI−HAの一定量を、様々な量のR4とインキュベートし、アガロースゲル電気泳動によって分析した(図2)。
R4−DNA複合体の形成
R4のDNAへの結合を確認するために、漸増量のR4及びDNAを含む遠心分離した混合物の上清におけるDNAの量を、260nmにおいて吸光度を測定することによって決定した(図3A)。DNAをその電荷の中和によって溶液から沈殿させるR4の能力は、溶液中のDNAの減少を生じさせ、それにしたがって、それら2つの間の会合を示すことになるだろう。遠心分離された上清におけるDNAの量は、0.1μg〜5μgのR4とインキュベートした場合、劇的には変化しなかった。しかしながら、7.5μgのR4を用いた場合、わずかな減少が生じ、これより多い量を含む溶液中において、DNAは検出されなかった。この結果は、存在するR4の量がDNAの溶液からの沈殿と比例することを示す。吸光度の測定値をまた、いかなる遠心分離された不溶性物質も再懸濁するために用いられた洗浄上清についても取った。これらの試料中にDNAがほとんど存在しないことは、R4−DNA沈殿物が安定であること、及びDNAがこの複合体から解離する可能性は低いことを示唆する。
R4(S2Pam2Cys)−DNAでのDCのトランスフェクション
R4構築物又はR4(S2Pam2Cys)構築物がトランスフェクションされたDNAの発現を誘導する能力があるかどうかを決定するために、マウスDC系、D1にpEGFPプラスミドをR4又はR4(S2Pam2Cys)を用いてトランスフェクションした(図4A)。これらの細胞における緑色蛍光タンパク質(GFP)の発現レベルを、市販されているトランスフェクション試薬FUGENEを用いてトランスフェクションしたD1細胞における緑色蛍光タンパク質の発現レベルと比較した。
ヒト単球由来樹状細胞(MoDC)のトランスフェクション
ヒト単球由来樹状細胞(MoDC)のトランスフェクションを増強するR4(S2Pam2Cys)の能力を調べた。MoDCにpEGFPプラスミドをトランスフェクションした実験において(図6A)、R4(S2Pam2Cys)のみが、GFPの発現を誘導することにおいて効率的であった。FUGENEか又はR4のいずれかを加えたpEGFPでの細胞のトランスフェクションは、相対的に非効率であり、これらの細胞におけるトランスフェクションレベルは、プラスミド単独で処理されたものと類似していた。この結果は、MoDCに、各トランスフェクション試薬と共にpCI−HAプラスミドをトランスフェクションした実験において、繰り返された(図6B)。
R4(S2Pam2Cys)−DNAでの非DC細胞系のトランスフェクション
DNAと複合体を形成した、R4構築物又はR4(S2Pam2Cys)構築物が非DC細胞系をトランスフェクションする能力があるかどうかを調べるために、ヒト肺上皮細胞系A549及びマウス肺胞マクロファージ細胞系MH−Sに、R4又はR4(S2Pam2Cys)を用いてpEGFPプラスミドをトランスフェクションした(図7A及び7B)。ヒト肺上皮細胞系A549(A)又はマウス肺胞マクロファージ細胞系MH−S(B)を、異なる希釈度のR4、R4Pam2Cys、又はFUGENEと複合体を形成した、緑色蛍光タンパク質をコードするプラスミドの非存在下又は存在下において2×105個の細胞/mlの濃度で、37℃、5%CO2で48時間、培養した。トランスフェクション効率については、フローサイトメーターで細胞の蛍光を測定することによって決定した。これらの細胞系の両方とも他の研究においてTLR−2を発現することが報告されている(Oshikawa 2003、Slevogt 2007)。未処理のA549又はMH−S細胞、及びDNAと共にR4で処理された細胞において、皆無かそれに近い蛍光が観察された。しかしながら、R4(S2Pam2Cys)及びDNAをトランスフェクションされたA549又はMH−S細胞は、pEGFPプラスミド及びFUGENE試薬をトランスフェクションされた細胞において観察された発現レベルと、より高くはないにしても、匹敵するレベルでGFPを発現した。
R4(S2Pam2Cys)−DNAで免疫処置されたマウスにおける特異的抗体産生
DNAと複合体を形成したR4(S2Pam2Cys)の抗体を誘導する能力を評価した。BALB/cマウス(週齢6〜8週間)に、単独か、又は1:3若しくは1:5のモル比でR4(S2Pam2Cys)と複合体を形成した、インフルエンザ血球凝集素をコードするDNAを含む50μgのDNAプラスミドを0日目及び28日目に尾の付け根に皮下接種した。陽性対照として、マウスをまた、スプリットPR8ウイルス(3μg/マウス)で鼻腔内に免疫処置した。非免疫処置マウスは陰性対照としての役割を果たした。最後の接種後14日目にマウスから血清を採取し、ELISAを行って、インフルエンザPR8ウイルスに特異的な抗体の存在を検出した。血清抗HA抗体をELISAによって測定した。非免疫処置マウス又は「ナイーブ」マウスにおいてHA特異的抗体は検出されなかったが、1:3及び1:5のモル比で混合された2つの用量のR4(S2Pam2Cys)及びDNAで免疫処置されたマウスにおいて、インフルエンザウイルスに結合することができる実質的レベルの抗体が検出された(図8)。
R4(S2Pam2Cys)−DNAで免疫処置されたマウスにおけるインフルエンザ核タンパク質特異的IFN−γ陽性CD8+T細胞の誘導
DNAと複合体を形成したR4(S2Pam2Cys)の細胞性免疫応答を誘導する能力を評価した。BALB/cマウス(週齢6〜8週間)に、単独か、又は1:1、1:2、若しくは1:5のNH3 +:PO4 −モル比でR4(S2Pam2Cys)と複合体を形成した、インフルエンザ核タンパク質をコードする20μgのDNAプラスミドを尾の付け根に皮下接種した。R4(S2Pam2Cys)を含む溶液の10μlのアリコートをDNAの溶液に2分間ごとに合計1時間、ゆっくり加えることによって、複合体形成を達成した。全ての接種材料を0.7MのNaClに溶解した。免疫処置後7日目又は10日目にマウスから脾臓及び鼠径リンパ節を摘出し、細胞内サイトカイン染色を行って核タンパク質147〜155に特異的なIFN−γ分泌CD8+T細胞の存在を検出した。それぞれの棒は2匹のマウスからの平均及び標準誤差を表す。
Alexopoulou L., Holt A. C., Medzhitov R. and Flavell R. A. (2001) Recognition of double−stranded RNA and activation of NF−kappaB by Toll−like receptor 3. Nature 413 (6857): 732−738
Asea A., Rehli M., Kabingu E., Boch J. A., Bare O., Auron P. E., Stevenson M. A. and Calderwood S. K. (2002) Novel signal transduction pathway utilized by extracellular HSP70: role of toll−like receptor (TLR) 2 and TLR4. J Biol Chem 277 (17): 15028−15034
