WO2011069486A1 - Aptamères d'arn se liant spécifiquement au récepteur soluble de l'interleukine 6 - Google Patents
Aptamères d'arn se liant spécifiquement au récepteur soluble de l'interleukine 6 Download PDFInfo
- Publication number
- WO2011069486A1 WO2011069486A1 PCT/DE2010/001412 DE2010001412W WO2011069486A1 WO 2011069486 A1 WO2011069486 A1 WO 2011069486A1 DE 2010001412 W DE2010001412 W DE 2010001412W WO 2011069486 A1 WO2011069486 A1 WO 2011069486A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- aptamer
- rna
- binding
- sil
- rna aptamer
- Prior art date
Links
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 114
- 108010038501 Interleukin-6 Receptors Proteins 0.000 title claims abstract description 38
- 102000010781 Interleukin-6 Receptors Human genes 0.000 title claims abstract description 7
- 108091008103 RNA aptamers Proteins 0.000 claims abstract description 58
- 239000003814 drug Substances 0.000 claims abstract description 10
- 208000015181 infectious disease Diseases 0.000 claims abstract description 7
- 229940039227 diagnostic agent Drugs 0.000 claims abstract description 6
- 239000000032 diagnostic agent Substances 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims description 27
- 125000003729 nucleotide group Chemical group 0.000 claims description 27
- 239000012634 fragment Substances 0.000 claims description 15
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 5
- 206010028980 Neoplasm Diseases 0.000 claims description 4
- 206010006187 Breast cancer Diseases 0.000 claims description 3
- 208000026310 Breast neoplasm Diseases 0.000 claims description 3
- 208000011231 Crohn disease Diseases 0.000 claims description 3
- 208000034578 Multiple myelomas Diseases 0.000 claims description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 3
- 206010040047 Sepsis Diseases 0.000 claims description 3
- 208000006673 asthma Diseases 0.000 claims description 3
- 125000001153 fluoro group Chemical group F* 0.000 claims description 3
- 230000004968 inflammatory condition Effects 0.000 claims description 3
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 3
- 201000006417 multiple sclerosis Diseases 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 208000031886 HIV Infections Diseases 0.000 claims description 2
- 208000037357 HIV infectious disease Diseases 0.000 claims description 2
- 201000011510 cancer Diseases 0.000 claims description 2
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 230000027455 binding Effects 0.000 abstract description 83
- 238000009739 binding Methods 0.000 abstract description 82
- 201000010099 disease Diseases 0.000 abstract description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 9
- 230000004054 inflammatory process Effects 0.000 abstract description 4
- 230000002265 prevention Effects 0.000 abstract description 2
- 229940124597 therapeutic agent Drugs 0.000 abstract description 2
- 229920002477 rna polymer Polymers 0.000 description 46
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 description 45
- 230000003993 interaction Effects 0.000 description 41
- 102000004889 Interleukin-6 Human genes 0.000 description 37
- 108090001005 Interleukin-6 Proteins 0.000 description 37
- 210000004027 cell Anatomy 0.000 description 37
- 229940100601 interleukin-6 Drugs 0.000 description 34
- 239000000499 gel Substances 0.000 description 25
- 102000004169 proteins and genes Human genes 0.000 description 25
- 108090000623 proteins and genes Proteins 0.000 description 25
- 102000005962 receptors Human genes 0.000 description 19
- 108020003175 receptors Proteins 0.000 description 19
- 239000012528 membrane Substances 0.000 description 17
- 239000000872 buffer Substances 0.000 description 15
- 101000599048 Homo sapiens Interleukin-6 receptor subunit alpha Proteins 0.000 description 14
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 14
- 108020004414 DNA Proteins 0.000 description 13
- 102000053602 DNA Human genes 0.000 description 13
- 238000010494 dissociation reaction Methods 0.000 description 13
- 230000005593 dissociations Effects 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 12
- 229920002401 polyacrylamide Polymers 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 11
- 102000039446 nucleic acids Human genes 0.000 description 11
- 108020004707 nucleic acids Proteins 0.000 description 11
- 150000007523 nucleic acids Chemical class 0.000 description 11
- 239000003446 ligand Substances 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 9
- 238000003556 assay Methods 0.000 description 9
- 238000000926 separation method Methods 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 8
- 102000004127 Cytokines Human genes 0.000 description 7
- 108090000695 Cytokines Proteins 0.000 description 7
- 238000000376 autoradiography Methods 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 239000000020 Nitrocellulose Substances 0.000 description 5
- 108020001507 fusion proteins Proteins 0.000 description 5
- 102000037865 fusion proteins Human genes 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 229920001220 nitrocellulos Polymers 0.000 description 5
- 108091035707 Consensus sequence Proteins 0.000 description 4
- 241001529936 Murinae Species 0.000 description 4
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000009870 specific binding Effects 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 239000012099 Alexa Fluor family Substances 0.000 description 3
- 108010062802 CD66 antigens Proteins 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- 102100024533 Carcinoembryonic antigen-related cell adhesion molecule 1 Human genes 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102000016943 Muramidase Human genes 0.000 description 3
- 108010014251 Muramidase Proteins 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 230000004570 RNA-binding Effects 0.000 description 3
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 239000013543 active substance Substances 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000009918 complex formation Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 229960000274 lysozyme Drugs 0.000 description 3
- 239000004325 lysozyme Substances 0.000 description 3
- 235000010335 lysozyme Nutrition 0.000 description 3
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 150000002972 pentoses Chemical class 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 238000004904 shortening Methods 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 230000000087 stabilizing effect Effects 0.000 description 3
- 229940035893 uracil Drugs 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 101710137500 T7 RNA polymerase Proteins 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical group O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 229940119059 actemra Drugs 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 102000003675 cytokine receptors Human genes 0.000 description 2