Billingham J., Breen C., Rawson J. O., Yarwood J., Mann B.E. (1997) Adsorption of polycations on clays: A comparative in situ study using 133Cs and 23Na solution phase NMR. Journal of Colloid and Interface Science 193:183−189
Bulut Y., Faure E., Thomas L., Karahashi H., Michelsen K. S., Equils O., Morrison S. G., Morrison R. P. and Arditi M. (2002) Chlamydiai heat shock protein 60 activates macrophages and endothelial cells through Toll−like receptor 4 and MD2 in a MyD88−dependent pathway. J.Immunol 168 (3); 1435−1440
Buschle M, Schmidt W, Zauner W, Mechtler K, Trska B, Kirlappos H, et al. (1997) Transloading of tumor antigen−derived peptides into antigen−presenting cells. Proc Natl Acad Sci USA 94(7):3256−3261
Chow J. C., Young D. W., Golenbock D. T., Christ W. J. and Gusovsky F. (1999) Toll−like receptor−4 mediates lipopolysaccharide−induced signal transduction. J−Biol Chem 274 (16): 10689−10692
Christiaens B, Grooten J, Reusens M, Joliot A, Goethals M, Vandekerckhove J, Procbiantz A, Rosseneu M. (2004) Membrane interaction and cellular internalization of penetratin peptides. Eur J Biochem. 271(6);1187−1197
Fawell S, Seery J, Daikh Y, Moore C, Chen LL, Pepinsky B, et al. (1994) Tat−mediated delivery of heterologous proteins into cells. Proc Natl Arad Sci USA 91(2);664− 668
Flacher V, Bouschbacher M, Verronese E, Massacrier C, Sisirak V, Berthier−Vergnes O, de Saint−Vis B, Caux C, Dezutter−Dambuyant C, Lebecque S, Valladeau J. (2006) Hiuman Langerhans cells express a specific TLR profile and differentially respond to viruses and Gram−positive bacteria. J Immunol. 177(11):7959−7967
Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K, et al. (2001) Argininerich peptides. An abundant source of membrane−permeable peptides having potential as carriers for intracellular protein delivery, J Biol Chem 276(8):5836−5840
Hayashi F., Smith K. D., Ozinsky A., Hawn T. R., Yi E. C., Goodlett D. R., Eng J. K., Akira S., Underhill D. M. and Aderem A. (2001) The innate immune response to bacterial flagellin is mediated by Toll−like receptor 5. Nature 410 (6832): 1099−1103
Heil F., Hemmi H., Hochrein H., Ampenberger F., Kirschning C., Akira S., Lipford G., Wagner H and Bauer S. (2004) Species−specific recognition of single−stranded RNA via toll−like receptor 7 and 8. Science 303 (5663): 1526−1529
Hemmi H., Kaisho T., Takeuchi O., Sato S., Sanjo H., Hoshino K., Horiuchi T., Tomizawa H., Takeda K. and Akira S. (2002) Small anti−viral compounds activate immune cells via the TLR7 MyD88−dependent signaling pathway. Nat Immunol 3 (2): 196−200
Hemmi H., Takeuchi O., Kawai T., Kaisho T., Sato S., Sanjo H., Matsumoto M., Hoshino K., Wagner H., Takeda K. and Akira S. (2000) A Toll−like receptor recognizes bacterial DNA. Nature 408 (6813): 740−745
Husebye H, Halaas (, Stenmark H, Tunheim G, Sandanger (, Bogen B, Brech A, Latz E, Espevik T. (2006) Endocytic pathways regulate Toll−like receptor 4 signaling and link innate and adaptive immunity. EMBO J. 25(4):683−692