- 108010057085 cytokine receptors Proteins 0.000 description 2
- 229940104302 cytosine Drugs 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical group O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 102000052611 human IL6 Human genes 0.000 description 2
- 238000007885 magnetic separation Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 241000894007 species Species 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
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 229960003989 tocilizumab Drugs 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- ASJSAQIRZKANQN-UHFFFAOYSA-N 2-deoxypentose Chemical compound OCC(O)C(O)CC=O ASJSAQIRZKANQN-UHFFFAOYSA-N 0.000 description 1
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101100338243 Caenorhabditis elegans hil-6 gene Proteins 0.000 description 1
- 108010067225 Cell Adhesion Molecules Proteins 0.000 description 1
- 102000016289 Cell Adhesion Molecules Human genes 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 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
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000701533 Escherichia virus T4 Species 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 108091081406 G-quadruplex Proteins 0.000 description 1
- 206010018001 Gastrointestinal perforation Diseases 0.000 description 1
- 239000001828 Gelatine Substances 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000980898 Homo sapiens Cell division cycle-associated protein 4 Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 102000016844 Immunoglobulin-like domains Human genes 0.000 description 1
- 108050006430 Immunoglobulin-like domains Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- -1 Linker amino acids Chemical class 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 1
- 206010046306 Upper respiratory tract infection Diseases 0.000 description 1
- 101710201961 Virion infectivity factor Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000001270 agonistic effect Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- FCPVYOBCFFNJFS-LQDWTQKMSA-M benzylpenicillin sodium Chemical compound [Na+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 FCPVYOBCFFNJFS-LQDWTQKMSA-M 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 102000044493 human CDCA4 Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 150000004712 monophosphates Chemical class 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- OCQZXMCGTAWGEQ-UHFFFAOYSA-N prop-2-enamide;n-[(prop-2-enoylamino)methyl]prop-2-enamide Chemical compound NC(=O)C=C.C=CC(=O)NCNC(=O)C=C OCQZXMCGTAWGEQ-UHFFFAOYSA-N 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 230000008060 renal absorption Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000020029 respiratory tract infectious disease Diseases 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/322—2'-R Modification
Definitions
- the invention relates to aptamers which specifically bind a target molecule.
- cytokine interleukin-6 IL-6
- IL-6 interleukin-6
- Cytokine IL-6 is known to be involved in a variety of diseases, e.g. inflammatory autoimmune diseases such as rheumatoid arthritis.
- inflammatory autoimmune diseases such as rheumatoid arthritis.
- membrane-bound cytokine receptors soluble forms also exist which consist of the extracellular domains of the membrane-like forms and also possess the ability to bind ligand, albeit at a reduced affinity to the membrane-like form. Soluble receptors can neutralize ligands, for example, by their comparatively high concentration and act in this way
- soluble interleukin-6 receptor sIL-6R
- the complex of EL-6 and sIL-6R causes dimerization of the membrane-bound gpl30 (Taga, T. et al. (1989) Cell, 58, 573-581 Mackiewicz, A. et al., (1992) J. Immunol., 149, 2021-2027; Scheller and Rose-John, Med. Microbiol Immunol. (2006), 195, 173-183; Rose-John et al. , (2006), JJ Leukocyte Biol. 80, 227-235; Jones et al.
- gpl30 is present in almost all organs, e.g. Heart, kidneys, spleen, liver, lung, placenta, and brain (Saito, M. et al., 1992, J. Immunol., 148, 4066-4071) .
- interleukin-6 may therefore also act on cells which do not express the membrane-bound mterleukin-6 receptor but the glycoprotein gp 130 (Hirota, H. et al., (1995) Proc Natl Acad., USA, 92, 4862-4866).
- tocilizumab (Actemra®) is a monoclonal antibody currently used to treat rheumatoid arthritis. Tocilizumab (Actemra®) exerts its action via the blockade of the human IL-6 receptor, resulting in the binding of IL-6 to the receptor and a consequent
- Other side effects include gastrointestinal perforation, hypersensitivity reactions including anaphylaxis, headache and
- Aptamers are artificial DNA or RNA molecules that contain another molecule, e.g. a protein, specifically bind. With high specificity and affinity as well as chemical
- aptamers have a low immunogenicity compared to antibodies.
- aptamers may be selected by a method termed SELEX (Evolutionary Evolutionary Ligations by Exponential Enrichment) (Ellington and Szostak (1990), Nature 346, 818-822, Gopinath (2007), Anal. Bioanal Chem 387, 171-182, WO 91/19813).
- the invention provides an RNA aptamer that specifically binds the human soluble interleukin-6 receptor.
- the soluble human mlelevikin-6 receptor (sIL-6R) has a molecular weight of about 50 kDa at 339 amino acids (AA).
- the amino acid sequence of sIL-6R is shown in SEQ ID NO: 11.
- Amino acid sequence of the sEL-6R is identical to that of the extracellular domain of the membrane-bound interleukin-6 receptor (IL-6R), so that the inventive
- aptamer also specifically binds the IL-6R. Any reference to the sIL-6R should therefore also include the IL-6R, unless expressly stated otherwise.
- the RNA aptamer according to the invention is affine and specific for the human sIL-6 receptor and is unlikely or not immunogenic. It provides a promising means to treat or prevent diseases involving the soluble IL-6 receptor. It is also simple and inexpensive to produce, durable and storable.
- the RNA aptamer of the invention can be used in a variety of ways to
- IL-6 and / or the soluble and / or the membrane-bound interleukin-6 receptor are directly or indirectly involved, to diagnose or influence. It can be used, for example, to prepare a drug or diagnostic agent that can be used to diagnose, prevent and / or treat inflammatory conditions or diseases, cancers or infections. Examples of diseases in which the inventive RNA aptamer
- RNA aptamer according to the invention can also be used to introduce other molecules, for example peptides, proteins, oligonucleotides, dyes, diagnostic agents, etc. into a cell. To do this, these molecules are prepared using techniques that are familiar to those skilled in the art are common, coupled to the RNA aptamer.
- the molecule coupled to the RNA aptamer according to the invention can then be introduced into the cell by means of the sIL-6R or the IL-6R and be released intracellularly.
- the molecules can also be biologically or medically active substances, so that the RNA aptamer can also have a function as part of a prodrug.
- RNA aptamer an isolated single-stranded RNA (ssRNA) comprising a target molecule, e.g. a protein that specifically binds.
- ssRNA isolated single-stranded RNA
- RNA aptamer ssRNA oligonucleotides having at most 150, preferably at most 130, at most 110, at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20 , at most 15 or at most 10 nucleotides understood.
- nucleotide are here in particular the basic building blocks of nucleic acids, i. organic molecules derived from a sugar residue, usually a pentose, e.g. Deoxyribose or ribose, an organic base (nucleobase) and phosphoric acid.
- a pentose e.g. Deoxyribose or ribose
- an organic base e.g. Deoxyribose or ribose
- phosphoric acid is regularly linked to the sugar via an ester bond, the sugar to the nucleobase via an N-glycosidic bond.
- RNA ribonucleic acid
- the phosphoric acid is usually present in RNA and DNA as monophosphate.
- a “target molecule” is understood here preferably to be non-RNA and non-DNA molecules, for example proteins, peptides and glycoproteins.
- binding is meant a bond that does not rely on the Watson-Crick pairing between nucleotides. It is preferably a non-covalent bond.
- a human sIL-6 receptor specifically binding RNA aptamer or a fragment thereof is here understood an RNA aptamer or RNA aptamer fragment, a related
- RNA aptamer or RNA aptamer fragment understood that has a dissociation constant of at most 1000 nM (nmol / 1), preferably at most 500 nM, more preferably at most 250 nM and particularly preferably at most 150 nM. this preferably refers to averages of a size determined using the one-site binding model described below using filter binding studies.
- the notion of hsIL-6 receptor specific binding also encompasses specific binding to the hIL-6 receptor, ie the membrane-bound human IL-6 receptor.
- a “diagnostic emitter 1" is understood here to mean a product that can be used in a diagnostic method.
- the term also encompasses products, for example
- RNA aptamer according to the invention comprises
- Nucleotide sequence ggkgnggcwguggwgwggg (SEQ ID NO: 1).
- the RNA aptamer according to the invention comprises the nucleotide sequence ggkgnggcwguggwgwggg (SEQ ID NO: 2), ggggnggcwguggwgwggg (SEQ ID NO: 3) or ggggnggcwguggwgwggg (SEQ ID NO: 4).
- the letters k, n and w are as defined above, h is a, c or u.
- sequence of SEQ ID NO: 2 differs from the sequence according to SEQ ID NO: 1 in that there is no g at position 5
- sequence of SEQ ID NO: 3 differs from the sequence according to SEQ ID NO: 1 in that that at position 3 is a g
- sequence of SEQ ID NO: 4 differs of the sequence according to SEQ ID NO: 1, characterized in that at position 3 is a g and at position 5 no g.
- the RNA aptamer according to the invention comprises one of the sequences indicated in SEQ ID NO: 5-10 or SEQ ID NO: 13-18 or a fragment thereof which specifically binds the human soluble interleukin-6 receptor.
- One or more, possibly also all bases of the nucleotides of the RNA aptamer or of the RNA aptamer fragment can be modified. This can be beneficial to
- the modification may be, for example, that the 2'-OH group of a nucleotide base is replaced by a fluoro, amino or methoxy group.
- the 2-OH group is advantageous by a methoxy group, in a pyrimidine base by a fluoro or
- RNA aptamer of the invention may also be combined with other compounds, e.g. Cholesterol or polyethylene glycol (PEG), or may be timed, for example, to increase bioavailability, reduce degradation or excretion.
- other compounds e.g. Cholesterol or polyethylene glycol (PEG)
- PEG polyethylene glycol
- dT deoxythymidine
- a fragment of an RNA aptamer according to the invention preferably comprises at least 14, 15, 16, 17, 18, 19 or at least 20, more preferably at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 and particularly preferably 60 consecutive nucleotides of any of the sequences given in SEQ ID NO: 5-10 or SEQ ID NO: 13-18.
- the fragment may also comprise at least 70, 80, 90, 100 or 106 contiguous nucleotides.
- a fragment comprises at least one of the sequences according to SEQ ID NO: 1-4. Examples of fragments within the meaning of the present invention are given in the sequences with SEQ ID NO: 19-21.
- the aptamer of the present invention non-competitively binds to human sIL-6R. This does not affect the binding of mterleukin 6 (IL-6) to sIL-6R.
- the signal transduction can be modified or prevented. This can be effected, for example, by attaching the gpl30 or the IL6 sterically or otherwise by means of an antibody directed against the RNA aptamer or, for example, by a molecule coupled to the RNA aptamer, for example a peptide, a nucleic acid or another compound, is hampered.
- the aptamer bound to the receptor in turn is bound by a binding molecule, for example an antibody directed against the aptamer.
- a binding molecule for example an antibody directed against the aptamer.
- an indirect mechanism can also be used so that, for example, a molecule coupled to the RNA aptamer (eg biotin or an antigen) is in turn bound by another molecule (eg avidin or an antibody). It may also be advantageous in diagnostic methods if the RNA aptamer binds non-competitively to the sIL-6R.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising an aptamer of the invention.
- the pharmaceutical composition moreover preferably comprises suitable carrier material, excipients and the like.
- the drug may also contain one or more other active ingredients.
- the active ingredients may also be coupled to the RNA aptamer, i. covalently or non-covalently bound.
- Suitable formulations and dosage forms are known to those skilled in the art or may be routinely prepared according to the prior art.