Mitchell DJ, Kim DT, Steinman L, Fathman CG, Rothbard JB. (2000) Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res 56(5):318−325
Morr M., Takeuchi O., Akira S., Simon M. M. and Muhlradt P. F. (2002) Differential recognition of structural details of bacterial lipopeptides by toll−like receptors. Eur J Immunol 32 (12): 3337−3347
Oehlke J, Scheller A, Wiesner B, Krause E, Beyermann M, Klauschenz E, et al. (1998) Cellular uptake of an alpha−helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non−endocytically. Biochim Biophys Acta 1414(1−2):127−139
Okusawa T., Fujita M., Nakamura J., Into T., Yasuda M., Yoshimura A., Hara Y., Hasebe A., Golenbock D. T., Morita M., Kuroki Y., Ogawa T. and Shibata K. (2004) Relationship between structures and biological activities of mycoplasmal diacylated lip opeptides and their recognition by toll−like receptors 2 and 6. Infect Immun 72(3): 1657−1665
Oshikawa K, Sugiyama Y. (2003) Regulation of toll−like receptor 2 and 4 gene expression in murine alveolar macrophages. Exp Lung Res 29(6):401−412
Otvos L, Jr., Cudic M, Chua BY, Deliyannis G, Jackson DC. (2004) An insect antibacterial peptide−based drug delivery system. Mol Pharm 1(3):220−232
Ozinsky A., Underhill D. M., Fontenot J. D., Hajjar A. M., Smith K. D., Wilson C. B., Schroeder L. and Aderem A. (2000) The repertoire for pattern recognition of pathogens
by the innate immune system is defined by cooperation between toll−like receptors. Proc Natl Acad Sci USA 97 (25): 13766−13771
Pepper M, Dzierszinski F, Wilson E, Tait E, FangQ, Yarovinsky F, Laufer TM, Roos D, Hunter CA. (2008) Plasmacytoid dendritic cells are activated by Toxoplasma gondii to present antigen and produce cytokines. J Immunol. 2008 180(9):6229−6236
Poltorak A., He X, Smirnova L, Liu M. Y., Van Huffel C., Du X., Birdwell D., Alejos E., Silva M., Galanos C., Freudenberg M., Ricciardi−Castagnoli P., Layton B. and Beutler B. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282 (5396): 2085−2088
Pooga M, Hallbrink M, Zorko M, Langel U. (1998) Cell penetration by transportan. Faseb J 12(1):67−77
Schjetne K. W., Thompson K. M., Nilsen N., Flo T. H., Fleckenstein B., Iversen J. G., Espevik T., and Bogen B. (2003) Cutting edge: link between innate and adaptive immunity: Toll−like receptor 2 internalizes antigen for presentation to CD4+ T cells and could be an efficient vaccine target J Immunol 171:32−36
Schwandner R., Dziarski R., Wesche H., Rothe M. and Kirschning C. J. (1999) Peptidoglycan− and lipoteichoic acid−induced cell activation is mediated by toll−like receptor 2. J Biol Chem 274 (25): 17406−17409
Slevogt H, Seybold J, Tiwari KN, Hocke AC, Jonatat C, Dietel S, Hippenstiel S, Singer BB, Bachmann S, Suttorp N, Opitz B. (2007) Moraxella catarrhalis is internalized in respiratory epithelial cells by a trigger like mechanism and initiates a TLR2− and partly NOD1−dependent inflammatory immune response. Cell Microbiol 9(3):694−707
Takeshita F, Leifer CA, Gursel I, Ishii KJ, Takeshita S, Gursel M, Klinman DM. (2001) Cutting edge: Role of Toll−like receptor 9 in CpG DNA−induced activation of human cells. J Immunol. 167(7):3555−3558