- Aptamers according to the invention can, for example, also be bound to nanoparticles which are loaded with other active substances, thereby allowing a targeted delivery of the active substances.
- the invention also relates to the use of a
- Aptamers according to the invention for the preparation of a medicament for the preparation of a medicament, as indicated above, preferably a medicament for the treatment of a condition or a disease associated with non-normal concentrations of the sIL-6R in body fluid, eg blood, or with a non-normal occurrence of IL-6R , or a diagnostic agent, for example, to diagnose a disease with the normal state deviating concentrations of sIL-6R in body fluid, such as blood, or are associated with a non-normal occurrence of IL-6R.
- the medicament is thereby preferably intended for the treatment of inflammatory conditions, cancer or infections, for example rheumatoid arthritis, sepsis, asthma, Crohn's disease, multiple sclerosis, depression, breast cancer, multiple myeloma or an HTV infection.
- inflammatory conditions for example rheumatoid arthritis, sepsis, asthma, Crohn's disease, multiple sclerosis, depression, breast cancer, multiple myeloma or an HTV infection.
- Fig. 1 Binding of the aptamer 16-3 to the target molecule sEL-6R.
- the proportion of bound RNA aptamer 16-3 (in%) was at defined sIL-6R concentrations in
- Filter binding studies were determined and plotted as a function of the sIL-6R concentration (logarithmic plot). The measurement points and error bars shown result from the averages and standard deviations of ten independent studies. The dissociation constants were calculated using a "one-site-modeling" model.
- Fig. 2 Gel shift assay for analyzing the interaction between aptamer 16-3 and sIL-6R.
- Fig. 3 Gel shift assay to analyze the specific interaction between aptamer 16-3 and the target molecule sIL-6R using two control proteins.
- the aptamer 16-3 was tested for interaction with the control proteins CEACAM1 (lanes 2-5: 10 nM - 300 nM) and lysozyme (lanes 7-10: 10 nM - 300 nM) by gel shift assay in a 5% , native PAA gel analyzed.
- the positive control used was an approach with sIL-6R (75 nM, lane 6).
- Fig. 4 gel-shift analysis of the specific interaction between aptamer 16-3 and the
- Target molecule sIL-6R with associated controls.
- additional competition between labeled and unlabelled aptamer (lanes 3 and 7) and labeled aptamer and unlabeled control RNA R24 (lane 8).
- Lane 4 shows the binding of an anti-His-AK to sIL-6R aptamer complexes
- lane 9 the interaction between aptamer and anti-His-AK.
- FIG. 5 Filter binding study of RNA aptamer 16-3 on sIL-6R in the presence and absence of IL-6.
- A The protein sIL-6R was in increasing concentration (0-300 nM) with radiolabelled aptamer 16-3 in the presence of 740 nM IL-6 (+ IL-6) or in the absence of IL-6 (-IL- 6) and filtered. The amounts of labeled RNA remaining on the filter are illustrated. To calculate the percentage of bound RNA, a defined amount (125%) of RNA was applied to the membrane in each case.
- RNA aptamer 16-3 The proportion of bound RNA aptamer 16-3 (in%) at defined sIL-6R concentrations in the presence of 740 nM IL-6 (+ IL-6) or in the absence of IL-6 (-IL-6 ) was determined by filter binding studies and plotted (logarithmic plot). The dissociation constants were calculated using a one-site binding model. It was a double determination.
- Fig. 6 Gel-shift assay for analysis of possible competition between aptamer 16-3 and IL-6 for binding to sIL-6R.
- the separation of the aptamer-protein mixtures was carried out in 5% native PAA gel. Detection was by autoradiography.
- Fig. 7 The fusion protein Hyper-IL-6. Schematically shown is the hyper IL-6 fusion construct consisting of the N- and C-terminal truncated sIL-6R (AS 113-323) and IL 6, which are linked together via a flexible linker. Linker amino acids are given in single letter code.
- Hyper-IL-6 and sgpl30Fc Interaction partners Hyper-IL-6 and sgpl30Fc.
- Hyper-IL-6 (lane 2), sgpl30Fc (lane 3) and both proteins in mixture (lane 4) were radiolabeled with aptamer 16-3 ( ⁇ 1 nM). incubated. After gel electrophoresis (5%, native PAA gel) and drying of the gel, the bands were detected by autoradiography.
- Fig. 9 Shortening of the aptamer 16-3 on the basis of the predicted secondary structure using the mfold web server program.
- A. In addition to the secondary structure of the "wild type" aptamer 16-3 (SEQ ID NO: 17), the variants 16-3 A, 16-3_B and 16-3_C (SEQ ID NO 19-21) based thereon are abbreviated.
- B. RNA sequences of the truncated variants of the sIL-6R-specific aptamer 16-3 in the 5 '-3' direction.
- Fig. 10 Binding of the aptamer 16-3 and truncated variants of sIL-6R. The proportion of bound RNA (in%) at defined sIL-6R concentrations was in
- Filter binding studies determined and presented as a function of the sIL-6R concentration (logarithmic plot). The measurement points and error bars shown represent mean values and standard deviations from at least three independent studies. The dissociation constants were calculated using a one-site binding model.
- variant 16 3B guanine residues possibly involved in the consensus sequence (in 16 3 B in normal writing) were replaced by uracil residues (bold).
- the mutation of the G at position 15 characterizes variant 16 3 B M1.
- additional variants were created: 16 3 B M2 - 16-3_B_M5.
- Subscript numbers exemplify the position of the individual bases.
- Fig. 12 Mutation analysis for the binding of aptamers 16-3, 16-3 B and five variants of hyper-IL-6. The proportion of relative RNA binding (in%) was determined at a defined sIL-6R concentration of 300 nM using filter binding studies. The measurement points and error bars shown represent mean values and standard deviations from two independent studies. The relative binding of the aptamer 16-3 B and the variants relates to the binding of the "wild-type" aptamer 16-3. Fig. 13 Gel shift assay for analysis of variants of aptamer 16-3B.