Takeuchi O., Kaufmann A., Grote K, Kawai T., Hoshino K., Morr M., Muhlradt P. F. and Akira S. (2000) Cutting edge: preferentially the R−stereoisomer of the mycoplasmal lipopeptide macrophage−activating lipopeptide−2 activates immune cells through a toll−like receptor 2− and MyD88−dependent signaling pathway. J Immunol 164 (2): 554−557
Takeuchi O., Sato S., Horiuchi T., Hoshino K., Takeda K., Dong Z., Modlin R. L. and Akira S. (2002) Cutting edge: role of Toll−like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol 169 (1): 10−14 .
Wagner E., Zenke M., Cotten M., Beug, H., Birnstiel M. L. (1990) Transferrinpolycation conjugates as carriers for DNA uptake into cells. Proc. Natl. Acad. Sci. USA 87:3410−3414
Claims (20)
- 少なくとも1個のTLRリガンドに連結した正荷電基を含む化合物。
- 前記正荷電基がTLRリガンドに共有結合している、請求項1に記載の化合物。
- 前記正荷電基が1個又は複数のアミノ酸を含む、請求項1又は請求項2に記載の化合物。
- 前記正荷電基が分岐ペプチド又は直鎖ペプチドである、請求項1〜3のいずれか一項に記載の化合物。
- 前記ペプチドが少なくとも1個のアルギニン残基又はリシン残基を含む、請求項4に記載の化合物。
- 前記ペプチドが少なくとも4個のアルギニン残基又は少なくとも4個のリシン残基を含む、請求項4又は請求項5に記載の化合物。
- 前記TLRリガンドが脂質、又はペプチドグリカン、又はリポタンパク質、又はリポ多糖を含む、請求項1〜6のいずれか一項に記載の化合物。
- 前記TLRリガンドがパルミトイル、ミリストイル、ステアロイル、ラウロイル、オクタノイル、又はデカノイルを含む、請求項1〜6のいずれか一項に記載の化合物。
- 前記TLRリガンドがPam2Cys、Pam3Cys、Ste2Cys、Lau2Cys、及びOct2Cysからなる群より選択される、請求項1〜6のいずれか一項に記載の化合物。
- 前記TLRリガンドがTLR−2か又はTLR−6のいずれかに結合する、請求項1〜9のいずれか一項に記載の化合物。
- 前記TLRリガンドがTLR−2に結合する、請求項1〜10のいずれか一項に記載の化合物。
- 核酸及び請求項1〜11のいずれか一項に記載の化合物を含む複合体であって、前記核酸と前記正荷電基との間の静電相互作用によって前記核酸が前記化合物と会合している、複合体。
- 少なくとも1個のTLRを発現する細胞を、請求項12に記載の複合体に接触させる段階を含む、トランスフェクション方法。
- 抗原に対する免疫応答を生じさせる方法であって、請求項12に記載の複合体を対象に投与する段階を含み、前記核酸が前記抗原又はそのエピトープをコードする、方法。
- 抗原に対する免疫応答を生じさせる方法であって、請求項12に記載の複合体をトランスフェクションされた細胞を対象に投与する段階を含み、前記核酸が前記抗原又はそのエピトープをコードする方法。
- TLRを発現する細胞で遺伝子の発現を抑圧する方法であって、請求項12に記載の複合体を対象に投与する段階を含み、前記核酸が、siRNA、shRNA、siRNAをコードするDNA、及びshRNAをコードするDNAからなる群より選択され、かつ前記siRNA又はshRNAが前記遺伝子をターゲットとする、方法。
- 対象における免疫応答の誘導のためのワクチンの製造における、請求項1〜12のいずれか一項に記載の化合物又は複合体の使用。
- 遺伝子異常又は遺伝子欠損をもつ対象の処置のための医薬の製造における、請求項1〜12のいずれか一項に記載の化合物又は複合体の使用。
- 遺伝子の異常な発現又は望ましくない発現を患っている対象の処置のための医薬の製造における、請求項1〜12のいずれか一項に記載の化合物又は複合体の使用。
- 細胞のトランスフェクションのための試薬の製造における、請求項1〜12のいずれか一項に記載の化合物又は複合体の使用。
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2007905530A AU2007905530A0 (en) | 2007-10-09 | A Method of Transfection and Compositions Therefor | |
AU2007905530 | 2007-10-09 | ||
AU2007905536 | 2007-10-09 | ||
AU2007905536A AU2007905536A0 (en) | 2007-10-09 | A Method of Transfection | |
PCT/AU2008/001501 WO2009046498A1 (en) | 2007-10-09 | 2008-10-09 | A method of transfection and compositions therefor |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2010539992A true JP2010539992A (ja) | 2010-12-24 |
JP5743315B2 JP5743315B2 (ja) | 2015-07-01 |
Family
ID=40548887
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2010528245A Active JP5743315B2 (ja) | 2007-10-09 | 2008-10-09 | トランスフェクション方法及びそのための組成物 |
Country Status (8)
Country | Link |
---|---|
US (1) | US9089508B2 (ja) |
EP (1) | EP2200638B1 (ja) |
JP (1) | JP5743315B2 (ja) |
AU (1) | AU2008310313C1 (ja) |
CA (1) | CA2702154C (ja) |
DK (1) | DK2200638T3 (ja) |
ES (1) | ES2575395T3 (ja) |
WO (1) | WO2009046498A1 (ja) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2009344150B2 (en) * | 2009-04-09 | 2014-10-09 | Innavac Pty Ltd | Immunogenic composition and uses thereof |
WO2010115229A1 (en) * | 2009-04-09 | 2010-10-14 | The University Of Melbourne | Immunogenic composition and uses thereof |
EP3520814A1 (en) | 2010-09-22 | 2019-08-07 | Ena Therapeutics Pty Ltd | Immunostimulating composition |
DE102011018499A1 (de) | 2011-04-23 | 2012-10-25 | Emc Microcollections Gmbh | Topische Nanopartikel-Vakzine zur Immunstimulation der dendritischen Zellen in der Haut |
WO2013117348A1 (en) * | 2012-02-09 | 2013-08-15 | Carsten Kirschning | Agonists and antagonists of toll-like receptor (tlr) 13 |
EP2674170B1 (en) | 2012-06-15 | 2014-11-19 | Invivogen | Novel compositions of TLR7 and/or TLR8 agonists conjugated to lipids |
EP2732825B1 (en) | 2012-11-19 | 2015-07-01 | Invivogen | Conjugates of a TLR7 and/or TLR8 agonist and a TLR2 agonist |
EP2769738B1 (en) | 2013-02-22 | 2016-07-20 | Invivogen | Conjugated TLR7 and/or TLR8 and TLR2 polycationic agonists |
US20220388950A1 (en) | 2019-06-26 | 2022-12-08 | Axelia Oncology Pty Ltd | Novel molecules |
EP4395761A1 (en) * | 2021-08-31 | 2024-07-10 | ENA Respiratory Pty Ltd | Allergy treatment |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004501657A (ja) * | 2000-06-28 | 2004-01-22 | マックス−デルブルック−セントラム フュール モレクラーレ メディツィン | トランスフェクション効率の改良方法 |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU772617B2 (en) | 1999-11-19 | 2004-05-06 | Csl Limited | Vaccine compositions |
CA2494193A1 (en) | 2002-08-12 | 2004-02-19 | The Council Of The Queensland Institute Of Medical Research | Novel immunogenic lipopeptides comprising t-helper and cytotoxic t lymphocyte (ctl) epitopes |
EP1543039B1 (en) * | 2002-08-12 | 2011-07-13 | The Council Of The Queensland Institute Of Medical Research | Novel immunogenic lipopeptides comprising t-helper and b-cell epitopes |
US20060002941A1 (en) * | 2004-01-23 | 2006-01-05 | Vievax Corp. | Compositions comprising immune response altering agents and methods of use |
AU2005295317B2 (en) | 2004-10-18 | 2011-10-13 | Globeimmune, Inc. | Yeast-based therapeutic for chronic hepatitis C infection |
EP1666056A1 (en) * | 2004-12-06 | 2006-06-07 | Bestewil Holding B.V. | Cancer vaccine vesicles |
ES2534332T3 (es) | 2006-03-07 | 2015-04-21 | Vaxinnate Corporation | Composiciones que incluyen hemaglutinina, métodos de preparación y métodos de uso de las mismas |
WO2010115229A1 (en) | 2009-04-09 | 2010-10-14 | The University Of Melbourne | Immunogenic composition and uses thereof |
-
2008
- 2008-10-09 US US12/682,415 patent/US9089508B2/en active Active
- 2008-10-09 AU AU2008310313A patent/AU2008310313C1/en active Active
- 2008-10-09 CA CA2702154A patent/CA2702154C/en not_active Expired - Fee Related
- 2008-10-09 ES ES08800135.9T patent/ES2575395T3/es active Active
- 2008-10-09 EP EP08800135.9A patent/EP2200638B1/en active Active
- 2008-10-09 JP JP2010528245A patent/JP5743315B2/ja active Active
- 2008-10-09 WO PCT/AU2008/001501 patent/WO2009046498A1/en active Application Filing