- Ligands by exponential enrichment in vitro selection process (Tuerk, C. and L. Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science, 1990. 249 (4968): p. 10) used.
- this process in steps of iterative cycles, (1) incubation of a nucleic acid library with the target molecule, (2) separation of binding from non-binding nucleic acids, and (3) amplification of binding species, i. an accumulation of binding
- RNA library high diversity about 10 13 different molecules
- on magnetic particles the on magnetic particles (Dynabeads ®) immobilized sIL-6R (the amino acid sequence of sIL-6R see SEQ ID NO: 11) soluble variant of the membrane-bound interleukin-6 receptor (IL-6R).
- Immobilization was biotinylated of sIL-6R and then immobilized interaction on the surface of streptavidin-coupled Dynabeads ® via a biotin-streptavidin.
- the protein-coupled particles were used for aptamer selection. After separation of non-binding nucleic acids by means of magnetic separation and washing steps, binding RNA species were eluted by heating and amplified by means of an RT-PCR reaction. Subsequent T7 transcription formed the transition to the following round of selection. After about 6-20 of these iterative cycles, the enriched library was cloned and sequenced. An RNA start library of sufficiently high sequence diversity was produced. A synthetically prepared ssDNA library Rl (Metabion, Kunststoff) was used for this purpose
- the ssDNA library Rl had the following basic structure: 5'-AATGCTAATACGACTCACTATAGGAAGAAAGAGGTCTGAGACATTCT-N60- CnCTGGAGTTGACGnGCTT-3 '
- the sequence of the ssDNA library Rl was characterized by a randomized region of 60 nucleotides flanked by 47 and 21 nucleotide constant regions at both the 3 'and 5' ends. These served to attach two
- the T7 primer region included the sequence of the T7 promoter (TAATACGACTCACTATAGG), which served as the recognition sequence of the T7 RNA polymerase, and transcribed the dsDNA into the ssRNA start library (106 nt, -3.6 x 10 13 molecules ) with the basic structure
- RNA molecules were transcribed into cDNA after addition of the RT primer RI by means of reverse transcription and in a
- RNA starting library Rl 500 pmol
- Dynabeads ® sIL-6R 100 pmol
- Selection Buffer B (10x PBS (0.137 M NaCl, 2.7 mM KCl, 6.5 mM Na 2 HP0 4 , 15 mM KH 2 PO 4 ); 3 mM MgCl 2 ; pH 7.4) for 30 min at room temperature (RT ).
- the binding to the target RNA molecules were separated by magnetic separation of non-binding molecules, discarded the supernatant and the RNA-target complexes washed once with 100 .mu.l x selection buffer B.
- the elution of binding nucleic acids was carried out after careful resuspension in 55 ⁇ aqua dest. under heat at 80 ° C for 3 min. This eluate was subjected directly to RT-PCR without further purification steps.
- RT-PCR was monitored by PAGE (10% native polyacrylamide (PAA) gel).
- PAGE 100% native polyacrylamide (PAA) gel.
- the RT-PCR product was directly subjected to in vitro T7 transcription using T7 RNA polymerase. Without further purification, 20 were incubated ⁇ of the transcription approach with the immobilized on Dynabeads ® sIL-6R. After separation of non-binding RN A molecules, the beads were washed twice with 100 ⁇ l lx selection buffer B, the number of washing steps increasing to four with increasing round of selection. After sixteen rounds, the selection was ended. The dsDNA library of round 9 and final round 16 decreased
- sequences given include both the 60 nucleotide randomized region and the flanking 5 'and 3' primer regions, except for the T7 promoter sequence (TAATACGACTCACTATAGG), of which only the two terminal G nucleotides are included.
- T7 promoter sequence TAATACGACTCACTATAGG
- the number in front of the hyphen indicates the selection round (9 or 16).
- Target molecule sIL-6R and to determine dissociation constants (Kd values) Filter binding studies performed under radioactive conditions. In addition, gel-shift studies using polyacrylamide gel electrophoresis (PAGE) and
- Dissociation constants were carried out by means of filter binding studies. For this purpose, a constant amount ( ⁇ 1 nM) of radioactively labeled RNA (the labeling was carried out by incorporation of ⁇ x- [ 32 P] -ATP (0.1 ⁇ / ⁇ , 100 nM) during T7 transcription) with increasing concentrations (1 nM to 300 nM) of protein incubated for 20 min at RT. Unless otherwise stated, the buffer used was the lx selection buffer B (1 ⁇ PBS (0.137 M NaCl, 2.7 mM KCl, 6.5 mM Na 2 HPO 4 , 15 mM KH 2 PO 4 ), 3 mM MgCl 2 ; pH 7.4).
- a nitrocellulose membrane was pretreated for 10 minutes with aminohexanoic acid buffer (20% methanol, 40 mM aminohexanoic acid (C 6 H 13 N0 2 )), clamped in a 96-well dot blot apparatus (Schleicher & Schull) and under vacuum twice with 200 ⁇ lx selection buffer B washed.
- aminohexanoic acid buffer (20% methanol, 40 mM aminohexanoic acid (C 6 H 13 N0 2 )
- the reactions were filtered through the nitrocellulose membrane. Protein binding RNA molecules were retained on the membrane. Non-binding RNA molecules were removed by washing four times with lx Selection Buffer B.
- the nitrocellulose membrane was dried and the amounts of radioactively labeled substances remaining on the membrane were quantified using a phosphorimager (BioRad). The evaluation of the data obtained was carried out using the software Quantity One ® (BioRad). For the plot of binding curves the program GraphPad Prism® (GraphPad Software, Inc., USA) was used. The dissociation constant (K d ) was determined according to a one-site binding model based on the following equation:
- Nitrocellulose membrane remaining amount of radiolabeled nucleic acids was quantified as described above and used to determine the dissociation constant.
- Table 1 Characteristics of aptamers 16-1, 16-2, 16-3 and 16-8.
- the aptamer 16-3 exhibited a very affinity binding to the sIL-6R with a significantly lower dissociation constant (K d ).
- K d dissociation constant
- PAGE Polyacrylamide gel electrophoresis Due to their size, aptamer-protein complexes formed under native conditions have a delayed migration behavior in the gel. The complexes therefore migrate more slowly than the corresponding free RNA molecules and are therefore detectable as so-called "shift" in the gel.
- Equal amounts ( ⁇ 1 nM) a- [ 32 P] radiolabelled RNA were incubated with increasing amounts of protein (1 nM - 300 nM) in lx selection buffer B (see above) for 30 min at RT. Samples were seeded with 6x DNA sample buffer (50% (w / v) sucrose; 1% (w / v) SDS; 0.1% (w / v) orange
- TEMED ( ⁇ , ⁇ , ⁇ ⁇ ⁇ '-tetramemyl-enylenediamine) 0.1% (v / v); APS (ammonium persulfate) 0.1% (w / v)).
- the electrophoretic separation was carried out at 80 V and 4 ° C in lx TBE. After completion of the electrophoresis, the gel was dried by applying a vacuum. The radioactive bands were detected by autoradiography.
- the aptamer 16-3 clearly showed an interaction with the sIL-6R, since the gel showed a "shift” caused by the complex formation (see Fig. 2).
- the intensity of this slowed band increased with increasing concentration of sIL-6R ( Figure 2, from left to right).
- Figure 2 In the area of gelatine is also a concentration-dependent
- RNA aptamers were specific for the target molecule sIL-6R.
- these proteins were, on the one hand, CEACAM1, a cell adhesion molecule which carried a C-terminal His tag analogously to sIL-6R.
- the other protein was lysozyme, an enzyme that can cleave polysaccharides of prokaryotic cell walls. Both proteins should not interact with the sIL-6R specific ribonucleic acids.
- RNA band retarded in their behavior was indeed due to a specific interaction between aptamer and sIL-6R.
- a> 1000-fold molar excess of unlabelled aptamer was added (lane 3, lane 7) ).
- the labeled and unlabeled RNA molecules competed for binding to the soluble IL-6R, resulting in the weakening of the retarded band (competition).
- Antibody complexes were evident in the gel by a supershift, ie an even stronger one delayed band compared to the aptamer-protein complex (lane 4).
- the anti-His antibody showed no interaction with the aptamer 16-3 alone ( Figure 4, lane 9).
- RNA aptamers In in vitro competition studies, the interaction between the selected RNA aptamers and the sIL-6R was investigated in the presence of its natural ligands IL-6 and gpl30 as well as of hyper-IL-6. 2.3.1 Competition analysis between aptamer 16-3 and interleukin-6 (IL-6)
- the aptamer 16-3 and IL-6 thus did not have the same binding site on the receptor.
- the concentration of IL-6 was increased to 1.5 ⁇ . Also, this high ligand concentration had no influence on the aptamer bond.
- Hyper-IL-6 is a bioactive fusion protein of IL-6 and sIL-6R, where both interaction partners are linked by a flexible polypeptide linker.
- This linker is composed of the 16 N-terminal non-helical amino acids of IL-6 and thirteen other amino acids, predominantly glycine and serine.
- the N-terminal immunoglobulin-like domain Dl and the C-terminus of the sIL-6R have been omitted, since both are not suitable for the
- the aptamer thus interacted with hyper-IL-6 at a region unequal to the sgpl30Fc binding site.
- the aptamer did not interact with sgpl30Fc alone ( Figure 8, lane 3).
- aptamer 16-3 was truncated to a 19mer (16-3A; SEQ ID NO: 19) which retained only the conserved, G-rich region of the aptamer.
- the truncated RNA 16-3B (47 nt; SEQ ID NO: 20) included, in addition to the G-rich region, the stabilizing, double-stranded region consisting of nine base pairs.
- the third truncated RNA, 63mer 16-3C (SEQ ID NO: 20), was characterized by the absence of the constant primer regions of the original sequence (the sequence contained three additional G nucleotides at the 5 'end of the randomized region of 60 nucleotides in length ). 16-3_A (19 nucleotides, see SEQ ID NO: 19):
- variant 16-3 B a Ka value of about 26.4 nM (Table 2) could be determined.
- the affinity of the 47mer 16-3 B was in the range of the "wild-type" aptamer. This significant shortening thus had no influence on the affinity for the receptor.
- the variant 16-3 A reduced to the G-rich region, which only consisted of 19 nucleotides, could bind the protein, but with reduced binding affinity (Kd ⁇ 446 nM) compared to 16-3.
- the absence of the primer regions in 16 3_C also weakened the affinity for the target protein (Kd - 117 nM).
- Mutation within the GG pairs involved in the quadruplex formation should result in loss of binding to the hyper-IL-6 protein.
- the mutational analyzes were in the form of filter binding studies as described above. The binding of the truncated variants was compared in each case with the binding portion of the aptamer 16-3. The result of the analysis is shown in FIG. 12.
- the aptamers 16-3 and 16-3 B resembled each other in their strong binding behavior towards hyper-IL-6.
- the exchange of defined guanine residues within the three mutants 16-3_B_ M3, 16-3_B_M4 and 16-3 B M5 led to one
- the results of the mutation analyzes are consistent with the assumption of a possible formation of a G-Quaclmplex Srruktur.
- the loss of function of the four mutants 16-3 B M1, 16-3_B_M3, 16-3_B_M4 and 16-3JB M5 indicates that the guanine residues 15, 21, 27 and 32 of the aptamer 16-3 B are essential for the aptamer bond and perhaps in the
- BAF / gpl30 / IL6R / TNF examined.
- the cell lines BAF / gpl30 and BAF / gp 130 / IL6R / TNF were derived from the murine pre-B cell line BAF / 3, an immortalized, the
- Bone marrow-derived pro-B cell line whose growth and proliferation depends on the presence of the cytokine IL-3. The growth of the BAF / 3 cells used here was dependent on IL-6. Cells of this cell line were probed with cDNA for gp 130
- BAF / gpl30 or the cDNAs for gpl30, TNF and IL-6R (BAF / gp 130 / IL6R / TNF) stably transfected.
- the BAF / gp 130 / IL-6R TNF cells were able to produce TNF, in addition to the production of gpl30 and IL-6R, but this was not essential in the context of subsequent aptamer studies.
- the BAF / gpl30 and BAF / gpl30 / IL-6R / TNF cell lines were cultured in 25 cm 2 bottles and kept in the incubator at 37 ° C.
- the culture medium DMEM Dulbecco's
- Hyper IL 6 (10 ng / ml) was additionally added to the BAF / gpl30 cells, the BAF / gpl30 / IL-6R / TNF cells additionally the cytokine IL 6 (10 ng / ml) and the antibiotic puromycin (12 ⁇ g / ml). ml).
- the growth of the cells was also observed in the absence of the cytokines, since without these stimuli cell growth was not possible.
- the presence of the interleukin 6 receptor on the surface of the BAF / gp 130 / IL6R / TNF cells was controlled using a murine monoclonal IL-6R specific IgG1 antibody (not shown).
- RNA molecules for 5 min at RT. Separation of unbound RNA molecules was followed by centrifugation of the cells at 500 g for 5 min. The pellet was washed twice with 500 ⁇ l of Selection Buffer B.
- Resuspension in 350 ⁇ l lx selection buffer B was the aptamer binding on
- BAF / gpl30 / IL6R / TNF cells were cultured in 25 cm 2 culture bottles (see above). For harvest, the cells were centrifuged for 5 min at 500 g and RT. The supernatant was discarded, the pellet was taken up in 5 ml of DMEM (without FCS) and the cell count determined using a Neubauer counting chamber.
- Fluorescence labeling of the RNA was carried out here with AlexaFluor® 647 (Invitrogen).
- the anti-hIL-6R antibody in combination with the goat anti-mouse secondary AK blocked the specific interaction of the aptamer with the BAF / gpl30 / IL6R / TNF cells.
- This result demonstrates simultaneously the specific binding of the aptamer to the native IL-6R on the surface of BAF / gpl30 / IL6R TNF cells, since the anti-hIL-6R antibody was directed against the IL-6R.
- Nonspecific binding to IL-6R deficient BAF / gpl30 control cells was not observed.
- RNA aptamer RNA aptamer
- randomized region plus 5 'and 3' primer regions randomized area plus 5 'and 3'
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
L'invention concerne des aptamères se liant spécifiquement à une molécule cible. Le but de l'invention est de fournir des agents diagnostiques et/ou thérapeutiques destinés à être utilisés pour le dépistage et/ou la prophylaxie ou le traitement de processus inflammatoires, de maladies cancéreuses et d'infections. A cet effet, les aptamères d'ARN de l'invention se lient spécifiquement au récepteur soluble humain de l'interleukine 6 (sIL-6R).
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE200910056944 DE102009056944A1 (de) | 2009-12-07 | 2009-12-07 | RNA-Aptamere, die den löslichen Interleukin-6-Rezeptor spezifisch binden |
DE102009056944.8 | 2009-12-07 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2011069486A1 true WO2011069486A1 (fr) | 2011-06-16 |
Family
ID=43708793
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DE2010/001412 WO2011069486A1 (fr) | 2009-12-07 | 2010-12-06 | Aptamères d'arn se liant spécifiquement au récepteur soluble de l'interleukine 6 |
Country Status (2)
Country | Link |
---|---|
DE (1) | DE102009056944A1 (fr) |
WO (1) | WO2011069486A1 (fr) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991019813A1 (fr) | 1990-06-11 | 1991-12-26 | The University Of Colorado Foundation, Inc. | Ligands d'acide nucleique |
-
2009
- 2009-12-07 DE DE200910056944 patent/DE102009056944A1/de not_active Withdrawn
-
2010
- 2010-12-06 WO PCT/DE2010/001412 patent/WO2011069486A1/fr active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991019813A1 (fr) | 1990-06-11 | 1991-12-26 | The University Of Colorado Foundation, Inc. | Ligands d'acide nucleique |
Non-Patent Citations (21)
Title |
---|
B. OSBORNE ET AL., CURR. OPIN. CHEM. BIOL., vol. 1, 1997, pages 5 - 9 |
CINDY MEYER: "Interleukin-6-Rezeptor-spezifische RNA-Aptamere", 28 January 2010 (2010-01-28), XP002631143, Retrieved from the Internet <URL:http://ediss.sub.uni-hamburg.de/volltexte/2010/4459/pdf/Dissertation_Cindy_Meyer.pdf> [retrieved on 20110317] * |
DAUSSE E ET AL: "Aptamers: a new class of oligonucleotides in the drug discovery pipeline?", CURRENT OPINION IN PHARMACOLOGY, ELSEVIER SCIENCE PUBLISHERS, NL, vol. 9, no. 5, 1 October 2009 (2009-10-01), pages 602 - 607, XP026665554, ISSN: 1471-4892, [retrieved on 20090828], DOI: DOI:10.1016/J.COPH.2009.07.006 * |
ELLINGTON, SZOSTAK, NATURE, vol. 346, 1990, pages 818 - 822 |
FISCHER, M. ET AL.: "I. A bioactive designer cytokine for human hematopoietic progenitor cell expansion", NAT BIOTECHNOL, vol. 15, no. 2, 1997, pages 142 - 5 |
GOPINATH, ANAL. BIOANAL. CHEM., vol. 387, 2007, pages 171 - 182 |
HIROTA, H. ET AL., PROC. NATL. ACAD. SCI. U.S.A, vol. 92, 1995, pages 4862 - 4866 |
JONES ET AL., THE FASEB JOURNAL, vol. 15, 2001, pages 43 - 57 |
JOSTOCK, T. ET AL.: "Soluble gp130 is the natural inhibitor of soluble interleukin-6 receptor transsignaling responses", EUR J BIOCHEM, vol. 268, no. 1, 2001, pages 160 - 7 |
MACKIEWICZ, A. ET AL., J. IMMUNOL., vol. 149, 1992, pages 2021 - 2027 |
MAJUMDER PAROMITA ET AL: "Aptamers: from bench side research towards patented molecules with therapeutic applications.", EXPERT OPINION ON THERAPEUTIC PATENTS NOV 2009 LNKD- PUBMED:19852719, vol. 19, no. 11, November 2009 (2009-11-01), pages 1603 - 1613, XP002628467, ISSN: 1744-7674 * |
ROSE-JOHN ET AL., J. J. LEUKOCYTE BIOL., vol. 80, 2006, pages 227 - 235 |
SAITO, M. ET AL., J. IMMUNOL., vol. 148, 1992, pages 4066 - 4071 |
SCHELLER, J., GRÖTZINGER, J., ROSE-JOHN, STEFAN: "Zytokinsignale über lösliche und membranständige Rezeptoren", BIOSPEKTRUM, vol. 13, 2007, pages 250 - 253 |
SCHELLER, ROSE-JOHN, MED. MICROBIOL. IMMUNOL., vol. 195, 2006, pages 173 - 183 |
SHINRIKI SATORU ET AL: "Humanized Anti-Interleukin-6 Receptor Antibody Suppresses Tumor Angiogenesis and In vivo Growth of Human Oral Squamous Cell Carcinoma", CLINICAL CANCER RESEARCH, vol. 15, no. 17, September 2009 (2009-09-01), pages 5426 - 5434, XP002628465, ISSN: 1078-0432 * |
TAGA, T. ET AL., CELL, vol. 58, 1989, pages 573 - 581 |
TUERK, C., L. GOLD: "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase", SCIENCE, vol. 249, no. 4968, 1990, pages 505 - 10 |
YAMAGUCHI JUNICHI ET AL: "cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions", NUCLEIC ACIDS RESEARCH, vol. 37, no. 16, September 2009 (2009-09-01), XP002631169, ISSN: 0305-1048 * |
YOKOTA SHUMPEI ET AL: "Clinical study of tocilizumab in children with systemic-onset juvenile idiopathic arthritis.", CLINICAL REVIEWS IN ALLERGY & IMMUNOLOGY JUN 2005 LNKD- PUBMED:16129907, vol. 28, no. 3, June 2005 (2005-06-01), pages 231 - 237, XP002628466, ISSN: 1080-0549 * |
ZUKER, M.: "Mfold web server for nucleic acid folding and hybridization prediction", NUCLEIC ACIDS RESEARCH, vol. 31, no. 13, 2003, pages 3406 - 3415 |
Also Published As
Publication number | Publication date |
---|---|
DE102009056944A1 (de) | 2011-06-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0934331B1 (fr) | Selection et evolution speculaires d'acides nucleiques | |
EP4008784A1 (fr) | Arn guide pour édition ciblée avec séquence de base fonctionnelle ajoutée à celui-ci | |
WO2021060527A1 (fr) | Oligonucléotide et procédé d'édition spécifique d'un site d'arn cible | |
US9044495B2 (en) | Nucleic acid ligands to LL37 | |
WO2003093504A1 (fr) | Procede d'amplification d'acides nucleiques | |
WO1999027133A1 (fr) | Procedure selex amelioree et aptamere anti-cd4 | |
CN108368509A (zh) | 改善了血中滞留性的抗il-17适体 | |
US20220251562A1 (en) | Anti-CD3 Aptamers for Use in Cell Targeting and Labeling | |
JP7333508B2 (ja) | オリゴヌクレオチド、その製造方法及び標的rnaの部位特異的編集方法 | |
EP1306382A1 (fr) | L-acides nucléiques modifiés | |
DE102007041476A1 (de) | Aptamere, die an ein an der Hämostase beteiligtes Zielmolekül binden | |
WO2011069486A1 (fr) | Aptamères d'arn se liant spécifiquement au récepteur soluble de l'interleukine 6 | |
DE69929699T2 (de) | Nukleinsäure, die spezifisch an ein ras zielprotein binden kann | |
CN116981773A (zh) | 用于编辑靶标rna的多聚腺苷酸化信号序列的指导rna | |
EP2876163B1 (fr) | Aptamères d'ADN se liant spécifiquement E et P-sélectines | |
WO2011069487A2 (fr) | Aptamères d'adn se liant spécifiquement au récepteur soluble de l'interleukine 6 | |
WO2020181130A2 (fr) | Conjugués de polynucléotides et leurs utilisations | |
US20110092575A1 (en) | Sirna of human osteopontin | |
US9850492B2 (en) | Aptamer to IL-17 and use thereof | |
US20240218351A1 (en) | Single stranded rna purification methods | |
EP1692284B1 (fr) | Apatameres a action anti-apoptotique | |
DE10122847A1 (de) | Enterotoxin B bindende Nukleinsäure | |
KR20240021235A (ko) | 단일 가닥 rna 정제 방법 | |
WO2024123646A2 (fr) | Oligonucléotides modifiés | |
WO2018075264A1 (fr) | Aptamères d'arn chimiquement modifiés et leurs utilisations |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 10803357 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1120100046964 Country of ref document: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 10803357 Country of ref document: EP Kind code of ref document: A1 |