- 2008-10-09 DK DK08800135.9T patent/DK2200638T3/en active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004501657A (ja) * | 2000-06-28 | 2004-01-22 | マックス−デルブルック−セントラム フュール モレクラーレ メディツィン | トランスフェクション効率の改良方法 |
Non-Patent Citations (6)
Title |
---|
JPN6013032741; PNAS vol.101 no.43, 2004, pp.15440-15445 * |
JPN6013032743; International Immunology vol.18 no.12, 2006, pp.1801-1813 * |
JPN6013032745; J Mol Med vol.82, 2004, pp.144-152 * |
JPN6015012910; Methods in Molecular Medicine vol.127, 2006, pp.159-169 * |
JPN6015012912; NATURE MEDICINE vol.9 no.3, 2003, pp.357-362 * |
JPN6015012915; PEPTIDES vol.29, 2008, pp.881-890 * |
Also Published As
Publication number | Publication date |
---|---|
ES2575395T3 (es) | 2016-06-28 |
WO2009046498A1 (en) | 2009-04-16 |
CA2702154A1 (en) | 2009-04-16 |
AU2008310313A1 (en) | 2009-04-16 |
JP5743315B2 (ja) | 2015-07-01 |
EP2200638B1 (en) | 2016-03-16 |
AU2008310313C1 (en) | 2015-02-12 |
US20100310595A1 (en) | 2010-12-09 |
AU2008310313B2 (en) | 2014-08-07 |
CA2702154C (en) | 2017-05-16 |
EP2200638A4 (en) | 2012-03-21 |
US9089508B2 (en) | 2015-07-28 |
EP2200638A1 (en) | 2010-06-30 |
DK2200638T3 (en) | 2016-06-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5743315B2 (ja) | トランスフェクション方法及びそのための組成物 | |
US20240092843A1 (en) | Peptides and methods for the treatment of diabetes | |
CN102448485B (zh) | 免疫原性组合物及其用途 | |
KR20210005046A (ko) | T-세포 유도 백신 조성물의 조합물 및 이의 용도 | |
CA2727973C (en) | Peptidyl diacylglycerides | |
TWI392502B (zh) | 聚己醣抗原及含新穎醣脂質佐劑之相關抗癌疫苗 | |
EP3800199B1 (en) | H3.3 ctl peptides and uses thereof | |
KR20240019135A (ko) | 작제물과 면역자극 화합물의 공동발현 | |
Nara et al. | An octavalent dendrimer of multiple antigenic peptide with a property of pan-coronavirus IgM induction improved clinical signs of feline infectious peritonitis in cats | |
WO2015130488A2 (en) | Mhc class i associated peptides for prevention and treatment of hepatitis b virus infection | |
WO2004098489A2 (en) | Compositions and methods for modulation of specific epitopes of hsp60 | |
EP4034550A1 (en) | Vaccine therapy for ran protein diseases | |
WO2013134292A2 (en) | Compositions and methods for mitigation of inflammasome activation | |
CA3163485A1 (en) | Teipp peptide variant and uses thereof | |
CN117903264A (zh) | 新冠病毒SARS-CoV-2 HLA-A2限制性表位肽及应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20111003 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20130702 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20130927 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20131004 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20131128 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20131205 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20131226 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20140805 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20141028 |
|
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: 20150331 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20150427 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 5743315 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |