US20240026436A1 - Methods and compositions for the amplification of mrna - Google Patents
Methods and compositions for the amplification of mrna Download PDFInfo
- Publication number
- US20240026436A1 US20240026436A1 US18/324,633 US202318324633A US2024026436A1 US 20240026436 A1 US20240026436 A1 US 20240026436A1 US 202318324633 A US202318324633 A US 202318324633A US 2024026436 A1 US2024026436 A1 US 2024026436A1
- Authority
- US
- United States
- Prior art keywords
- primer
- rna
- mrna
- cdna
- 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.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 134
- 108020004999 messenger RNA Proteins 0.000 title claims description 118
- 239000000203 mixture Substances 0.000 title abstract description 72
- 230000003321 amplification Effects 0.000 title abstract description 21
- 238000003199 nucleic acid amplification method Methods 0.000 title abstract description 21
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 152
- 210000004443 dendritic cell Anatomy 0.000 claims abstract description 132
- 239000002299 complementary DNA Substances 0.000 claims abstract description 82
- 238000000338 in vitro Methods 0.000 claims abstract description 53
- 238000013518 transcription Methods 0.000 claims abstract description 29
- 230000035897 transcription Effects 0.000 claims abstract description 28
- 150000007523 nucleic acids Chemical group 0.000 claims description 107
- 102000039446 nucleic acids Human genes 0.000 claims description 85
- 108020004707 nucleic acids Proteins 0.000 claims description 85
- 206010028980 Neoplasm Diseases 0.000 claims description 81
- 210000004027 cell Anatomy 0.000 claims description 70
- 108020004414 DNA Proteins 0.000 claims description 40
- 239000000427 antigen Substances 0.000 claims description 32
- 108091007433 antigens Proteins 0.000 claims description 32
- 102000036639 antigens Human genes 0.000 claims description 32
- 108091034117 Oligonucleotide Proteins 0.000 claims description 27
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 24
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 21
- 210000004881 tumor cell Anatomy 0.000 claims description 14
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 10
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 9
- 230000036961 partial effect Effects 0.000 claims description 9
- 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 claims description 8
- 239000013592 cell lysate Substances 0.000 claims description 8
- 230000028993 immune response Effects 0.000 claims description 8
- 230000002194 synthesizing effect Effects 0.000 claims description 8
- 230000001419 dependent effect Effects 0.000 claims description 5
- 238000003752 polymerase chain reaction Methods 0.000 claims description 4
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 claims description 3
- 102100029764 DNA-directed DNA/RNA polymerase mu Human genes 0.000 claims description 3
- 239000011541 reaction mixture Substances 0.000 claims description 3
- 108091081021 Sense strand Proteins 0.000 claims description 2
- 230000002463 transducing effect Effects 0.000 claims description 2
- 102100034343 Integrase Human genes 0.000 claims 2
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims 1
- 239000006166 lysate Substances 0.000 abstract description 51
- 238000010839 reverse transcription Methods 0.000 abstract description 23
- 238000011068 loading method Methods 0.000 abstract description 15
- 238000003786 synthesis reaction Methods 0.000 abstract description 13
- 230000015572 biosynthetic process Effects 0.000 abstract description 12
- 239000012634 fragment Substances 0.000 abstract description 2
- 230000000638 stimulation Effects 0.000 abstract description 2
- 238000010804 cDNA synthesis Methods 0.000 description 55
- 108020004635 Complementary DNA Proteins 0.000 description 53
- 210000001519 tissue Anatomy 0.000 description 35
- -1 nucleoside triphosphates Chemical class 0.000 description 31
- 239000002243 precursor Substances 0.000 description 30
- 239000002773 nucleotide Substances 0.000 description 27
- 125000003729 nucleotide group Chemical group 0.000 description 27
- 239000000758 substrate Substances 0.000 description 25
- 238000006243 chemical reaction Methods 0.000 description 24
- 239000000523 sample Substances 0.000 description 23
- 238000002360 preparation method Methods 0.000 description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 21
- 230000000295 complement effect Effects 0.000 description 20
- 239000000243 solution Substances 0.000 description 18
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 17
- 230000000890 antigenic effect Effects 0.000 description 17
- 102100031780 Endonuclease Human genes 0.000 description 16
- 238000011282 treatment Methods 0.000 description 16
- 229940045513 CTLA4 antagonist Drugs 0.000 description 15
- 239000003795 chemical substances by application Substances 0.000 description 15
- 239000006228 supernatant Substances 0.000 description 15
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 14
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 14
- 201000011510 cancer Diseases 0.000 description 14
- 230000008569 process Effects 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- 238000005119 centrifugation Methods 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 12
- 238000004519 manufacturing process Methods 0.000 description 12
- 108091033319 polynucleotide Proteins 0.000 description 12
- 102000040430 polynucleotide Human genes 0.000 description 12
- 239000002157 polynucleotide Substances 0.000 description 12
- 210000001744 T-lymphocyte Anatomy 0.000 description 11
- 230000014759 maintenance of location Effects 0.000 description 11
- 239000000872 buffer Substances 0.000 description 10
- 210000000265 leukocyte Anatomy 0.000 description 10
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 9
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 9
- 108010083644 Ribonucleases Proteins 0.000 description 9
- 102000006382 Ribonucleases Human genes 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 230000014509 gene expression Effects 0.000 description 9
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 8
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 8
- 238000000684 flow cytometry Methods 0.000 description 8
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 8
- 230000001965 increasing effect Effects 0.000 description 8
- 239000008188 pellet Substances 0.000 description 8
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 8
- 238000001262 western blot Methods 0.000 description 8
- 102000004388 Interleukin-4 Human genes 0.000 description 7
- 108090000978 Interleukin-4 Proteins 0.000 description 7
- 229940028885 interleukin-4 Drugs 0.000 description 7
- 230000003834 intracellular effect Effects 0.000 description 7
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 7
- 102000004127 Cytokines Human genes 0.000 description 6
- 108090000695 Cytokines Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 6
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 6
- 102100027268 Interferon-stimulated gene 20 kDa protein Human genes 0.000 description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 101710163270 Nuclease Proteins 0.000 description 6
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 6
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000001356 surgical procedure Methods 0.000 description 6
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 5
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 5
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 5
- 102100022297 Integrin alpha-X Human genes 0.000 description 5
- 239000013614 RNA sample Substances 0.000 description 5
- 230000021839 RNA stabilization Effects 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000003501 co-culture Methods 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 230000035800 maturation Effects 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 210000004882 non-tumor cell Anatomy 0.000 description 5
- 239000002777 nucleoside Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 210000002381 plasma Anatomy 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 230000002441 reversible effect Effects 0.000 description 5
- 102100035793 CD83 antigen Human genes 0.000 description 4
- 108010053770 Deoxyribonucleases Proteins 0.000 description 4
- 102000016911 Deoxyribonucleases Human genes 0.000 description 4
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 4
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 4
- 102000003816 Interleukin-13 Human genes 0.000 description 4
- 108090000176 Interleukin-13 Proteins 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000002659 cell therapy Methods 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 238000012239 gene modification Methods 0.000 description 4
- 230000005017 genetic modification Effects 0.000 description 4
- 235000013617 genetically modified food Nutrition 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- 238000010348 incorporation Methods 0.000 description 4
- 210000004379 membrane Anatomy 0.000 description 4
- 239000002679 microRNA Substances 0.000 description 4
- 210000001616 monocyte Anatomy 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- 238000002271 resection Methods 0.000 description 4
- 239000003161 ribonuclease inhibitor Substances 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- WYWHKKSPHMUBEB-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 4
- 239000003104 tissue culture media Substances 0.000 description 4
- 239000001226 triphosphate Substances 0.000 description 4
- 235000011178 triphosphate Nutrition 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- FZWGECJQACGGTI-UHFFFAOYSA-N 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one Chemical compound NC1=NC(O)=C2N(C)C=NC2=N1 FZWGECJQACGGTI-UHFFFAOYSA-N 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 108010017826 DNA Polymerase I Proteins 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 229910052693 Europium Inorganic materials 0.000 description 3
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 3
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 3
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 3
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 3
- 206010027476 Metastases Diseases 0.000 description 3
- 238000002123 RNA extraction Methods 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
- 229960000643 adenine Drugs 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 210000000612 antigen-presenting cell Anatomy 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 229930195731 calicheamicin Natural products 0.000 description 3
- HXCHCVDVKSCDHU-LULTVBGHSA-N calicheamicin Chemical compound C1[C@H](OC)[C@@H](NCC)CO[C@H]1O[C@H]1[C@H](O[C@@H]2C\3=C(NC(=O)OC)C(=O)C[C@](C/3=C/CSSSC)(O)C#C\C=C/C#C2)O[C@H](C)[C@@H](NO[C@@H]2O[C@H](C)[C@@H](SC(=O)C=3C(=C(OC)C(O[C@H]4[C@@H]([C@H](OC)[C@@H](O)[C@H](C)O4)O)=C(I)C=3C)OC)[C@@H](O)C2)[C@@H]1O HXCHCVDVKSCDHU-LULTVBGHSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 150000001768 cations Chemical class 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000002512 chemotherapy Methods 0.000 description 3
- 230000000139 costimulatory effect Effects 0.000 description 3
- 238000011498 curative surgery Methods 0.000 description 3
- 229940104302 cytosine Drugs 0.000 description 3
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 3
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 3
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 3
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 3
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 3
- 229960002949 fluorouracil Drugs 0.000 description 3
- 229960002584 gefitinib Drugs 0.000 description 3
- XGALLCVXEZPNRQ-UHFFFAOYSA-N gefitinib Chemical compound C=12C=C(OCCCN3CCOCC3)C(OC)=CC2=NC=NC=1NC1=CC=C(F)C(Cl)=C1 XGALLCVXEZPNRQ-UHFFFAOYSA-N 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000001415 gene therapy Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 3
- 108091070501 miRNA Proteins 0.000 description 3
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 3
- 150000003833 nucleoside derivatives Chemical class 0.000 description 3
- 230000037452 priming Effects 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 210000002307 prostate Anatomy 0.000 description 3
- XKMLYUALXHKNFT-UHFFFAOYSA-N rGTP Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O XKMLYUALXHKNFT-UHFFFAOYSA-N 0.000 description 3
- 238000001959 radiotherapy Methods 0.000 description 3
- 239000011535 reaction buffer Substances 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 229940113082 thymine Drugs 0.000 description 3
- 230000002103 transcriptional effect Effects 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 229940035893 uracil Drugs 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- HPZMWTNATZPBIH-UHFFFAOYSA-N 1-methyladenine Chemical compound CN1C=NC2=NC=NC2=C1N HPZMWTNATZPBIH-UHFFFAOYSA-N 0.000 description 2
- RFLVMTUMFYRZCB-UHFFFAOYSA-N 1-methylguanine Chemical compound O=C1N(C)C(N)=NC2=C1N=CN2 RFLVMTUMFYRZCB-UHFFFAOYSA-N 0.000 description 2
- 101800001779 2'-O-methyltransferase Proteins 0.000 description 2
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 description 2
- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 description 2
- OVONXEQGWXGFJD-UHFFFAOYSA-N 4-sulfanylidene-1h-pyrimidin-2-one Chemical compound SC=1C=CNC(=O)N=1 OVONXEQGWXGFJD-UHFFFAOYSA-N 0.000 description 2
- RYVNIFSIEDRLSJ-UHFFFAOYSA-N 5-(hydroxymethyl)cytosine Chemical compound NC=1NC(=O)N=CC=1CO RYVNIFSIEDRLSJ-UHFFFAOYSA-N 0.000 description 2
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 2
- CKOMXBHMKXXTNW-UHFFFAOYSA-N 6-methyladenine Chemical compound CNC1=NC=NC2=C1N=CN2 CKOMXBHMKXXTNW-UHFFFAOYSA-N 0.000 description 2
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 2
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- GAGWJHPBXLXJQN-UORFTKCHSA-N Capecitabine Chemical compound C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 GAGWJHPBXLXJQN-UORFTKCHSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- XULFJDKZVHTRLG-JDVCJPALSA-N DOSPA trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F.CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)CCNC(=O)C(CCCNCCCN)NCCCN)OCCCCCCCC\C=C/CCCCCCCC XULFJDKZVHTRLG-JDVCJPALSA-N 0.000 description 2
- 108010092160 Dactinomycin Proteins 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 2
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 2
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 2
- 101150106931 IFNG gene Proteins 0.000 description 2
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 2
- 229930010555 Inosine Natural products 0.000 description 2
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 239000005411 L01XE02 - Gefitinib Substances 0.000 description 2
- 102000043131 MHC class II family Human genes 0.000 description 2
- 108091054438 MHC class II family Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- HYVABZIGRDEKCD-UHFFFAOYSA-N N(6)-dimethylallyladenine Chemical compound CC(C)=CCNC1=NC=NC2=C1N=CN2 HYVABZIGRDEKCD-UHFFFAOYSA-N 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 229940122907 Phosphatase inhibitor Drugs 0.000 description 2
- 108091036407 Polyadenylation Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102000039471 Small Nuclear RNA Human genes 0.000 description 2
- 230000029662 T-helper 1 type immune response Effects 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000030741 antigen processing and presentation Effects 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- VSRXQHXAPYXROS-UHFFFAOYSA-N azanide;cyclobutane-1,1-dicarboxylic acid;platinum(2+) Chemical compound [NH2-].[NH2-].[Pt+2].OC(=O)C1(C(O)=O)CCC1 VSRXQHXAPYXROS-UHFFFAOYSA-N 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 235000011089 carbon dioxide Nutrition 0.000 description 2
- 229960004562 carboplatin Drugs 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 230000000973 chemotherapeutic effect Effects 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
- 238000010367 cloning Methods 0.000 description 2
- 238000002648 combination therapy Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- JSRLJPSBLDHEIO-SHYZEUOFSA-N dUMP Chemical compound O1[C@H](COP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 JSRLJPSBLDHEIO-SHYZEUOFSA-N 0.000 description 2
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 229940029030 dendritic cell vaccine Drugs 0.000 description 2
- 238000000432 density-gradient centrifugation Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- CHPZKNULDCNCBW-UHFFFAOYSA-N gallium nitrate Chemical compound [Ga+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O CHPZKNULDCNCBW-UHFFFAOYSA-N 0.000 description 2
- 239000013029 homogenous suspension Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 229960001101 ifosfamide Drugs 0.000 description 2
- HOMGKSMUEGBAAB-UHFFFAOYSA-N ifosfamide Chemical compound ClCCNP1(=O)OCCCN1CCCl HOMGKSMUEGBAAB-UHFFFAOYSA-N 0.000 description 2
- YLMAHDNUQAMNNX-UHFFFAOYSA-N imatinib methanesulfonate Chemical compound CS(O)(=O)=O.C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 YLMAHDNUQAMNNX-UHFFFAOYSA-N 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 229960003786 inosine Drugs 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 2
- DRAVOWXCEBXPTN-UHFFFAOYSA-N isoguanine Chemical compound NC1=NC(=O)NC2=C1NC=N2 DRAVOWXCEBXPTN-UHFFFAOYSA-N 0.000 description 2
- BCFGMOOMADDAQU-UHFFFAOYSA-N lapatinib Chemical compound O1C(CNCCS(=O)(=O)C)=CC=C1C1=CC=C(N=CN=C2NC=3C=C(Cl)C(OCC=4C=C(F)C=CC=4)=CC=3)C2=C1 BCFGMOOMADDAQU-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229960001428 mercaptopurine Drugs 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 230000011987 methylation Effects 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 229960001156 mitoxantrone Drugs 0.000 description 2
- 239000002991 molded plastic Substances 0.000 description 2
- KVKFRMCSXWQSNT-UHFFFAOYSA-N n,n'-dimethylethane-1,2-diamine Chemical compound CNCCNC KVKFRMCSXWQSNT-UHFFFAOYSA-N 0.000 description 2
- QZGIWPZCWHMVQL-UIYAJPBUSA-N neocarzinostatin chromophore Chemical compound O1[C@H](C)[C@H](O)[C@H](O)[C@@H](NC)[C@H]1O[C@@H]1C/2=C/C#C[C@H]3O[C@@]3([C@@H]3OC(=O)OC3)C#CC\2=C[C@H]1OC(=O)C1=C(O)C=CC2=C(C)C=C(OC)C=C12 QZGIWPZCWHMVQL-UIYAJPBUSA-N 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 125000004430 oxygen atom Chemical group O* 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- CPTBDICYNRMXFX-UHFFFAOYSA-N procarbazine Chemical compound CNNCC1=CC=C(C(=O)NC(C)C)C=C1 CPTBDICYNRMXFX-UHFFFAOYSA-N 0.000 description 2
- 229960000624 procarbazine Drugs 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 239000003909 protein kinase inhibitor Substances 0.000 description 2
- 150000003212 purines Chemical class 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
- 150000003230 pyrimidines Chemical class 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- CYOHGALHFOKKQC-UHFFFAOYSA-N selumetinib Chemical compound OCCONC(=O)C=1C=C2N(C)C=NC2=C(F)C=1NC1=CC=C(Br)C=C1Cl CYOHGALHFOKKQC-UHFFFAOYSA-N 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- PVYJZLYGTZKPJE-UHFFFAOYSA-N streptonigrin Chemical compound C=1C=C2C(=O)C(OC)=C(N)C(=O)C2=NC=1C(C=1N)=NC(C(O)=O)=C(C)C=1C1=CC=C(OC)C(OC)=C1O PVYJZLYGTZKPJE-UHFFFAOYSA-N 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 125000004434 sulfur atom Chemical group 0.000 description 2
- 210000004243 sweat Anatomy 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 229960003087 tioguanine Drugs 0.000 description 2
- 238000003151 transfection method Methods 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000012130 whole-cell lysate Substances 0.000 description 2
- NNJPGOLRFBJNIW-HNNXBMFYSA-N (-)-demecolcine Chemical compound C1=C(OC)C(=O)C=C2[C@@H](NC)CCC3=CC(OC)=C(OC)C(OC)=C3C2=C1 NNJPGOLRFBJNIW-HNNXBMFYSA-N 0.000 description 1
- YWTBGJGMTBHQTM-IBGZPJMESA-N (2S)-1-(1H-indol-3-yl)-3-[[5-(3-methyl-2H-indazol-5-yl)-3-pyridinyl]oxy]-2-propanamine Chemical compound C1=CC=C2C(C[C@H](N)COC=3C=NC=C(C=3)C3=CC=C4NN=C(C4=C3)C)=CNC2=C1 YWTBGJGMTBHQTM-IBGZPJMESA-N 0.000 description 1
- WDQLRUYAYXDIFW-RWKIJVEZSA-N (2r,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-4-[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-[[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxymethyl]oxan-2-yl]oxy-6-(hydroxymethyl)oxane-2,3,5-triol Chemical compound O[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)O1 WDQLRUYAYXDIFW-RWKIJVEZSA-N 0.000 description 1
- FLWWDYNPWOSLEO-HQVZTVAUSA-N (2s)-2-[[4-[1-(2-amino-4-oxo-1h-pteridin-6-yl)ethyl-methylamino]benzoyl]amino]pentanedioic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1C(C)N(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FLWWDYNPWOSLEO-HQVZTVAUSA-N 0.000 description 1
- CGMTUJFWROPELF-YPAAEMCBSA-N (3E,5S)-5-[(2S)-butan-2-yl]-3-(1-hydroxyethylidene)pyrrolidine-2,4-dione Chemical compound CC[C@H](C)[C@@H]1NC(=O)\C(=C(/C)O)C1=O CGMTUJFWROPELF-YPAAEMCBSA-N 0.000 description 1
- TVIRNGFXQVMMGB-OFWIHYRESA-N (3s,6r,10r,13e,16s)-16-[(2r,3r,4s)-4-chloro-3-hydroxy-4-phenylbutan-2-yl]-10-[(3-chloro-4-methoxyphenyl)methyl]-6-methyl-3-(2-methylpropyl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetrone Chemical compound C1=C(Cl)C(OC)=CC=C1C[C@@H]1C(=O)NC[C@@H](C)C(=O)O[C@@H](CC(C)C)C(=O)O[C@H]([C@H](C)[C@@H](O)[C@@H](Cl)C=2C=CC=CC=2)C/C=C/C(=O)N1 TVIRNGFXQVMMGB-OFWIHYRESA-N 0.000 description 1
- XRBSKUSTLXISAB-XVVDYKMHSA-N (5r,6r,7r,8r)-8-hydroxy-7-(hydroxymethyl)-5-(3,4,5-trimethoxyphenyl)-5,6,7,8-tetrahydrobenzo[f][1,3]benzodioxole-6-carboxylic acid Chemical compound COC1=C(OC)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@H](O)[C@@H](CO)[C@@H]2C(O)=O)=C1 XRBSKUSTLXISAB-XVVDYKMHSA-N 0.000 description 1
- XRBSKUSTLXISAB-UHFFFAOYSA-N (7R,7'R,8R,8'R)-form-Podophyllic acid Natural products COC1=C(OC)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C(CO)C2C(O)=O)=C1 XRBSKUSTLXISAB-UHFFFAOYSA-N 0.000 description 1
- AESVUZLWRXEGEX-DKCAWCKPSA-N (7S,9R)-7-[(2S,4R,5R,6R)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione iron(3+) Chemical compound [Fe+3].COc1cccc2C(=O)c3c(O)c4C[C@@](O)(C[C@H](O[C@@H]5C[C@@H](N)[C@@H](O)[C@@H](C)O5)c4c(O)c3C(=O)c12)C(=O)CO AESVUZLWRXEGEX-DKCAWCKPSA-N 0.000 description 1
- JXVAMODRWBNUSF-KZQKBALLSA-N (7s,9r,10r)-7-[(2r,4s,5s,6s)-5-[[(2s,4as,5as,7s,9s,9ar,10ar)-2,9-dimethyl-3-oxo-4,4a,5a,6,7,9,9a,10a-octahydrodipyrano[4,2-a:4',3'-e][1,4]dioxin-7-yl]oxy]-4-(dimethylamino)-6-methyloxan-2-yl]oxy-10-[(2s,4s,5s,6s)-4-(dimethylamino)-5-hydroxy-6-methyloxan-2 Chemical compound O([C@@H]1C2=C(O)C=3C(=O)C4=CC=CC(O)=C4C(=O)C=3C(O)=C2[C@@H](O[C@@H]2O[C@@H](C)[C@@H](O[C@@H]3O[C@@H](C)[C@H]4O[C@@H]5O[C@@H](C)C(=O)C[C@@H]5O[C@H]4C3)[C@H](C2)N(C)C)C[C@]1(O)CC)[C@H]1C[C@H](N(C)C)[C@H](O)[C@H](C)O1 JXVAMODRWBNUSF-KZQKBALLSA-N 0.000 description 1
- INAUWOVKEZHHDM-PEDBPRJASA-N (7s,9s)-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-7-[(2r,4s,5s,6s)-5-hydroxy-6-methyl-4-morpholin-4-yloxan-2-yl]oxy-4-methoxy-8,10-dihydro-7h-tetracene-5,12-dione;hydrochloride Chemical compound Cl.N1([C@H]2C[C@@H](O[C@@H](C)[C@H]2O)O[C@H]2C[C@@](O)(CC=3C(O)=C4C(=O)C=5C=CC=C(C=5C(=O)C4=C(O)C=32)OC)C(=O)CO)CCOCC1 INAUWOVKEZHHDM-PEDBPRJASA-N 0.000 description 1
- RCFNNLSZHVHCEK-IMHLAKCZSA-N (7s,9s)-7-(4-amino-6-methyloxan-2-yl)oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7h-tetracene-5,12-dione;hydrochloride Chemical compound [Cl-].O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)C1CC([NH3+])CC(C)O1 RCFNNLSZHVHCEK-IMHLAKCZSA-N 0.000 description 1
- NOPNWHSMQOXAEI-PUCKCBAPSA-N (7s,9s)-7-[(2r,4s,5s,6s)-4-(2,3-dihydropyrrol-1-yl)-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7h-tetracene-5,12-dione Chemical compound N1([C@H]2C[C@@H](O[C@@H](C)[C@H]2O)O[C@H]2C[C@@](O)(CC=3C(O)=C4C(=O)C=5C=CC=C(C=5C(=O)C4=C(O)C=32)OC)C(=O)CO)CCC=C1 NOPNWHSMQOXAEI-PUCKCBAPSA-N 0.000 description 1
- FPVKHBSQESCIEP-UHFFFAOYSA-N (8S)-3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol Natural products C1C(O)C(CO)OC1N1C(NC=NCC2O)=C2N=C1 FPVKHBSQESCIEP-UHFFFAOYSA-N 0.000 description 1
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 1
- AGNGYMCLFWQVGX-AGFFZDDWSA-N (e)-1-[(2s)-2-amino-2-carboxyethoxy]-2-diazonioethenolate Chemical compound OC(=O)[C@@H](N)CO\C([O-])=C\[N+]#N AGNGYMCLFWQVGX-AGFFZDDWSA-N 0.000 description 1
- FONKWHRXTPJODV-DNQXCXABSA-N 1,3-bis[2-[(8s)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-3h-pyrrolo[3,2-e]indole-6-carbonyl]-1h-indol-5-yl]urea Chemical compound C1([C@H](CCl)CN2C(=O)C=3NC4=CC=C(C=C4C=3)NC(=O)NC=3C=C4C=C(NC4=CC=3)C(=O)N3C4=CC(O)=C5NC=C(C5=C4[C@H](CCl)C3)C)=C2C=C(O)C2=C1C(C)=CN2 FONKWHRXTPJODV-DNQXCXABSA-N 0.000 description 1
- SATCOUWSAZBIJO-UHFFFAOYSA-N 1-methyladenine Natural products N=C1N(C)C=NC2=C1NC=N2 SATCOUWSAZBIJO-UHFFFAOYSA-N 0.000 description 1
- WJNGQIYEQLPJMN-IOSLPCCCSA-N 1-methylinosine Chemical compound C1=NC=2C(=O)N(C)C=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O WJNGQIYEQLPJMN-IOSLPCCCSA-N 0.000 description 1
- MXHRCPNRJAMMIM-SHYZEUOFSA-N 2'-deoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-SHYZEUOFSA-N 0.000 description 1
- BTOTXLJHDSNXMW-POYBYMJQSA-N 2,3-dideoxyuridine Chemical compound O1[C@H](CO)CC[C@@H]1N1C(=O)NC(=O)C=C1 BTOTXLJHDSNXMW-POYBYMJQSA-N 0.000 description 1
- BOMZMNZEXMAQQW-UHFFFAOYSA-N 2,5,11-trimethyl-6h-pyrido[4,3-b]carbazol-2-ium-9-ol;acetate Chemical compound CC([O-])=O.C[N+]1=CC=C2C(C)=C(NC=3C4=CC(O)=CC=3)C4=C(C)C2=C1 BOMZMNZEXMAQQW-UHFFFAOYSA-N 0.000 description 1
- OOVTUOCTLAERQD-OJMBIDBESA-N 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(2r,3s)-2-(hydroxymethyl)-1-methylpyrrolidin-3-yl]chromen-4-one;hydrochloride Chemical compound Cl.OC[C@@H]1N(C)CC[C@H]1C1=C(O)C=C(O)C2=C1OC(C=1C(=CC=CC=1)Cl)=CC2=O OOVTUOCTLAERQD-OJMBIDBESA-N 0.000 description 1
- SGAKLDIYNFXTCK-UHFFFAOYSA-N 2-[(2,4-dioxo-1h-pyrimidin-5-yl)methylamino]acetic acid Chemical compound OC(=O)CNCC1=CNC(=O)NC1=O SGAKLDIYNFXTCK-UHFFFAOYSA-N 0.000 description 1
- YSAJFXWTVFGPAX-UHFFFAOYSA-N 2-[(2,4-dioxo-1h-pyrimidin-5-yl)oxy]acetic acid Chemical compound OC(=O)COC1=CNC(=O)NC1=O YSAJFXWTVFGPAX-UHFFFAOYSA-N 0.000 description 1
- SVBOROZXXYRWJL-UHFFFAOYSA-N 2-[(4-oxo-2-sulfanylidene-1h-pyrimidin-5-yl)methylamino]acetic acid Chemical compound OC(=O)CNCC1=CNC(=S)NC1=O SVBOROZXXYRWJL-UHFFFAOYSA-N 0.000 description 1
- QCXJFISCRQIYID-IAEPZHFASA-N 2-amino-1-n-[(3s,6s,7r,10s,16s)-3-[(2s)-butan-2-yl]-7,11,14-trimethyl-2,5,9,12,15-pentaoxo-10-propan-2-yl-8-oxa-1,4,11,14-tetrazabicyclo[14.3.0]nonadecan-6-yl]-4,6-dimethyl-3-oxo-9-n-[(3s,6s,7r,10s,16s)-7,11,14-trimethyl-2,5,9,12,15-pentaoxo-3,10-di(propa Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N=C2C(C(=O)N[C@@H]3C(=O)N[C@H](C(N4CCC[C@H]4C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]3C)=O)[C@@H](C)CC)=C(N)C(=O)C(C)=C2O2)C2=C(C)C=C1 QCXJFISCRQIYID-IAEPZHFASA-N 0.000 description 1
- XQCZBXHVTFVIFE-UHFFFAOYSA-N 2-amino-4-hydroxypyrimidine Chemical compound NC1=NC=CC(O)=N1 XQCZBXHVTFVIFE-UHFFFAOYSA-N 0.000 description 1
- YKUFMYSNUQLIQS-UHFFFAOYSA-N 2-amino-5-methyl-1h-pyrimidin-6-one Chemical compound CC1=CNC(N)=NC1=O YKUFMYSNUQLIQS-UHFFFAOYSA-N 0.000 description 1
- FDAYLTPAFBGXAB-UHFFFAOYSA-N 2-chloro-n,n-bis(2-chloroethyl)ethanamine Chemical compound ClCCN(CCCl)CCCl FDAYLTPAFBGXAB-UHFFFAOYSA-N 0.000 description 1
- VNBAOSVONFJBKP-UHFFFAOYSA-N 2-chloro-n,n-bis(2-chloroethyl)propan-1-amine;hydrochloride Chemical compound Cl.CC(Cl)CN(CCCl)CCCl VNBAOSVONFJBKP-UHFFFAOYSA-N 0.000 description 1
- XMSMHKMPBNTBOD-UHFFFAOYSA-N 2-dimethylamino-6-hydroxypurine Chemical compound N1C(N(C)C)=NC(=O)C2=C1N=CN2 XMSMHKMPBNTBOD-UHFFFAOYSA-N 0.000 description 1
- SMADWRYCYBUIKH-UHFFFAOYSA-N 2-methyl-7h-purin-6-amine Chemical compound CC1=NC(N)=C2NC=NC2=N1 SMADWRYCYBUIKH-UHFFFAOYSA-N 0.000 description 1
- YIMDLWDNDGKDTJ-QLKYHASDSA-N 3'-deamino-3'-(3-cyanomorpholin-4-yl)doxorubicin Chemical compound N1([C@H]2C[C@@H](O[C@@H](C)[C@H]2O)O[C@H]2C[C@@](O)(CC=3C(O)=C4C(=O)C=5C=CC=C(C=5C(=O)C4=C(O)C=32)OC)C(=O)CO)CCOCC1C#N YIMDLWDNDGKDTJ-QLKYHASDSA-N 0.000 description 1
- NDMPLJNOPCLANR-UHFFFAOYSA-N 3,4-dihydroxy-15-(4-hydroxy-18-methoxycarbonyl-5,18-seco-ibogamin-18-yl)-16-methoxy-1-methyl-6,7-didehydro-aspidospermidine-3-carboxylic acid methyl ester Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 NDMPLJNOPCLANR-UHFFFAOYSA-N 0.000 description 1
- PWMYMKOUNYTVQN-UHFFFAOYSA-N 3-(8,8-diethyl-2-aza-8-germaspiro[4.5]decan-2-yl)-n,n-dimethylpropan-1-amine Chemical compound C1C[Ge](CC)(CC)CCC11CN(CCCN(C)C)CC1 PWMYMKOUNYTVQN-UHFFFAOYSA-N 0.000 description 1
- WEVYNIUIFUYDGI-UHFFFAOYSA-N 3-[6-[4-(trifluoromethoxy)anilino]-4-pyrimidinyl]benzamide Chemical compound NC(=O)C1=CC=CC(C=2N=CN=C(NC=3C=CC(OC(F)(F)F)=CC=3)C=2)=C1 WEVYNIUIFUYDGI-UHFFFAOYSA-N 0.000 description 1
- KOLPWZCZXAMXKS-UHFFFAOYSA-N 3-methylcytosine Chemical compound CN1C(N)=CC=NC1=O KOLPWZCZXAMXKS-UHFFFAOYSA-N 0.000 description 1
- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 description 1
- GJAKJCICANKRFD-UHFFFAOYSA-N 4-acetyl-4-amino-1,3-dihydropyrimidin-2-one Chemical compound CC(=O)C1(N)NC(=O)NC=C1 GJAKJCICANKRFD-UHFFFAOYSA-N 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- MQJSSLBGAQJNER-UHFFFAOYSA-N 5-(methylaminomethyl)-1h-pyrimidine-2,4-dione Chemical compound CNCC1=CNC(=O)NC1=O MQJSSLBGAQJNER-UHFFFAOYSA-N 0.000 description 1
- WPYRHVXCOQLYLY-UHFFFAOYSA-N 5-[(methoxyamino)methyl]-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CONCC1=CNC(=S)NC1=O WPYRHVXCOQLYLY-UHFFFAOYSA-N 0.000 description 1
- IDPUKCWIGUEADI-UHFFFAOYSA-N 5-[bis(2-chloroethyl)amino]uracil Chemical compound ClCCN(CCCl)C1=CNC(=O)NC1=O IDPUKCWIGUEADI-UHFFFAOYSA-N 0.000 description 1
- NMUSYJAQQFHJEW-KVTDHHQDSA-N 5-azacytidine Chemical compound O=C1N=C(N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NMUSYJAQQFHJEW-KVTDHHQDSA-N 0.000 description 1
- LQLQRFGHAALLLE-UHFFFAOYSA-N 5-bromouracil Chemical compound BrC1=CNC(=O)NC1=O LQLQRFGHAALLLE-UHFFFAOYSA-N 0.000 description 1
- JDBGXEHEIRGOBU-UHFFFAOYSA-N 5-hydroxymethyluracil Chemical compound OCC1=CNC(=O)NC1=O JDBGXEHEIRGOBU-UHFFFAOYSA-N 0.000 description 1
- OFJNVANOCZHTMW-UHFFFAOYSA-N 5-hydroxyuracil Chemical compound OC1=CNC(=O)NC1=O OFJNVANOCZHTMW-UHFFFAOYSA-N 0.000 description 1
- KELXHQACBIUYSE-UHFFFAOYSA-N 5-methoxy-1h-pyrimidine-2,4-dione Chemical compound COC1=CNC(=O)NC1=O KELXHQACBIUYSE-UHFFFAOYSA-N 0.000 description 1
- ZLAQATDNGLKIEV-UHFFFAOYSA-N 5-methyl-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CC1=CNC(=S)NC1=O ZLAQATDNGLKIEV-UHFFFAOYSA-N 0.000 description 1
- HSPHKCOAUOJLIO-UHFFFAOYSA-N 6-(aziridin-1-ylamino)-1h-pyrimidin-2-one Chemical compound N1C(=O)N=CC=C1NN1CC1 HSPHKCOAUOJLIO-UHFFFAOYSA-N 0.000 description 1
- DCPSTSVLRXOYGS-UHFFFAOYSA-N 6-amino-1h-pyrimidine-2-thione Chemical compound NC1=CC=NC(S)=N1 DCPSTSVLRXOYGS-UHFFFAOYSA-N 0.000 description 1
- NLLCDONDZDHLCI-UHFFFAOYSA-N 6-amino-5-hydroxy-1h-pyrimidin-2-one Chemical compound NC=1NC(=O)N=CC=1O NLLCDONDZDHLCI-UHFFFAOYSA-N 0.000 description 1
- WYXSYVWAUAUWLD-SHUUEZRQSA-N 6-azauridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=N1 WYXSYVWAUAUWLD-SHUUEZRQSA-N 0.000 description 1
- 229960005538 6-diazo-5-oxo-L-norleucine Drugs 0.000 description 1
- YCWQAMGASJSUIP-YFKPBYRVSA-N 6-diazo-5-oxo-L-norleucine Chemical compound OC(=O)[C@@H](N)CCC(=O)C=[N+]=[N-] YCWQAMGASJSUIP-YFKPBYRVSA-N 0.000 description 1
- WLCZTRVUXYALDD-IBGZPJMESA-N 7-[[(2s)-2,6-bis(2-methoxyethoxycarbonylamino)hexanoyl]amino]heptoxy-methylphosphinic acid Chemical compound COCCOC(=O)NCCCC[C@H](NC(=O)OCCOC)C(=O)NCCCCCCCOP(C)(O)=O WLCZTRVUXYALDD-IBGZPJMESA-N 0.000 description 1
- ZGXJTSGNIOSYLO-UHFFFAOYSA-N 88755TAZ87 Chemical compound NCC(=O)CCC(O)=O ZGXJTSGNIOSYLO-UHFFFAOYSA-N 0.000 description 1
- HDZZVAMISRMYHH-UHFFFAOYSA-N 9beta-Ribofuranosyl-7-deazaadenin Natural products C1=CC=2C(N)=NC=NC=2N1C1OC(CO)C(O)C1O HDZZVAMISRMYHH-UHFFFAOYSA-N 0.000 description 1
- BUROJSBIWGDYCN-GAUTUEMISA-N AP 23573 Chemical compound C1C[C@@H](OP(C)(C)=O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 BUROJSBIWGDYCN-GAUTUEMISA-N 0.000 description 1
- 208000035657 Abasia Diseases 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- 208000036764 Adenocarcinoma of the esophagus Diseases 0.000 description 1
- CEIZFXOZIQNICU-UHFFFAOYSA-N Alternaria alternata Crofton-weed toxin Natural products CCC(C)C1NC(=O)C(C(C)=O)=C1O CEIZFXOZIQNICU-UHFFFAOYSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 101100519158 Arabidopsis thaliana PCR2 gene Proteins 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical class C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 1
- MLDQJTXFUGDVEO-UHFFFAOYSA-N BAY-43-9006 Chemical compound C1=NC(C(=O)NC)=CC(OC=2C=CC(NC(=O)NC=3C=C(C(Cl)=CC=3)C(F)(F)F)=CC=2)=C1 MLDQJTXFUGDVEO-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- VGGGPCQERPFHOB-MCIONIFRSA-N Bestatin Chemical compound CC(C)C[C@H](C(O)=O)NC(=O)[C@@H](O)[C@H](N)CC1=CC=CC=C1 VGGGPCQERPFHOB-MCIONIFRSA-N 0.000 description 1
- 229940122361 Bisphosphonate Drugs 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- MBABCNBNDNGODA-LTGLSHGVSA-N Bullatacin Natural products O=C1C(C[C@H](O)CCCCCCCCCC[C@@H](O)[C@@H]2O[C@@H]([C@@H]3O[C@H]([C@@H](O)CCCCCCCCCC)CC3)CC2)=C[C@H](C)O1 MBABCNBNDNGODA-LTGLSHGVSA-N 0.000 description 1
- KGGVWMAPBXIMEM-ZRTAFWODSA-N Bullatacinone Chemical compound O1[C@@H]([C@@H](O)CCCCCCCCCC)CC[C@@H]1[C@@H]1O[C@@H]([C@H](O)CCCCCCCCCC[C@H]2OC(=O)[C@H](CC(C)=O)C2)CC1 KGGVWMAPBXIMEM-ZRTAFWODSA-N 0.000 description 1
- KGGVWMAPBXIMEM-JQFCFGFHSA-N Bullatacinone Natural products O=C(C[C@H]1C(=O)O[C@H](CCCCCCCCCC[C@H](O)[C@@H]2O[C@@H]([C@@H]3O[C@@H]([C@@H](O)CCCCCCCCCC)CC3)CC2)C1)C KGGVWMAPBXIMEM-JQFCFGFHSA-N 0.000 description 1
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 description 1
- GAGWJHPBXLXJQN-UHFFFAOYSA-N Capecitabine Natural products C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1C1C(O)C(O)C(C)O1 GAGWJHPBXLXJQN-UHFFFAOYSA-N 0.000 description 1
- SHHKQEUPHAENFK-UHFFFAOYSA-N Carboquone Chemical compound O=C1C(C)=C(N2CC2)C(=O)C(C(COC(N)=O)OC)=C1N1CC1 SHHKQEUPHAENFK-UHFFFAOYSA-N 0.000 description 1
- AOCCBINRVIKJHY-UHFFFAOYSA-N Carmofur Chemical compound CCCCCCNC(=O)N1C=C(F)C(=O)NC1=O AOCCBINRVIKJHY-UHFFFAOYSA-N 0.000 description 1
- DLGOEMSEDOSKAD-UHFFFAOYSA-N Carmustine Chemical compound ClCCNC(=O)N(N=O)CCCl DLGOEMSEDOSKAD-UHFFFAOYSA-N 0.000 description 1
- JWBOIMRXGHLCPP-UHFFFAOYSA-N Chloditan Chemical compound C=1C=CC=C(Cl)C=1C(C(Cl)Cl)C1=CC=C(Cl)C=C1 JWBOIMRXGHLCPP-UHFFFAOYSA-N 0.000 description 1
- XCDXSSFOJZZGQC-UHFFFAOYSA-N Chlornaphazine Chemical compound C1=CC=CC2=CC(N(CCCl)CCCl)=CC=C21 XCDXSSFOJZZGQC-UHFFFAOYSA-N 0.000 description 1
- MKQWTWSXVILIKJ-LXGUWJNJSA-N Chlorozotocin Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](C=O)NC(=O)N(N=O)CCCl MKQWTWSXVILIKJ-LXGUWJNJSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 229930188224 Cryptophycin Natural products 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-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
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- ZBNZXTGUTAYRHI-UHFFFAOYSA-N Dasatinib Chemical compound C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1Cl ZBNZXTGUTAYRHI-UHFFFAOYSA-N 0.000 description 1
- WEAHRLBPCANXCN-UHFFFAOYSA-N Daunomycin Natural products CCC1(O)CC(OC2CC(N)C(O)C(C)O2)c3cc4C(=O)c5c(OC)cccc5C(=O)c4c(O)c3C1 WEAHRLBPCANXCN-UHFFFAOYSA-N 0.000 description 1
- NNJPGOLRFBJNIW-UHFFFAOYSA-N Demecolcine Natural products C1=C(OC)C(=O)C=C2C(NC)CCC3=CC(OC)=C(OC)C(OC)=C3C2=C1 NNJPGOLRFBJNIW-UHFFFAOYSA-N 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 108010002156 Depsipeptides Proteins 0.000 description 1
- AUGQEEXBDZWUJY-ZLJUKNTDSA-N Diacetoxyscirpenol Chemical compound C([C@]12[C@]3(C)[C@H](OC(C)=O)[C@@H](O)[C@H]1O[C@@H]1C=C(C)CC[C@@]13COC(=O)C)O2 AUGQEEXBDZWUJY-ZLJUKNTDSA-N 0.000 description 1
- AUGQEEXBDZWUJY-UHFFFAOYSA-N Diacetoxyscirpenol Natural products CC(=O)OCC12CCC(C)=CC1OC1C(O)C(OC(C)=O)C2(C)C11CO1 AUGQEEXBDZWUJY-UHFFFAOYSA-N 0.000 description 1
- 229930193152 Dynemicin Natural products 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
- 241000196324 Embryophyta Species 0.000 description 1
- AFMYMMXSQGUCBK-UHFFFAOYSA-N Endynamicin A Natural products C1#CC=CC#CC2NC(C=3C(=O)C4=C(O)C=CC(O)=C4C(=O)C=3C(O)=C3)=C3C34OC32C(C)C(C(O)=O)=C(OC)C41 AFMYMMXSQGUCBK-UHFFFAOYSA-N 0.000 description 1
- SAMRUMKYXPVKPA-VFKOLLTISA-N Enocitabine Chemical compound O=C1N=C(NC(=O)CCCCCCCCCCCCCCCCCCCCC)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 SAMRUMKYXPVKPA-VFKOLLTISA-N 0.000 description 1
- 102000050554 Eph Family Receptors Human genes 0.000 description 1
- 108091008815 Eph receptors Proteins 0.000 description 1
- HTIJFSOGRVMCQR-UHFFFAOYSA-N Epirubicin Natural products COc1cccc2C(=O)c3c(O)c4CC(O)(CC(OC5CC(N)C(=O)C(C)O5)c4c(O)c3C(=O)c12)C(=O)CO HTIJFSOGRVMCQR-UHFFFAOYSA-N 0.000 description 1
- OBMLHUPNRURLOK-XGRAFVIBSA-N Epitiostanol Chemical compound C1[C@@H]2S[C@@H]2C[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 OBMLHUPNRURLOK-XGRAFVIBSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229930189413 Esperamicin Natural products 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- HKVAMNSJSFKALM-GKUWKFKPSA-N Everolimus Chemical compound C1C[C@@H](OCCO)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 HKVAMNSJSFKALM-GKUWKFKPSA-N 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- KGPGFQWBCSZGEL-ZDUSSCGKSA-N GSK690693 Chemical compound C=12N(CC)C(C=3C(=NON=3)N)=NC2=C(C#CC(C)(C)O)N=CC=1OC[C@H]1CCCNC1 KGPGFQWBCSZGEL-ZDUSSCGKSA-N 0.000 description 1
- 108010014905 Glycogen Synthase Kinase 3 Proteins 0.000 description 1
- 102000002254 Glycogen Synthase Kinase 3 Human genes 0.000 description 1
- 101100341519 Homo sapiens ITGAX gene Proteins 0.000 description 1
- 101000984753 Homo sapiens Serine/threonine-protein kinase B-raf Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- VSNHCAURESNICA-UHFFFAOYSA-N Hydroxyurea Chemical compound NC(=O)NO VSNHCAURESNICA-UHFFFAOYSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- MPBVHIBUJCELCL-UHFFFAOYSA-N Ibandronate Chemical compound CCCCCN(C)CCC(O)(P(O)(O)=O)P(O)(O)=O MPBVHIBUJCELCL-UHFFFAOYSA-N 0.000 description 1
- XDXDZDZNSLXDNA-TZNDIEGXSA-N Idarubicin Chemical compound C1[C@H](N)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2C[C@@](O)(C(C)=O)C1 XDXDZDZNSLXDNA-TZNDIEGXSA-N 0.000 description 1
- XDXDZDZNSLXDNA-UHFFFAOYSA-N Idarubicin Natural products C1C(N)C(O)C(C)OC1OC1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2CC(O)(C(C)=O)C1 XDXDZDZNSLXDNA-UHFFFAOYSA-N 0.000 description 1
- 108091082332 JAK family Proteins 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 239000005517 L01XE01 - Imatinib Substances 0.000 description 1
- 239000005551 L01XE03 - Erlotinib Substances 0.000 description 1
- 239000002147 L01XE04 - Sunitinib Substances 0.000 description 1
- 239000005511 L01XE05 - Sorafenib Substances 0.000 description 1
- 239000002067 L01XE06 - Dasatinib Substances 0.000 description 1
- 239000002136 L01XE07 - Lapatinib Substances 0.000 description 1
- 239000005536 L01XE08 - Nilotinib Substances 0.000 description 1
- 239000003798 L01XE11 - Pazopanib Substances 0.000 description 1
- 239000002118 L01XE12 - Vandetanib Substances 0.000 description 1
- 239000002145 L01XE14 - Bosutinib Substances 0.000 description 1
- 239000002146 L01XE16 - Crizotinib Substances 0.000 description 1
- 239000002144 L01XE18 - Ruxolitinib Substances 0.000 description 1
- CZQHHVNHHHRRDU-UHFFFAOYSA-N LY294002 Chemical compound C1=CC=C2C(=O)C=C(N3CCOCC3)OC2=C1C1=CC=CC=C1 CZQHHVNHHHRRDU-UHFFFAOYSA-N 0.000 description 1
- 229920001491 Lentinan Polymers 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- GQYIWUVLTXOXAJ-UHFFFAOYSA-N Lomustine Chemical compound ClCCN(N=O)C(=O)NC1CCCCC1 GQYIWUVLTXOXAJ-UHFFFAOYSA-N 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 102000043129 MHC class I family Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 229940124640 MK-2206 Drugs 0.000 description 1
- ULDXWLCXEDXJGE-UHFFFAOYSA-N MK-2206 Chemical compound C=1C=C(C=2C(=CC=3C=4N(C(NN=4)=O)C=CC=3N=2)C=2C=CC=CC=2)C=CC=1C1(N)CCC1 ULDXWLCXEDXJGE-UHFFFAOYSA-N 0.000 description 1
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- VJRAUFKOOPNFIQ-UHFFFAOYSA-N Marcellomycin Natural products C12=C(O)C=3C(=O)C4=C(O)C=CC(O)=C4C(=O)C=3C=C2C(C(=O)OC)C(CC)(O)CC1OC(OC1C)CC(N(C)C)C1OC(OC1C)CC(O)C1OC1CC(O)C(O)C(C)O1 VJRAUFKOOPNFIQ-UHFFFAOYSA-N 0.000 description 1
- 229930126263 Maytansine Natural products 0.000 description 1
- IVDYZAAPOLNZKG-KWHRADDSSA-N Mepitiostane Chemical compound O([C@@H]1[C@]2(CC[C@@H]3[C@@]4(C)C[C@H]5S[C@H]5C[C@@H]4CC[C@H]3[C@@H]2CC1)C)C1(OC)CCCC1 IVDYZAAPOLNZKG-KWHRADDSSA-N 0.000 description 1
- VFKZTMPDYBFSTM-KVTDHHQDSA-N Mitobronitol Chemical compound BrC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CBr VFKZTMPDYBFSTM-KVTDHHQDSA-N 0.000 description 1
- 108090000744 Mitogen-Activated Protein Kinase Kinases Proteins 0.000 description 1
- 102000004232 Mitogen-Activated Protein Kinase Kinases Human genes 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 239000005462 Mubritinib Substances 0.000 description 1
- SGSSKEDGVONRGC-UHFFFAOYSA-N N(2)-methylguanine Chemical compound O=C1NC(NC)=NC2=C1N=CN2 SGSSKEDGVONRGC-UHFFFAOYSA-N 0.000 description 1
- PJKKQFAEFWCNAQ-UHFFFAOYSA-N N(4)-methylcytosine Chemical compound CNC=1C=CNC(=O)N=1 PJKKQFAEFWCNAQ-UHFFFAOYSA-N 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- SYNHCENRCUAUNM-UHFFFAOYSA-N Nitrogen mustard N-oxide hydrochloride Chemical compound Cl.ClCC[N+]([O-])(C)CCCl SYNHCENRCUAUNM-UHFFFAOYSA-N 0.000 description 1
- 206010030137 Oesophageal adenocarcinoma Diseases 0.000 description 1
- 229930187135 Olivomycin Natural products 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- 102000038030 PI3Ks Human genes 0.000 description 1
- QIUASFSNWYMDFS-NILGECQDSA-N PX-866 Chemical compound CC(=O)O[C@@H]1C[C@]2(C)C(=O)CC[C@H]2C2=C1[C@@]1(C)[C@@H](COC)OC(=O)\C(=C\N(CC=C)CC=C)C1=C(O)C2=O QIUASFSNWYMDFS-NILGECQDSA-N 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- VREZDOWOLGNDPW-ALTGWBOUSA-N Pancratistatin Chemical compound C1=C2[C@H]3[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O)[C@@H]3NC(=O)C2=C(O)C2=C1OCO2 VREZDOWOLGNDPW-ALTGWBOUSA-N 0.000 description 1
- VREZDOWOLGNDPW-MYVCAWNPSA-N Pancratistatin Natural products O=C1N[C@H]2[C@H](O)[C@H](O)[C@H](O)[C@H](O)[C@@H]2c2c1c(O)c1OCOc1c2 VREZDOWOLGNDPW-MYVCAWNPSA-N 0.000 description 1
- 108010057150 Peplomycin Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- KMSKQZKKOZQFFG-HSUXVGOQSA-N Pirarubicin Chemical compound O([C@H]1[C@@H](N)C[C@@H](O[C@H]1C)O[C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1CCCCO1 KMSKQZKKOZQFFG-HSUXVGOQSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- HFVNWDWLWUCIHC-GUPDPFMOSA-N Prednimustine Chemical compound O=C([C@@]1(O)CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)[C@@H](O)C[C@@]21C)COC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 HFVNWDWLWUCIHC-GUPDPFMOSA-N 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 108091008611 Protein Kinase B Proteins 0.000 description 1
- 102000016971 Proto-Oncogene Proteins c-kit Human genes 0.000 description 1
- 108010014608 Proto-Oncogene Proteins c-kit Proteins 0.000 description 1
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Natural products N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 102100033810 RAC-alpha serine/threonine-protein kinase Human genes 0.000 description 1
- 239000012162 RNA isolation reagent Substances 0.000 description 1
- 108010065868 RNA polymerase SP6 Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- OWPCHSCAPHNHAV-UHFFFAOYSA-N Rhizoxin Natural products C1C(O)C2(C)OC2C=CC(C)C(OC(=O)C2)CC2CC2OC2C(=O)OC1C(C)C(OC)C(C)=CC=CC(C)=CC1=COC(C)=N1 OWPCHSCAPHNHAV-UHFFFAOYSA-N 0.000 description 1
- 108091028664 Ribonucleotide Proteins 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
- 241000283984 Rodentia Species 0.000 description 1
- NSFWWJIQIKBZMJ-YKNYLIOZSA-N Roridin A Chemical compound C([C@]12[C@]3(C)[C@H]4C[C@H]1O[C@@H]1C=C(C)CC[C@@]13COC(=O)[C@@H](O)[C@H](C)CCO[C@H](\C=C\C=C/C(=O)O4)[C@H](O)C)O2 NSFWWJIQIKBZMJ-YKNYLIOZSA-N 0.000 description 1
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 1
- 101150001535 SRC gene Proteins 0.000 description 1
- 102100027103 Serine/threonine-protein kinase B-raf Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229920000519 Sizofiran Polymers 0.000 description 1
- 108020004688 Small Nuclear RNA Proteins 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 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
- 230000006044 T cell activation Effects 0.000 description 1
- BXFOFFBJRFZBQZ-QYWOHJEZSA-N T-2 toxin Chemical compound C([C@@]12[C@]3(C)[C@H](OC(C)=O)[C@@H](O)[C@H]1O[C@H]1[C@]3(COC(C)=O)C[C@@H](C(=C1)C)OC(=O)CC(C)C)O2 BXFOFFBJRFZBQZ-QYWOHJEZSA-N 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 210000000173 T-lymphoid precursor cell Anatomy 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 description 1
- 102000013530 TOR Serine-Threonine Kinases Human genes 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- CBPNZQVSJQDFBE-FUXHJELOSA-N Temsirolimus Chemical compound C1C[C@@H](OC(=O)C(C)(CO)CO)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 CBPNZQVSJQDFBE-FUXHJELOSA-N 0.000 description 1
- CGMTUJFWROPELF-UHFFFAOYSA-N Tenuazonic acid Natural products CCC(C)C1NC(=O)C(=C(C)/O)C1=O CGMTUJFWROPELF-UHFFFAOYSA-N 0.000 description 1
- FOCVUCIESVLUNU-UHFFFAOYSA-N Thiotepa Chemical compound C1CN1P(N1CC1)(=S)N1CC1 FOCVUCIESVLUNU-UHFFFAOYSA-N 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 108010001244 Tli polymerase Proteins 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- UMILHIMHKXVDGH-UHFFFAOYSA-N Triethylene glycol diglycidyl ether Chemical compound C1OC1COCCOCCOCCOCC1CO1 UMILHIMHKXVDGH-UHFFFAOYSA-N 0.000 description 1
- 108091008605 VEGF receptors Proteins 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 1
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 1
- 241001441550 Zeiformes Species 0.000 description 1
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 1
- SPJCRMJCFSJKDE-ZWBUGVOYSA-N [(3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-yl] 2-[4-[bis(2-chloroethyl)amino]phenyl]acetate Chemical compound O([C@@H]1CC2=CC[C@H]3[C@@H]4CC[C@@H]([C@]4(CC[C@@H]3[C@@]2(C)CC1)C)[C@H](C)CCCC(C)C)C(=O)CC1=CC=C(N(CCCl)CCCl)C=C1 SPJCRMJCFSJKDE-ZWBUGVOYSA-N 0.000 description 1
- IFJUINDAXYAPTO-UUBSBJJBSA-N [(8r,9s,13s,14s,17s)-17-[2-[4-[4-[bis(2-chloroethyl)amino]phenyl]butanoyloxy]acetyl]oxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-3-yl] benzoate Chemical compound C([C@@H]1[C@@H](C2=CC=3)CC[C@]4([C@H]1CC[C@@H]4OC(=O)COC(=O)CCCC=1C=CC(=CC=1)N(CCCl)CCCl)C)CC2=CC=3OC(=O)C1=CC=CC=C1 IFJUINDAXYAPTO-UUBSBJJBSA-N 0.000 description 1
- XZSRRNFBEIOBDA-CFNBKWCHSA-N [2-[(2s,4s)-4-[(2r,4s,5s,6s)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-3,4-dihydro-1h-tetracen-2-yl]-2-oxoethyl] 2,2-diethoxyacetate Chemical compound O([C@H]1C[C@](CC2=C(O)C=3C(=O)C4=CC=CC(OC)=C4C(=O)C=3C(O)=C21)(O)C(=O)COC(=O)C(OCC)OCC)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 XZSRRNFBEIOBDA-CFNBKWCHSA-N 0.000 description 1
- PSHJFSXQZBLAMV-UHFFFAOYSA-K [Eu+3].CC(O)=O.CC(O)=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.NCCNCCN Chemical compound [Eu+3].CC(O)=O.CC(O)=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.NCCNCCN PSHJFSXQZBLAMV-UHFFFAOYSA-K 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- ZOZKYEHVNDEUCO-XUTVFYLZSA-N aceglatone Chemical compound O1C(=O)[C@H](OC(C)=O)[C@@H]2OC(=O)[C@@H](OC(=O)C)[C@@H]21 ZOZKYEHVNDEUCO-XUTVFYLZSA-N 0.000 description 1
- 229950002684 aceglatone Drugs 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 229930183665 actinomycin Natural products 0.000 description 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229960001570 ademetionine Drugs 0.000 description 1
- 229950004955 adozelesin Drugs 0.000 description 1
- BYRVKDUQDLJUBX-JJCDCTGGSA-N adozelesin Chemical compound C1=CC=C2OC(C(=O)NC=3C=C4C=C(NC4=CC=3)C(=O)N3C[C@H]4C[C@]44C5=C(C(C=C43)=O)NC=C5C)=CC2=C1 BYRVKDUQDLJUBX-JJCDCTGGSA-N 0.000 description 1
- 229960001686 afatinib Drugs 0.000 description 1
- ULXXDDBFHOBEHA-CWDCEQMOSA-N afatinib Chemical compound N1=CN=C2C=C(O[C@@H]3COCC3)C(NC(=O)/C=C/CN(C)C)=CC2=C1NC1=CC=C(F)C(Cl)=C1 ULXXDDBFHOBEHA-CWDCEQMOSA-N 0.000 description 1
- 229940045714 alkyl sulfonate alkylating agent Drugs 0.000 description 1
- 150000008052 alkyl sulfonates Chemical class 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 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
- IBOLVNKLOOYDDG-UHFFFAOYSA-N alpha-putrescinylthymine Chemical compound NCCCCNCC1=CNC(=O)NC1=O IBOLVNKLOOYDDG-UHFFFAOYSA-N 0.000 description 1
- 229960000473 altretamine Drugs 0.000 description 1
- 229950010817 alvocidib Drugs 0.000 description 1
- BIIVYFLTOXDAOV-YVEFUNNKSA-N alvocidib Chemical compound O[C@@H]1CN(C)CC[C@@H]1C1=C(O)C=C(O)C2=C1OC(C=1C(=CC=CC=1)Cl)=CC2=O BIIVYFLTOXDAOV-YVEFUNNKSA-N 0.000 description 1
- 229960002749 aminolevulinic acid Drugs 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 238000012197 amplification kit Methods 0.000 description 1
- 229960001220 amsacrine Drugs 0.000 description 1
- XCPGHVQEEXUHNC-UHFFFAOYSA-N amsacrine Chemical compound COC1=CC(NS(C)(=O)=O)=CC=C1NC1=C(C=CC=C2)C2=NC2=CC=CC=C12 XCPGHVQEEXUHNC-UHFFFAOYSA-N 0.000 description 1
- BBDAGFIXKZCXAH-CCXZUQQUSA-N ancitabine Chemical compound N=C1C=CN2[C@@H]3O[C@H](CO)[C@@H](O)[C@@H]3OC2=N1 BBDAGFIXKZCXAH-CCXZUQQUSA-N 0.000 description 1
- 229950000242 ancitabine Drugs 0.000 description 1
- 239000003098 androgen Substances 0.000 description 1
- 229940030486 androgens Drugs 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 229940045687 antimetabolites folic acid analogs Drugs 0.000 description 1
- 229940045988 antineoplastic drug protein kinase inhibitors Drugs 0.000 description 1
- 238000002617 apheresis Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 150000008209 arabinosides Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 229960003005 axitinib Drugs 0.000 description 1
- RITAVMQDGBJQJZ-FMIVXFBMSA-N axitinib Chemical compound CNC(=O)C1=CC=CC=C1SC1=CC=C(C(\C=C\C=2N=CC=CC=2)=NN2)C2=C1 RITAVMQDGBJQJZ-FMIVXFBMSA-N 0.000 description 1
- 229960002756 azacitidine Drugs 0.000 description 1
- 229950011321 azaserine Drugs 0.000 description 1
- 150000001541 aziridines Chemical class 0.000 description 1
- 108010028263 bacteriophage T3 RNA polymerase Proteins 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 229960000397 bevacizumab Drugs 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 229950008548 bisantrene Drugs 0.000 description 1
- 150000004663 bisphosphonates Chemical class 0.000 description 1
- 229950006844 bizelesin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical class N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 206010006007 bone sarcoma Diseases 0.000 description 1
- 229960003736 bosutinib Drugs 0.000 description 1
- UBPYILGKFZZVDX-UHFFFAOYSA-N bosutinib Chemical compound C1=C(Cl)C(OC)=CC(NC=2C3=CC(OC)=C(OCCCN4CCN(C)CC4)C=C3N=CC=2C#N)=C1Cl UBPYILGKFZZVDX-UHFFFAOYSA-N 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- KQNZDYYTLMIZCT-KQPMLPITSA-N brefeldin A Chemical compound O[C@@H]1\C=C\C(=O)O[C@@H](C)CCC\C=C\[C@@H]2C[C@H](O)C[C@H]21 KQNZDYYTLMIZCT-KQPMLPITSA-N 0.000 description 1
- JUMGSHROWPPKFX-UHFFFAOYSA-N brefeldin-A Natural products CC1CCCC=CC2(C)CC(O)CC2(C)C(O)C=CC(=O)O1 JUMGSHROWPPKFX-UHFFFAOYSA-N 0.000 description 1
- 229960005520 bryostatin Drugs 0.000 description 1
- MJQUEDHRCUIRLF-TVIXENOKSA-N bryostatin 1 Chemical compound C([C@@H]1CC(/[C@@H]([C@@](C(C)(C)/C=C/2)(O)O1)OC(=O)/C=C/C=C/CCC)=C\C(=O)OC)[C@H]([C@@H](C)O)OC(=O)C[C@H](O)C[C@@H](O1)C[C@H](OC(C)=O)C(C)(C)[C@]1(O)C[C@@H]1C\C(=C\C(=O)OC)C[C@H]\2O1 MJQUEDHRCUIRLF-TVIXENOKSA-N 0.000 description 1
- MUIWQCKLQMOUAT-AKUNNTHJSA-N bryostatin 20 Natural products COC(=O)C=C1C[C@@]2(C)C[C@]3(O)O[C@](C)(C[C@@H](O)CC(=O)O[C@](C)(C[C@@]4(C)O[C@](O)(CC5=CC(=O)O[C@]45C)C(C)(C)C=C[C@@](C)(C1)O2)[C@@H](C)O)C[C@H](OC(=O)C(C)(C)C)C3(C)C MUIWQCKLQMOUAT-AKUNNTHJSA-N 0.000 description 1
- MBABCNBNDNGODA-LUVUIASKSA-N bullatacin Chemical compound O1[C@@H]([C@@H](O)CCCCCCCCCC)CC[C@@H]1[C@@H]1O[C@@H]([C@H](O)CCCCCCCCCC[C@@H](O)CC=2C(O[C@@H](C)C=2)=O)CC1 MBABCNBNDNGODA-LUVUIASKSA-N 0.000 description 1
- 229960002092 busulfan Drugs 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 108700002839 cactinomycin Proteins 0.000 description 1
- 229950009908 cactinomycin Drugs 0.000 description 1
- 229910052792 caesium Inorganic materials 0.000 description 1
- TVFDJXOCXUVLDH-UHFFFAOYSA-N caesium atom Chemical compound [Cs] TVFDJXOCXUVLDH-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- IVFYLRMMHVYGJH-PVPPCFLZSA-N calusterone Chemical compound C1C[C@]2(C)[C@](O)(C)CC[C@H]2[C@@H]2[C@@H](C)CC3=CC(=O)CC[C@]3(C)[C@H]21 IVFYLRMMHVYGJH-PVPPCFLZSA-N 0.000 description 1
- 229950009823 calusterone Drugs 0.000 description 1
- 229940127093 camptothecin Drugs 0.000 description 1
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 description 1
- 229960004117 capecitabine Drugs 0.000 description 1
- 229960002115 carboquone Drugs 0.000 description 1
- 229930188550 carminomycin Natural products 0.000 description 1
- XREUEWVEMYWFFA-CSKJXFQVSA-N carminomycin Chemical compound C1[C@H](N)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1C2=C(O)C(C(=O)C3=C(O)C=CC=C3C3=O)=C3C(O)=C2C[C@@](O)(C(C)=O)C1 XREUEWVEMYWFFA-CSKJXFQVSA-N 0.000 description 1
- XREUEWVEMYWFFA-UHFFFAOYSA-N carminomycin I Natural products C1C(N)C(O)C(C)OC1OC1C2=C(O)C(C(=O)C3=C(O)C=CC=C3C3=O)=C3C(O)=C2CC(O)(C(C)=O)C1 XREUEWVEMYWFFA-UHFFFAOYSA-N 0.000 description 1
- 229960003261 carmofur Drugs 0.000 description 1
- 229960005243 carmustine Drugs 0.000 description 1
- 229950001725 carubicin Drugs 0.000 description 1
- 229950007509 carzelesin Drugs 0.000 description 1
- BBZDXMBRAFTCAA-AREMUKBSSA-N carzelesin Chemical compound C1=2NC=C(C)C=2C([C@H](CCl)CN2C(=O)C=3NC4=CC=C(C=C4C=3)NC(=O)C3=CC4=CC=C(C=C4O3)N(CC)CC)=C2C=C1OC(=O)NC1=CC=CC=C1 BBZDXMBRAFTCAA-AREMUKBSSA-N 0.000 description 1
- 108010047060 carzinophilin Proteins 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 108091092328 cellular RNA Proteins 0.000 description 1
- 210000003756 cervix mucus Anatomy 0.000 description 1
- 229960005395 cetuximab Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 210000003467 cheek Anatomy 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000012829 chemotherapy agent Substances 0.000 description 1
- 229960004630 chlorambucil Drugs 0.000 description 1
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 1
- 229950008249 chlornaphazine Drugs 0.000 description 1
- 229960001480 chlorozotocin Drugs 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 210000004252 chorionic villi Anatomy 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- ACSIXWWBWUQEHA-UHFFFAOYSA-N clodronic acid Chemical compound OP(O)(=O)C(Cl)(Cl)P(O)(O)=O ACSIXWWBWUQEHA-UHFFFAOYSA-N 0.000 description 1
- 229960002286 clodronic acid Drugs 0.000 description 1
- 239000008119 colloidal silica Substances 0.000 description 1
- 238000009096 combination chemotherapy Methods 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229960005061 crizotinib Drugs 0.000 description 1
- KTEIFNKAUNYNJU-GFCCVEGCSA-N crizotinib Chemical compound O([C@H](C)C=1C(=C(F)C=CC=1Cl)Cl)C(C(=NC=1)N)=CC=1C(=C1)C=NN1C1CCNCC1 KTEIFNKAUNYNJU-GFCCVEGCSA-N 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000002681 cryosurgery Methods 0.000 description 1
- 108010089438 cryptophycin 1 Proteins 0.000 description 1
- PSNOPSMXOBPNNV-VVCTWANISA-N cryptophycin 1 Chemical compound C1=C(Cl)C(OC)=CC=C1C[C@@H]1C(=O)NC[C@@H](C)C(=O)O[C@@H](CC(C)C)C(=O)O[C@H]([C@H](C)[C@@H]2[C@H](O2)C=2C=CC=CC=2)C/C=C/C(=O)N1 PSNOPSMXOBPNNV-VVCTWANISA-N 0.000 description 1
- 108010090203 cryptophycin 8 Proteins 0.000 description 1
- PSNOPSMXOBPNNV-UHFFFAOYSA-N cryptophycin-327 Natural products C1=C(Cl)C(OC)=CC=C1CC1C(=O)NCC(C)C(=O)OC(CC(C)C)C(=O)OC(C(C)C2C(O2)C=2C=CC=CC=2)CC=CC(=O)N1 PSNOPSMXOBPNNV-UHFFFAOYSA-N 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 229960003901 dacarbazine Drugs 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 229960002448 dasatinib Drugs 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229960005052 demecolcine Drugs 0.000 description 1
- 230000017858 demethylation Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- MXHRCPNRJAMMIM-UHFFFAOYSA-N desoxyuridine Natural products C1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-UHFFFAOYSA-N 0.000 description 1
- 229950003913 detorubicin Drugs 0.000 description 1
- WVYXNIXAMZOZFK-UHFFFAOYSA-N diaziquone Chemical compound O=C1C(NC(=O)OCC)=C(N2CC2)C(=O)C(NC(=O)OCC)=C1N1CC1 WVYXNIXAMZOZFK-UHFFFAOYSA-N 0.000 description 1
- 229950002389 diaziquone Drugs 0.000 description 1
- UMGXUWVIJIQANV-UHFFFAOYSA-M didecyl(dimethyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCC[N+](C)(C)CCCCCCCCCC UMGXUWVIJIQANV-UHFFFAOYSA-M 0.000 description 1
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 description 1
- 239000003534 dna topoisomerase inhibitor Substances 0.000 description 1
- 229960003668 docetaxel Drugs 0.000 description 1
- AMRJKAQTDDKMCE-UHFFFAOYSA-N dolastatin Chemical compound CC(C)C(N(C)C)C(=O)NC(C(C)C)C(=O)N(C)C(C(C)C)C(OC)CC(=O)N1CCCC1C(OC)C(C)C(=O)NC(C=1SC=CN=1)CC1=CC=CC=C1 AMRJKAQTDDKMCE-UHFFFAOYSA-N 0.000 description 1
- 229930188854 dolastatin Natural products 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- ZWAOHEXOSAUJHY-ZIYNGMLESA-N doxifluridine Chemical compound O[C@@H]1[C@H](O)[C@@H](C)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ZWAOHEXOSAUJHY-ZIYNGMLESA-N 0.000 description 1
- 229950005454 doxifluridine Drugs 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- NOTIQUSPUUHHEH-UXOVVSIBSA-N dromostanolone propionate Chemical compound C([C@@H]1CC2)C(=O)[C@H](C)C[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H](OC(=O)CC)[C@@]2(C)CC1 NOTIQUSPUUHHEH-UXOVVSIBSA-N 0.000 description 1
- 229950004683 drostanolone propionate Drugs 0.000 description 1
- 229960005501 duocarmycin Drugs 0.000 description 1
- VQNATVDKACXKTF-XELLLNAOSA-N duocarmycin Chemical compound COC1=C(OC)C(OC)=C2NC(C(=O)N3C4=CC(=O)C5=C([C@@]64C[C@@H]6C3)C=C(N5)C(=O)OC)=CC2=C1 VQNATVDKACXKTF-XELLLNAOSA-N 0.000 description 1
- 229930184221 duocarmycin Natural products 0.000 description 1
- AFMYMMXSQGUCBK-AKMKHHNQSA-N dynemicin a Chemical compound C1#C\C=C/C#C[C@@H]2NC(C=3C(=O)C4=C(O)C=CC(O)=C4C(=O)C=3C(O)=C3)=C3[C@@]34O[C@]32[C@@H](C)C(C(O)=O)=C(OC)[C@H]41 AFMYMMXSQGUCBK-AKMKHHNQSA-N 0.000 description 1
- FSIRXIHZBIXHKT-MHTVFEQDSA-N edatrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CC(CC)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FSIRXIHZBIXHKT-MHTVFEQDSA-N 0.000 description 1
- 229950006700 edatrexate Drugs 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- XOPYFXBZMVTEJF-PDACKIITSA-N eleutherobin Chemical compound C(/[C@H]1[C@H](C(=CC[C@@H]1C(C)C)C)C[C@@H]([C@@]1(C)O[C@@]2(C=C1)OC)OC(=O)\C=C\C=1N=CN(C)C=1)=C2\CO[C@@H]1OC[C@@H](O)[C@@H](O)[C@@H]1OC(C)=O XOPYFXBZMVTEJF-PDACKIITSA-N 0.000 description 1
- XOPYFXBZMVTEJF-UHFFFAOYSA-N eleutherobin Natural products C1=CC2(OC)OC1(C)C(OC(=O)C=CC=1N=CN(C)C=1)CC(C(=CCC1C(C)C)C)C1C=C2COC1OCC(O)C(O)C1OC(C)=O XOPYFXBZMVTEJF-UHFFFAOYSA-N 0.000 description 1
- 229950000549 elliptinium acetate Drugs 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- JOZGNYDSEBIJDH-UHFFFAOYSA-N eniluracil Chemical compound O=C1NC=C(C#C)C(=O)N1 JOZGNYDSEBIJDH-UHFFFAOYSA-N 0.000 description 1
- 229950010213 eniluracil Drugs 0.000 description 1
- 229950011487 enocitabine Drugs 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 229960001904 epirubicin Drugs 0.000 description 1
- 229950002973 epitiostanol Drugs 0.000 description 1
- 229930013356 epothilone Natural products 0.000 description 1
- 150000003883 epothilone derivatives Chemical class 0.000 description 1
- 229960001433 erlotinib Drugs 0.000 description 1
- AAKJLRGGTJKAMG-UHFFFAOYSA-N erlotinib Chemical compound C=12C=C(OCCOC)C(OCCOC)=CC2=NC=NC=1NC1=CC=CC(C#C)=C1 AAKJLRGGTJKAMG-UHFFFAOYSA-N 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 208000028653 esophageal adenocarcinoma Diseases 0.000 description 1
- 229950002017 esorubicin Drugs 0.000 description 1
- ITSGNOIFAJAQHJ-BMFNZSJVSA-N esorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)C[C@H](C)O1 ITSGNOIFAJAQHJ-BMFNZSJVSA-N 0.000 description 1
- LJQQFQHBKUKHIS-WJHRIEJJSA-N esperamicin Chemical compound O1CC(NC(C)C)C(OC)CC1OC1C(O)C(NOC2OC(C)C(SC)C(O)C2)C(C)OC1OC1C(\C2=C/CSSSC)=C(NC(=O)OC)C(=O)C(OC3OC(C)C(O)C(OC(=O)C=4C(=CC(OC)=C(OC)C=4)NC(=O)C(=C)OC)C3)C2(O)C#C\C=C/C#C1 LJQQFQHBKUKHIS-WJHRIEJJSA-N 0.000 description 1
- 229960001842 estramustine Drugs 0.000 description 1
- FRPJXPJMRWBBIH-RBRWEJTLSA-N estramustine Chemical compound ClCCN(CCCl)C(=O)OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 FRPJXPJMRWBBIH-RBRWEJTLSA-N 0.000 description 1
- QSRLNKCNOLVZIR-KRWDZBQOSA-N ethyl (2s)-2-[[2-[4-[bis(2-chloroethyl)amino]phenyl]acetyl]amino]-4-methylsulfanylbutanoate Chemical compound CCOC(=O)[C@H](CCSC)NC(=O)CC1=CC=C(N(CCCl)CCCl)C=C1 QSRLNKCNOLVZIR-KRWDZBQOSA-N 0.000 description 1
- 229960005237 etoglucid Drugs 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 229960005167 everolimus Drugs 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229960000961 floxuridine Drugs 0.000 description 1
- ODKNJVUHOIMIIZ-RRKCRQDMSA-N floxuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ODKNJVUHOIMIIZ-RRKCRQDMSA-N 0.000 description 1
- 229960000390 fludarabine Drugs 0.000 description 1
- GIUYCYHIANZCFB-FJFJXFQQSA-N fludarabine phosphate Chemical compound C1=NC=2C(N)=NC(F)=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O GIUYCYHIANZCFB-FJFJXFQQSA-N 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 150000002224 folic acids Chemical class 0.000 description 1
- 229950005309 fostamatinib Drugs 0.000 description 1
- GKDRMWXFWHEQQT-UHFFFAOYSA-N fostamatinib Chemical compound COC1=C(OC)C(OC)=CC(NC=2N=C(NC=3N=C4N(COP(O)(O)=O)C(=O)C(C)(C)OC4=CC=3)C(F)=CN=2)=C1 GKDRMWXFWHEQQT-UHFFFAOYSA-N 0.000 description 1
- 229960004783 fotemustine Drugs 0.000 description 1
- YAKWPXVTIGTRJH-UHFFFAOYSA-N fotemustine Chemical compound CCOP(=O)(OCC)C(C)NC(=O)N(CCCl)N=O YAKWPXVTIGTRJH-UHFFFAOYSA-N 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 229940044658 gallium nitrate Drugs 0.000 description 1
- 229960005277 gemcitabine Drugs 0.000 description 1
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 1
- 229940045109 genistein Drugs 0.000 description 1
- TZBJGXHYKVUXJN-UHFFFAOYSA-N genistein Natural products C1=CC(O)=CC=C1C1=COC2=CC(O)=CC(O)=C2C1=O TZBJGXHYKVUXJN-UHFFFAOYSA-N 0.000 description 1
- 235000006539 genistein Nutrition 0.000 description 1
- ZCOLJUOHXJRHDI-CMWLGVBASA-N genistein 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 ZCOLJUOHXJRHDI-CMWLGVBASA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- UUVWYPNAQBNQJQ-UHFFFAOYSA-N hexamethylmelamine Chemical compound CN(C)C1=NC(N(C)C)=NC(N(C)C)=N1 UUVWYPNAQBNQJQ-UHFFFAOYSA-N 0.000 description 1
- 238000001794 hormone therapy Methods 0.000 description 1
- 229960001330 hydroxycarbamide Drugs 0.000 description 1
- 230000003463 hyperproliferative effect Effects 0.000 description 1
- 229940015872 ibandronate Drugs 0.000 description 1
- 229960000908 idarubicin Drugs 0.000 description 1
- 229960003445 idelalisib Drugs 0.000 description 1
- IFSDAJWBUCMOAH-HNNXBMFYSA-N idelalisib Chemical compound C1([C@@H](NC=2C=3N=CNC=3N=CN=2)CC)=NC2=CC=CC(F)=C2C(=O)N1C1=CC=CC=C1 IFSDAJWBUCMOAH-HNNXBMFYSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 229960002411 imatinib Drugs 0.000 description 1
- KTUFNOKKBVMGRW-UHFFFAOYSA-N imatinib Chemical compound C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 KTUFNOKKBVMGRW-UHFFFAOYSA-N 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- DBIGHPPNXATHOF-UHFFFAOYSA-N improsulfan Chemical compound CS(=O)(=O)OCCCNCCCOS(C)(=O)=O DBIGHPPNXATHOF-UHFFFAOYSA-N 0.000 description 1
- 229950008097 improsulfan Drugs 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000010212 intracellular staining Methods 0.000 description 1
- 229960005386 ipilimumab Drugs 0.000 description 1
- 229960004768 irinotecan Drugs 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 229940043355 kinase inhibitor Drugs 0.000 description 1
- 210000001821 langerhans cell Anatomy 0.000 description 1
- 229960004891 lapatinib Drugs 0.000 description 1
- 238000002430 laser surgery Methods 0.000 description 1
- 229940115286 lentinan Drugs 0.000 description 1
- 229960003784 lenvatinib Drugs 0.000 description 1
- WOSKHXYHFSIKNG-UHFFFAOYSA-N lenvatinib Chemical compound C=12C=C(C(N)=O)C(OC)=CC2=NC=CC=1OC(C=C1Cl)=CC=C1NC(=O)NC1CC1 WOSKHXYHFSIKNG-UHFFFAOYSA-N 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229960002247 lomustine Drugs 0.000 description 1
- YROQEQPFUCPDCP-UHFFFAOYSA-N losoxantrone Chemical compound OCCNCCN1N=C2C3=CC=CC(O)=C3C(=O)C3=C2C1=CC=C3NCCNCCO YROQEQPFUCPDCP-UHFFFAOYSA-N 0.000 description 1
- 229950008745 losoxantrone Drugs 0.000 description 1
- 210000004880 lymph fluid Anatomy 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- MQXVYODZCMMZEM-ZYUZMQFOSA-N mannomustine Chemical compound ClCCNC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CNCCCl MQXVYODZCMMZEM-ZYUZMQFOSA-N 0.000 description 1
- 229950008612 mannomustine Drugs 0.000 description 1
- WKPWGQKGSOKKOO-RSFHAFMBSA-N maytansine Chemical compound CO[C@@H]([C@@]1(O)C[C@](OC(=O)N1)([C@H]([C@@H]1O[C@@]1(C)[C@@H](OC(=O)[C@H](C)N(C)C(C)=O)CC(=O)N1C)C)[H])\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 WKPWGQKGSOKKOO-RSFHAFMBSA-N 0.000 description 1
- 229960004961 mechlorethamine Drugs 0.000 description 1
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical compound ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 229950009246 mepitiostane Drugs 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- VJRAUFKOOPNFIQ-TVEKBUMESA-N methyl (1r,2r,4s)-4-[(2r,4s,5s,6s)-5-[(2s,4s,5s,6s)-5-[(2s,4s,5s,6s)-4,5-dihydroxy-6-methyloxan-2-yl]oxy-4-hydroxy-6-methyloxan-2-yl]oxy-4-(dimethylamino)-6-methyloxan-2-yl]oxy-2-ethyl-2,5,7,10-tetrahydroxy-6,11-dioxo-3,4-dihydro-1h-tetracene-1-carboxylat Chemical compound O([C@H]1[C@@H](O)C[C@@H](O[C@H]1C)O[C@H]1[C@H](C[C@@H](O[C@H]1C)O[C@H]1C[C@]([C@@H](C2=CC=3C(=O)C4=C(O)C=CC(O)=C4C(=O)C=3C(O)=C21)C(=O)OC)(O)CC)N(C)C)[C@H]1C[C@H](O)[C@H](O)[C@H](C)O1 VJRAUFKOOPNFIQ-TVEKBUMESA-N 0.000 description 1
- IZAGSTRIDUNNOY-UHFFFAOYSA-N methyl 2-[(2,4-dioxo-1h-pyrimidin-5-yl)oxy]acetate Chemical compound COC(=O)COC1=CNC(=O)NC1=O IZAGSTRIDUNNOY-UHFFFAOYSA-N 0.000 description 1
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 1
- 229960003775 miltefosine Drugs 0.000 description 1
- PQLXHQMOHUQAKB-UHFFFAOYSA-N miltefosine Chemical compound CCCCCCCCCCCCCCCCOP([O-])(=O)OCC[N+](C)(C)C PQLXHQMOHUQAKB-UHFFFAOYSA-N 0.000 description 1
- 229960005485 mitobronitol Drugs 0.000 description 1
- 229960003539 mitoguazone Drugs 0.000 description 1
- MXWHMTNPTTVWDM-NXOFHUPFSA-N mitoguazone Chemical compound NC(N)=N\N=C(/C)\C=N\N=C(N)N MXWHMTNPTTVWDM-NXOFHUPFSA-N 0.000 description 1
- VFKZTMPDYBFSTM-GUCUJZIJSA-N mitolactol Chemical compound BrC[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)CBr VFKZTMPDYBFSTM-GUCUJZIJSA-N 0.000 description 1
- 229950010913 mitolactol Drugs 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 229960000350 mitotane Drugs 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000007193 modulation by symbiont of host erythrocyte aggregation Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229950002212 mubritinib Drugs 0.000 description 1
- ZTFBIUXIQYRUNT-MDWZMJQESA-N mubritinib Chemical compound C1=CC(C(F)(F)F)=CC=C1\C=C\C1=NC(COC=2C=CC(CCCCN3N=NC=C3)=CC=2)=CO1 ZTFBIUXIQYRUNT-MDWZMJQESA-N 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 229960000951 mycophenolic acid Drugs 0.000 description 1
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 1
- XJVXMWNLQRTRGH-UHFFFAOYSA-N n-(3-methylbut-3-enyl)-2-methylsulfanyl-7h-purin-6-amine Chemical compound CSC1=NC(NCCC(C)=C)=C2NC=NC2=N1 XJVXMWNLQRTRGH-UHFFFAOYSA-N 0.000 description 1
- NJSMWLQOCQIOPE-OCHFTUDZSA-N n-[(e)-[10-[(e)-(4,5-dihydro-1h-imidazol-2-ylhydrazinylidene)methyl]anthracen-9-yl]methylideneamino]-4,5-dihydro-1h-imidazol-2-amine Chemical compound N1CCN=C1N\N=C\C(C1=CC=CC=C11)=C(C=CC=C2)C2=C1\C=N\NC1=NCCN1 NJSMWLQOCQIOPE-OCHFTUDZSA-N 0.000 description 1
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229940086322 navelbine Drugs 0.000 description 1
- 210000005170 neoplastic cell Anatomy 0.000 description 1
- 229960001346 nilotinib Drugs 0.000 description 1
- HHZIURLSWUIHRB-UHFFFAOYSA-N nilotinib Chemical compound C1=NC(C)=CN1C1=CC(NC(=O)C=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)=CC(C(F)(F)F)=C1 HHZIURLSWUIHRB-UHFFFAOYSA-N 0.000 description 1
- 229960001420 nimustine Drugs 0.000 description 1
- VFEDRRNHLBGPNN-UHFFFAOYSA-N nimustine Chemical compound CC1=NC=C(CNC(=O)N(CCCl)N=O)C(N)=N1 VFEDRRNHLBGPNN-UHFFFAOYSA-N 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 229960003301 nivolumab Drugs 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- CZDBNBLGZNWKMC-MWQNXGTOSA-N olivomycin Chemical class O([C@@H]1C[C@@H](O[C@H](C)[C@@H]1O)OC=1C=C2C=C3C[C@H]([C@@H](C(=O)C3=C(O)C2=C(O)C=1)O[C@H]1O[C@@H](C)[C@H](O)[C@@H](OC2O[C@@H](C)[C@H](O)[C@@H](O)C2)C1)[C@H](OC)C(=O)[C@@H](O)[C@@H](C)O)[C@H]1C[C@H](O)[C@H](OC)[C@H](C)O1 CZDBNBLGZNWKMC-MWQNXGTOSA-N 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 229940127084 other anti-cancer agent Drugs 0.000 description 1
- 229960001756 oxaliplatin Drugs 0.000 description 1
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 238000011499 palliative surgery Methods 0.000 description 1
- VREZDOWOLGNDPW-UHFFFAOYSA-N pancratistatine Natural products C1=C2C3C(O)C(O)C(O)C(O)C3NC(=O)C2=C(O)C2=C1OCO2 VREZDOWOLGNDPW-UHFFFAOYSA-N 0.000 description 1
- 229960001972 panitumumab Drugs 0.000 description 1
- 229960000639 pazopanib Drugs 0.000 description 1
- CUIHSIWYWATEQL-UHFFFAOYSA-N pazopanib Chemical compound C1=CC2=C(C)N(C)N=C2C=C1N(C)C(N=1)=CC=NC=1NC1=CC=C(C)C(S(N)(=O)=O)=C1 CUIHSIWYWATEQL-UHFFFAOYSA-N 0.000 description 1
- 229960003407 pegaptanib Drugs 0.000 description 1
- 229960002621 pembrolizumab Drugs 0.000 description 1
- 229960002340 pentostatin Drugs 0.000 description 1
- FPVKHBSQESCIEP-JQCXWYLXSA-N pentostatin Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC[C@H]2O)=C2N=C1 FPVKHBSQESCIEP-JQCXWYLXSA-N 0.000 description 1
- QIMGFXOHTOXMQP-GFAGFCTOSA-N peplomycin Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCCN[C@@H](C)C=1C=CC=CC=1)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1NC=NC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C QIMGFXOHTOXMQP-GFAGFCTOSA-N 0.000 description 1
- 229950003180 peplomycin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- SZFPYBIJACMNJV-UHFFFAOYSA-N perifosine Chemical compound CCCCCCCCCCCCCCCCCCOP([O-])(=O)OC1CC[N+](C)(C)CC1 SZFPYBIJACMNJV-UHFFFAOYSA-N 0.000 description 1
- 229950010632 perifosine Drugs 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 1
- 229960000952 pipobroman Drugs 0.000 description 1
- NJBFOOCLYDNZJN-UHFFFAOYSA-N pipobroman Chemical compound BrCCC(=O)N1CCN(C(=O)CCBr)CC1 NJBFOOCLYDNZJN-UHFFFAOYSA-N 0.000 description 1
- NUKCGLDCWQXYOQ-UHFFFAOYSA-N piposulfan Chemical compound CS(=O)(=O)OCCC(=O)N1CCN(C(=O)CCOS(C)(=O)=O)CC1 NUKCGLDCWQXYOQ-UHFFFAOYSA-N 0.000 description 1
- 229950001100 piposulfan Drugs 0.000 description 1
- 229960001221 pirarubicin Drugs 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 210000003281 pleural cavity Anatomy 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 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
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 229960004694 prednimustine Drugs 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- WOLQREOUPKZMEX-UHFFFAOYSA-N pteroyltriglutamic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(=O)NC(CCC(=O)NC(CCC(O)=O)C(O)=O)C(O)=O)C(O)=O)C=C1 WOLQREOUPKZMEX-UHFFFAOYSA-N 0.000 description 1
- IGFXRKMLLMBKSA-UHFFFAOYSA-N purine Chemical compound N1=C[N]C2=NC=NC2=C1 IGFXRKMLLMBKSA-UHFFFAOYSA-N 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 230000003439 radiotherapeutic effect Effects 0.000 description 1
- 229960003876 ranibizumab Drugs 0.000 description 1
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 description 1
- BMKDZUISNHGIBY-UHFFFAOYSA-N razoxane Chemical compound C1C(=O)NC(=O)CN1C(C)CN1CC(=O)NC(=O)C1 BMKDZUISNHGIBY-UHFFFAOYSA-N 0.000 description 1
- 229960000460 razoxane Drugs 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- OWPCHSCAPHNHAV-LMONGJCWSA-N rhizoxin Chemical compound C/C([C@H](OC)[C@@H](C)[C@@H]1C[C@H](O)[C@]2(C)O[C@@H]2/C=C/[C@@H](C)[C@]2([H])OC(=O)C[C@@](C2)(C[C@@H]2O[C@H]2C(=O)O1)[H])=C\C=C\C(\C)=C\C1=COC(C)=N1 OWPCHSCAPHNHAV-LMONGJCWSA-N 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 229960001302 ridaforolimus Drugs 0.000 description 1
- 229950004892 rodorubicin Drugs 0.000 description 1
- MBABCNBNDNGODA-WPZDJQSSSA-N rolliniastatin 1 Natural products O1[C@@H]([C@@H](O)CCCCCCCCCC)CC[C@H]1[C@H]1O[C@@H]([C@H](O)CCCCCCCCCC[C@@H](O)CC=2C(O[C@@H](C)C=2)=O)CC1 MBABCNBNDNGODA-WPZDJQSSSA-N 0.000 description 1
- IMUQLZLGWJSVMV-UOBFQKKOSA-N roridin A Natural products CC(O)C1OCCC(C)C(O)C(=O)OCC2CC(=CC3OC4CC(OC(=O)C=C/C=C/1)C(C)(C23)C45CO5)C IMUQLZLGWJSVMV-UOBFQKKOSA-N 0.000 description 1
- VHXNKPBCCMUMSW-FQEVSTJZSA-N rubitecan Chemical compound C1=CC([N+]([O-])=O)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VHXNKPBCCMUMSW-FQEVSTJZSA-N 0.000 description 1
- 229960000215 ruxolitinib Drugs 0.000 description 1
- HFNKQEVNSGCOJV-OAHLLOKOSA-N ruxolitinib Chemical compound C1([C@@H](CC#N)N2N=CC(=C2)C=2C=3C=CNC=3N=CN=2)CCCC1 HFNKQEVNSGCOJV-OAHLLOKOSA-N 0.000 description 1
- 229950009919 saracatinib Drugs 0.000 description 1
- OUKYUETWWIPKQR-UHFFFAOYSA-N saracatinib Chemical compound C1CN(C)CCN1CCOC1=CC(OC2CCOCC2)=C(C(NC=2C(=CC=C3OCOC3=2)Cl)=NC=N2)C2=C1 OUKYUETWWIPKQR-UHFFFAOYSA-N 0.000 description 1
- 229930182947 sarcodictyin Natural products 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 229950010746 selumetinib Drugs 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 229960002930 sirolimus Drugs 0.000 description 1
- 229950001403 sizofiran Drugs 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 108091029842 small nuclear ribonucleic acid Proteins 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229960003787 sorafenib Drugs 0.000 description 1
- 229950006315 spirogermanium Drugs 0.000 description 1
- ICXJVZHDZFXYQC-UHFFFAOYSA-N spongistatin 1 Natural products OC1C(O2)(O)CC(O)C(C)C2CCCC=CC(O2)CC(O)CC2(O2)CC(OC)CC2CC(=O)C(C)C(OC(C)=O)C(C)C(=C)CC(O2)CC(C)(O)CC2(O2)CC(OC(C)=O)CC2CC(=O)OC2C(O)C(CC(=C)CC(O)C=CC(Cl)=C)OC1C2C ICXJVZHDZFXYQC-UHFFFAOYSA-N 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000011301 standard therapy Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229960001052 streptozocin Drugs 0.000 description 1
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229960001796 sunitinib Drugs 0.000 description 1
- WINHZLLDWRZWRT-ATVHPVEESA-N sunitinib Chemical compound CCN(CC)CCNC(=O)C1=C(C)NC(\C=C/2C3=CC(F)=CC=C3NC\2=O)=C1C WINHZLLDWRZWRT-ATVHPVEESA-N 0.000 description 1
- 238000011477 surgical intervention Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- URLYINUFLXOMHP-HTVVRFAVSA-N tcn-p Chemical compound C=12C3=NC=NC=1N(C)N=C(N)C2=CN3[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O URLYINUFLXOMHP-HTVVRFAVSA-N 0.000 description 1
- 210000001138 tear Anatomy 0.000 description 1
- 229960000235 temsirolimus Drugs 0.000 description 1
- QFJCIRLUMZQUOT-UHFFFAOYSA-N temsirolimus Natural products C1CC(O)C(OC)CC1CC(C)C1OC(=O)C2CCCCN2C(=O)C(=O)C(O)(O2)C(C)CCC2CC(OC)C(C)=CC=CC=CC(C)CC(C)C(=O)C(OC)C(O)C(C)=CC(C)C(=O)C1 QFJCIRLUMZQUOT-UHFFFAOYSA-N 0.000 description 1
- NRUKOCRGYNPUPR-QBPJDGROSA-N teniposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@@H](OC[C@H]4O3)C=3SC=CC=3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 NRUKOCRGYNPUPR-QBPJDGROSA-N 0.000 description 1
- 229960001278 teniposide Drugs 0.000 description 1
- BPEWUONYVDABNZ-DZBHQSCQSA-N testolactone Chemical compound O=C1C=C[C@]2(C)[C@H]3CC[C@](C)(OC(=O)CC4)[C@@H]4[C@@H]3CCC2=C1 BPEWUONYVDABNZ-DZBHQSCQSA-N 0.000 description 1
- 229960005353 testolactone Drugs 0.000 description 1
- 238000005382 thermal cycling Methods 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 229960001196 thiotepa Drugs 0.000 description 1
- YFTWHEBLORWGNI-UHFFFAOYSA-N tiamiprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC(N)=NC2=C1NC=N2 YFTWHEBLORWGNI-UHFFFAOYSA-N 0.000 description 1
- 229950011457 tiamiprine Drugs 0.000 description 1
- 229940044693 topoisomerase inhibitor Drugs 0.000 description 1
- 229960000303 topotecan Drugs 0.000 description 1
- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 229960000575 trastuzumab Drugs 0.000 description 1
- 229950001353 tretamine Drugs 0.000 description 1
- IUCJMVBFZDHPDX-UHFFFAOYSA-N tretamine Chemical compound C1CN1C1=NC(N2CC2)=NC(N2CC2)=N1 IUCJMVBFZDHPDX-UHFFFAOYSA-N 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- 229960004560 triaziquone Drugs 0.000 description 1
- PXSOHRWMIRDKMP-UHFFFAOYSA-N triaziquone Chemical compound O=C1C(N2CC2)=C(N2CC2)C(=O)C=C1N1CC1 PXSOHRWMIRDKMP-UHFFFAOYSA-N 0.000 description 1
- 229930013292 trichothecene Natural products 0.000 description 1
- 150000003327 trichothecene derivatives Chemical class 0.000 description 1
- 229960001670 trilostane Drugs 0.000 description 1
- KVJXBPDAXMEYOA-CXANFOAXSA-N trilostane Chemical compound OC1=C(C#N)C[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@@]32O[C@@H]31 KVJXBPDAXMEYOA-CXANFOAXSA-N 0.000 description 1
- NOYPYLRCIDNJJB-UHFFFAOYSA-N trimetrexate Chemical compound COC1=C(OC)C(OC)=CC(NCC=2C(=C3C(N)=NC(N)=NC3=CC=2)C)=C1 NOYPYLRCIDNJJB-UHFFFAOYSA-N 0.000 description 1
- 229960001099 trimetrexate Drugs 0.000 description 1
- 125000002264 triphosphate group Chemical group [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 229960000875 trofosfamide Drugs 0.000 description 1
- UMKFEPPTGMDVMI-UHFFFAOYSA-N trofosfamide Chemical compound ClCCN(CCCl)P1(=O)OCCCN1CCCl UMKFEPPTGMDVMI-UHFFFAOYSA-N 0.000 description 1
- HDZZVAMISRMYHH-LITAXDCLSA-N tubercidin Chemical compound C1=CC=2C(N)=NC=NC=2N1[C@@H]1O[C@@H](CO)[C@H](O)[C@H]1O HDZZVAMISRMYHH-LITAXDCLSA-N 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 229950009811 ubenimex Drugs 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 229960001055 uracil mustard Drugs 0.000 description 1
- 229960000241 vandetanib Drugs 0.000 description 1
- UHTHHESEBZOYNR-UHFFFAOYSA-N vandetanib Chemical compound COC1=CC(C(/N=CN2)=N/C=3C(=CC(Br)=CC=3)F)=C2C=C1OCC1CCN(C)CC1 UHTHHESEBZOYNR-UHFFFAOYSA-N 0.000 description 1
- 229950000578 vatalanib Drugs 0.000 description 1
- YCOYDOIWSSHVCK-UHFFFAOYSA-N vatalanib Chemical compound C1=CC(Cl)=CC=C1NC(C1=CC=CC=C11)=NN=C1CC1=CC=NC=C1 YCOYDOIWSSHVCK-UHFFFAOYSA-N 0.000 description 1
- 229960003862 vemurafenib Drugs 0.000 description 1
- GPXBXXGIAQBQNI-UHFFFAOYSA-N vemurafenib Chemical compound CCCS(=O)(=O)NC1=CC=C(F)C(C(=O)C=2C3=CC(=CN=C3NC=2)C=2C=CC(Cl)=CC=2)=C1F GPXBXXGIAQBQNI-UHFFFAOYSA-N 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 229960003048 vinblastine Drugs 0.000 description 1
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- 229960004355 vindesine Drugs 0.000 description 1
- UGGWPQSBPIFKDZ-KOTLKJBCSA-N vindesine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(N)=O)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1N=C1[C]2C=CC=C1 UGGWPQSBPIFKDZ-KOTLKJBCSA-N 0.000 description 1
- GBABOYUKABKIAF-IELIFDKJSA-N vinorelbine Chemical compound C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC([C@]23[C@H]([C@@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC GBABOYUKABKIAF-IELIFDKJSA-N 0.000 description 1
- 229960002066 vinorelbine Drugs 0.000 description 1
- CILBMBUYJCWATM-PYGJLNRPSA-N vinorelbine ditartrate Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O.OC(=O)[C@H](O)[C@@H](O)C(O)=O.C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC([C@]23[C@H]([C@@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC CILBMBUYJCWATM-PYGJLNRPSA-N 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- 229940053867 xeloda Drugs 0.000 description 1
- 229950009268 zinostatin Drugs 0.000 description 1
- 229960000641 zorubicin Drugs 0.000 description 1
- FBTUMDXHSRTGRV-ALTNURHMSA-N zorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(\C)=N\NC(=O)C=1C=CC=CC=1)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 FBTUMDXHSRTGRV-ALTNURHMSA-N 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4615—Dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4637—Other peptides or polypeptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2521/00—Reaction characterised by the enzymatic activity
- C12Q2521/10—Nucleotidyl transfering
- C12Q2521/107—RNA dependent DNA polymerase,(i.e. reverse transcriptase)
Definitions
- the present invention relates generally to the field of molecular biology. More particularly, it concerns reverse transcription of mRNA and amplification of those products.
- DNA can be replicated and amplified in vitro by the polymerase chain reaction, in which primers are hybridized to opposite ends, and opposite strands of a target nucleic acids, and the primers are extended by a DNA dependent DNA polymerase.
- RNA which generally exists transiently in vivo, can be reverse transcribed by a RNA dependent DNA polymerase (reverse transcriptase) in order to generate a DNA copy, which is far more stable than RNA.
- reverse transcriptase reverse transcriptase
- the DNA which is generated from reverse transcription referred to as complementary DNA or cDNA, can then be amplified, sequenced, transcribed back into RNA, incorporated into a vector for cloning, or any combination thereof.
- RNA reverse transcribes into DNA.
- researchers take advantage of the poly(A) tail which exists on the 3′ end of mRNA. This allows researchers to use primers with a poly(T) sequence at the 3′ end of the primer, and makes capturing 3′ sequence information from mRNA very simple.
- the second primer corresponding to the 5′ end of the target sequence, comprises either random or targeted sequences, as in standard PCR.
- a third option for the 5′ primer is to take advantage of the inherent template switching activity of some reverse transcriptase enzymes and hybridize the second primer to a polynucleotide overhang generated by the reverse transcriptase, and continue to synthesize the strand complementary to the second primer (see, for example, U.S. Pat. No. 5,962,271).
- the complementary DNA strand can then be copied using a standard PCR reaction to amplify the sequence.
- the primers used in cDNA synthesis and amplification may include a promoter so that the cDNA can be transcribed using a process of in vitro transcription to generate large quantities of target mRNA. RNA generated by in vitro transcription can then be used to induce antigen presentation on dendritic cells.
- the methods for reverse transcription and in vitro transcription suffer from high bias during the reverse transcription process, and inefficient in vitro transcription, demonstrating a need for improved methods and compositions to enhance these processes.
- an oligonucleotide comprising the sequence of SEQ ID NO: 1.
- the oligonucleotide is further defined as a primer.
- the oligonucleotide comprises less than 40 nucleotides.
- the oligonucleotide consists of SEQ ID NO: 1.
- composition comprising a first primer and a second primer, wherein the first primer comprises a nucleic acid sequence of SEQ ID NO: 1, and wherein the second primer comprises a nucleic acid sequence of SEQ ID NO: 2.
- the composition is further defined as a nucleic acid amplification reaction mixture.
- the composition further comprises a third primer, wherein the third primer comprises a nucleic acid sequence of SEQ ID NO: 3.
- a method of synthesizing a first cDNA strand from a template mRNA comprising the steps of: a) hybridizing a first primer to the template mRNA, wherein the first primer comprises a nucleic acid sequence of SEQ ID NO: 2; b) extending the first primer with a reverse transcriptase that has terminal transferase and template switching activity to generate a partial first cDNA strand with an oligo(C) overhang; c) hybridizing a second primer to the oligo(C) overhang of the partial first cDNA strand, wherein the second primer comprises a nucleic acid sequence of SEQ ID NO: 1; and d) extending the partial first cDNA strand from the oligo(C) overhang using the second primer as the template, thereby generating a first cDNA strand.
- the method further comprises steps e) separating the template mRNA and first cDNA strand; f) hybridizing a third primer to the first cDNA strand; and g) extending the third primer to generate a second cDNA strand, thereby generating a double stranded cDNA.
- the second primer comprises a nucleic acid sequence of SEQ ID NO: 1.
- the third primer comprises a nucleic acid sequence of SEQ ID NO: 3.
- the template mRNA is obtained from a sample.
- the sample is a tumor sample.
- the tumor sample is stored in an RNA stabilization solution after removal from a subject.
- the RNA stabilization solution is RNALATER®.
- the method further comprises synthesizing RNA from the double stranded cDNA.
- the synthesizing RNA from the double stranded cDNA comprises in vitro transcription.
- the in vitro transcription comprises adding a fourth primer, a fifth primer, and an RNA polymerase, and synthesizing RNA from the double stranded cDNA with the RNA polymerase.
- the fourth primer comprises a nucleic acid sequence of SEQ ID NO: 1.
- the fourth primer comprises a nucleic acid sequence of SEQ ID NO: 3.
- the method further comprises capping the RNA.
- the method further comprises amplifying the double stranded cDNA.
- the second primer comprises the nucleic acid sequence of SEQ ID NO: 1.
- the third primer comprises the nucleic acid sequence of SEQ ID NO: 3.
- amplifying the double stranded cDNA comprises adding a DNA dependent DNA polymerase, a fourth primer, and a fifth primer and amplifying the cDNA by polymerase chain reaction.
- the fourth primer comprises the nucleic acid sequence of SEQ ID NO: 1.
- the fifth primer comprises the nucleic acid sequence of SEQ ID NO: 3.
- the method additionally comprises in vitro transcribing the amplified cDNA to generate sense-strand amplified mRNA.
- the second primer comprises the nucleic acid sequence of SEQ ID NO: 1.
- the third primer comprises the nucleic acid sequence of SEQ ID NO: 3.
- the in vitro transcribing of the amplified cDNA comprises adding primers having the sequence of SEQ ID NO: 1 and SEQ ID NO: 3 to the amplified cDNA, and hybridizing said primers to the amplified cDNA.
- the in vitro transcribing of the amplified cDNA comprises adding an RNA polymerase to the amplified cDNA, and extending the hybridized primers to generate RNA.
- the method further comprises capping the amplified RNA.
- a further embodiment of the invention provides, a method for transducing a dendritic cell population comprising contacting the dendritic cell population with a nucleic acid encoding one or more antigens, wherein the nucleic acid comprises RNA generated by any of the methods and aspects described above.
- the method further comprises contacting the dendritic cell population with a tumor cell lysate.
- the tumor cell lysate comprises a tumor antigen with an epitope having a sequence that overlaps a minimum of 5 amino acids with the sequence of the nucleic acid encoding one or more antigens.
- a method for providing an immune response in a subject having a diseased cell population comprising: a) obtaining a primed dendritic cell population produced by the method according to claim 36 or 37 ; and b) administering an effective amount of the primed dendritic cell population to the subject.
- the antigen-primed dendritic cell has been primed with an antigen associated with a cancer, an autoimmune disease or an infectious disease.
- the antigen-primed dendritic cell has been primed with at least one tumor antigen.
- the method further comprises administering an immune checkpoint inhibitor.
- the immune checkpoint inhibitor is a CTLA-4 antagonist.
- the immune checkpoint inhibitor is ipilimumab, pembrolizumab or nivolumab.
- kits comprising an oligonucleotide primer of claim 1 , an oligonucleotide primer of SEQ ID NO:2 and/or an oligonucleotide primer of SEQ ID NO:3.
- the kit may additionally comprise nucleic acids isolated from cancer cells, a DNA polymerase, and/or an RNA stabilization solution.
- the RNA stabilization solution is RNALATER®.
- the kit is further defined as a cDNA synthesis kit.
- the kit further comprises dNTPs, MgCl 2 , reverse transcriptase, and/or RNase inhibitor.
- essentially free in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts.
- the total amount of the specified component resulting from any unintended contamination of a composition is preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
- a” or “an” may mean one or more.
- the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
- “another” or “a further” may mean at least a second or more.
- the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
- FIG. 1 Freshly harvested tumor samples yield better quality RNA than frozen archived samples. Pictured, RNA isolated from either frozen or fresh tumor tissue was run on a non-denaturing 1% agarose gel. From left to right: L: ladder; 1: frozen tumor sample RNA; 2 frozen tumor sample RNA; 3: Frozen tumor sample RNA; 4: Fresh tumor sample RNA.
- FIG. 2 Comparison of two different methods of RNA isolation. Pictured, RNA samples were run on a non-denaturing 1% agarose gel. Lane 1: RNA isolated from 30 mg of tumor tissue by column purification. Lane 2: RNA isolated from 30 mg of tumor tissue by guanidinium thiocyanate/phenol/chloroform purification.
- FIG. 3 Schematic showing the process of reverse transcription used in the present invention, indicating the primers used.
- VKWD primer has the nucleotide sequence of SEQ ID NO: 1.
- CDS64T+oligo primer has the nucleotide sequence of SEQ ID NO: 2.
- T7 power switch primer has the nucleotide sequence of SEQ ID NO: 3.
- FIG. 4 Comparison of methods of reverse transcription followed by in vitro transcription. Pictured, 4 different protocols were used to generate in vitro transcribed RNA from identical starting material. Lane 1, uncapped transcripts generated by the Slagter-Jager method (Slagter-Jager et al., 2013). Lane 2, capped transcripts generated by the Slagter-Jager method (Slagter-Jager et al., 2013). Lane 3, uncapped RNA generated by the methods described herein. Lane 4, capped RNA generated by the methods described herein.
- FIG. 5 Comparison of mRNA generated and capped by two different methods. 5 ug of in vitro transcribed mRNA was run on a 1% agarose, non-denaturing gel. Lane 1, mRNA generated by previous method (Slagter-Jager et al., 2013) and capped post-transcription. Lane 2, mRNA generated by the present methods and capped co-transcriptionally.
- FIG. 6 IFN ⁇ production is significantly enhanced by T cells cocultured with dendritic cells loaded with capped mRNA and lysates. Pictured, relative amounts of IFN- ⁇ secretion as measured by ELISA for cells loaded with uncapped or capped mRNA generated by the previous method (Argos (Slagter-Jager)) or the present methods (VKWD), and lysates, or with mismatched mRNA and lysates.
- FIG. 7 Antigen specific CD8 + CD25 + T cell production is enhanced when co-cultured with dendritic cells homologously loaded with capped mRNA and lysate. Pictured, flow cytometric analysis of T cells for antigen specific activation markers following re-stimulation with dendritic cells loaded with the mRNA shown below the data.
- FIG. 8 Western blot analysis of dendritic cell lysates.
- Lane 1 unloaded dendritic cells.
- Lane 2 dendritic cells loaded with uncapped mRNA generated using the Slagter-Jager method.
- Lane 3 dendritic cells loaded with capped mRNA generated using the Slagter-Jager method.
- Lane 4 dendritic cells loaded with uncapped mRNA generated using the methods described herein.
- Lane 5 dendritic cells loaded with capped mRNA generated using the methods described herein.
- Lane 6 dendritic cells loaded with mismatched mRNA and lysate.
- Lane 7 dendritic cells loaded with mRNA generated using the methods described herein and capped co-transcriptionally.
- FIG. 9 Homologous antigenic loading with in vitro transcribed and amplified mRNA leads to enhanced retention of CTLA4. Pictured are flow cytometric analyses of dendritic cells either unloaded, or loaded with the indicated: mRNA, Lysate, 2 ⁇ (mRNA and Lysate), and MM (mismatched mRNA and lysate).
- FIG. 10 Enhanced retention and reduced release of CTLA4 from the homologously loaded dendritic cells. Pictured are western blots detecting CTLA4 in the supernatant or the lysate of dendritic cell cultures. Dendritic cells were either unloaded (UL) or loaded with mRNA, lysate, both, or mismatched mRNA and lysate. mRNA was isolated by either a column based method or guanidinium thiocyanate/phenol/chloroform method.
- FIG. 11 IL-12 transcript levels are elevated in dendritic cells homologously loaded with lysates and in vitro transcribed mRNA. Shown are IL-12a and IL-12b levels as detected by ELISA for dendritic cells loaded with the indicated RNA and/or lysate. The left axis indicates arbitrary transcriptional units normalized to unloaded control DC and set at 1.0.
- FIG. 12 Flow chart describing the preparation of a dendritic cell vaccine.
- FIG. 13 A-B Amplified mRNA generates TH1 immune responses in vitro with the same efficiency as native poly-A mRNA when loaded into DC with homologous lysate.
- A Monocyte-derived human DC were loaded with a) native poly A tumor mRNA and homologous/heterologous lysate or 2) capped IVT-amplified mRNA and homologous/heterologous lysate. DC were then cocultured with T cells and the percent of activated CD8+CD25+Ifng+ cells were analyzed by flow. T cells cocultured with unloaded DC or DC loaded with uncapped mRNA served as controls.
- Reverse transcription is a process in which a RNA dependent DNA polymerase synthesizes DNA using RNA as a template, and has become increasingly important as a tool in molecular biology for understanding gene expression and sequencing cellular RNA.
- Using a process of in vitro transcription and amplification following reverse transcription allows for the production of large quantities of RNA from a template RNA.
- the present disclosure provides methods for decreasing the bias of the reverse transcription process, and provides methods for increasing amplification efficiency of the reverse transcribed molecules.
- the present studies found the nucleotide composition and sequence of the primers to be important to the reverse transcription process, as well as the incorporation of a promoter sequence in the primers, for efficient in vitro transcription.
- compositions and methods for the synthesis, amplification, and in vitro transcription of full-length cDNA, or cDNA fragments may comprise contacting RNA with a primer which can anneal to the poly(A) tail of mRNA, a suitable enzyme which possesses reverse transcriptase activity, and a template switching oligonucleotide under conditions sufficient to permit the template-dependent extension of the primer a cDNA complementary to the mRNA template.
- the template switching oligonucleotide can hybridize to a reverse transcriptase generated overhang at the 3′ end of the newly generated cDNA, and allow for the reverse transcriptase to continue to synthesize the complement of the template switching oligonucleotide on the 3′ end of the cDNA. Subsequent amplification can introduce a promoter sequence for in vitro transcription. In vitro transcribed RNA can then be loaded into dendritic cells with homologous lysate for immune stimulation. Thus, further embodiments provide methods for loading of dendritic cells and use of the dendritic cell vaccine for the treatment of diseases, such as cancer.
- Amplification refers to an in vitro process for increasing the number of copies of a nucleotide sequence or sequences. Nucleic acid amplification results in the incorporation of nucleotides into DNA or RNA. As used herein, one amplification reaction may consist of many rounds of DNA replication. For example, one PCR reaction may consist of 30-100 “cycles” of denaturation and replication.
- PCR Polymerase chain reaction
- PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates.
- the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument.
- Primer means an oligonucleotide, either natural or synthetic that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3′ end along the template so that an extended duplex is formed.
- the sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Usually primers are extended by a DNA polymerase.
- Primers are generally of a length compatible with its use in synthesis of primer extension products, and are usually are in the range of between 8 to 100 nucleotides in length, such as 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to 40, and so on, more typically in the range of between 18-40, 20-35, 21-30 nucleotides long, and any length between the stated ranges.
- Typical primers can be in the range of between 10-50 nucleotides long, such as 15-45, 18-40, 20-30, 21-25 and so on, and any length between the stated ranges.
- the primers are usually not more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.
- the term “in the absence of exogenous manipulation” as used herein refers to there being modification of a nucleic acid molecule without changing the solution in which the nucleic acid molecule is being modified. In specific embodiments, it occurs in the absence of the hand of man or in the absence of a machine that changes solution conditions, which may also be referred to as buffer conditions. However, changes in temperature may occur during the modification.
- nucleoside is a base-sugar combination, i.e., a nucleotide lacking a phosphate. It is recognized in the art that there is a certain inter-changeability in usage of the terms nucleoside and nucleotide.
- the nucleotide deoxyuridine triphosphate, dUTP is a deoxyribonucleoside triphosphate. After incorporation into DNA, it serves as a DNA monomer, formally being deoxyuridylate, i.e., dUMP or deoxyuridine monophosphate.
- dUMP deoxyuridylate
- deoxyuridine monophosphate One may say that one incorporates dUTP into DNA even though there is no dUTP moiety in the resultant DNA. Similarly, one may say that one incorporates deoxyuridine into DNA even though that is only a part of the substrate molecule.
- Nucleotide is a term of art that refers to a base-sugar-phosphate combination. Nucleotides are the monomeric units of nucleic acid polymers, i.e., of DNA and RNA. The term includes ribonucleotide triphosphates, such as rATP, rCTP, rGTP, or rUTP, and deoxyribonucleotide triphosphates, such as dATP, dCTP, dUTP, dGTP, or dTTP.
- ribonucleotide triphosphates such as rATP, rCTP, rGTP, or rUTP
- deoxyribonucleotide triphosphates such as dATP, dCTP, dUTP, dGTP, or dTTP.
- nucleic acid or “polynucleotide” will generally refer to at least one molecule or strand of DNA, RNA, DNA-RNA chimera or a derivative or analog thereof, comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine “A,” guanine “G,” thymine “T” and cytosine “C”) or RNA (e.g., A, G, uracil “U” and C).
- nucleobase such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine “A,” guanine “G,” thymine “T” and cytosine “C”) or RNA (e.g., A, G, uracil “U” and C).
- nucleic acid encompasses the terms “oligonucleotide” and “polynucleotide.” “Oligonucleotide,” as used herein, refers collectively and interchangeably to two terms of art, “oligonucleotide” and “polynucleotide.” Note that although oligonucleotide and polynucleotide are distinct terms of art, there is no exact dividing line between them and they are used interchangeably herein.
- adaptor may also be used interchangeably with the terms “oligonucleotide” and “polynucleotide.”
- the term “adaptor” can indicate a linear adaptor (either single stranded or double stranded) or a stem-loop adaptor. These definitions generally refer to at least one single-stranded molecule, but in specific embodiments will also encompass at least one additional strand that is partially, substantially, or fully complementary to at least one single-stranded molecule.
- a nucleic acid may encompass at least one double-stranded molecule or at least one triple-stranded molecule that comprises one or more complementary strand(s) or “complement(s)” of a particular sequence comprising a strand of the molecule.
- a single stranded nucleic acid may be denoted by the prefix “ss,” and a double-stranded nucleic acid by the prefix “ds.”
- Oligonucleotide refers collectively and interchangeably to two terms of art, “oligonucleotide” and “polynucleotide.” Note that although oligonucleotide and polynucleotide are distinct terms of art, there is no exact dividing line between them and they are used interchangeably herein.
- the term “adaptor” may also be used interchangeably with the terms “oligonucleotide” and “polynucleotide.”
- nucleic acid molecule or “nucleic acid target molecule” refers to any single-stranded or double-stranded nucleic acid molecule including standard canonical bases, hypermodified bases, non-natural bases, or any combination of the bases thereof.
- the nucleic acid molecule contains the four canonical DNA bases—adenine, cytosine, guanine, and thymine, and/or the four canonical RNA bases—adenine, cytosine, guanine, and uracil. Uracil can be substituted for thymine when the nucleoside contains a 2′-deoxyribose group.
- the nucleic acid molecule can be transformed from RNA into DNA and from DNA into RNA.
- mRNA can be created into complementary DNA (cDNA) using reverse transcriptase and DNA can be created into RNA using RNA polymerase.
- a nucleic acid molecule can be of biological or synthetic origin. Examples of nucleic acid molecules include genomic DNA, cDNA, RNA, a DNA/RNA hybrid, amplified DNA, a pre-existing nucleic acid library, etc.
- a nucleic acid may be obtained from a human sample, such as blood, serum, plasma, cerebrospinal fluid, cheek scrapings, biopsy, semen, urine, feces, saliva, sweat, etc.
- a nucleic acid molecule may be subjected to various treatments, such as repair treatments and fragmenting treatments. Fragmenting treatments include mechanical, sonic, and hydrodynamic shearing. Repair treatments include nick repair via extension and/or ligation, polishing to create blunt ends, removal of damaged bases, such as deaminated, derivatized, abasic, or crosslinked nucleotides, etc.
- a nucleic acid molecule of interest may also be subjected to chemical modification (e.g., bisulfite conversion, methylation/demethylation), extension, amplification (e.g., PCR, isothermal, etc.), etc.
- “Analogous” forms of purines and pyrimidines are well known in the art, and include, but are not limited to aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyladenine, uracil-5-
- the nucleic acid molecule can also contain one or more hypermodified bases, for example and without limitation, 5-hydroxymethyluracil, 5-hydroxyuracil, ⁇ -putrescinylthymine, 5-hydroxymethylcytosine, 5-hydroxycytosine, 5-methylcytosine, N 4 -methyl cytosine, 2-aminoadenine, ⁇ -carbamoylmethyladenine, N 6 -methyladenine, inosine, xanthine, hypoxanthine, 2,6-diaminpurine, and N 7 -methylguanine.
- hypermodified bases for example and without limitation, 5-hydroxymethyluracil, 5-hydroxyuracil, ⁇ -putrescinylthymine, 5-hydroxymethylcytosine, 5-hydroxycytosine, 5-methylcytosine, N 4 -methyl cytosine, 2-aminoadenine, ⁇ -carbamoylmethyladenine, N 6 -methyladenine, inosine, xanthin
- the nucleic acid molecule can also contain one or more non-natural bases, for example and without limitation, 7-deaza-7-hydroxymethyladenine, 7-deaza-7-hydroxymethylguanine, isocytosine (isoC), 5-methylisocytosine, and isoguanine (isoG).
- non-natural bases for example and without limitation, 7-deaza-7-hydroxymethyladenine, 7-deaza-7-hydroxymethylguanine, isocytosine (isoC), 5-methylisocytosine, and isoguanine (isoG).
- the nucleic acid molecule containing only canonical, hypermodified, non-natural bases, or any combinations the bases thereof can also contain, for example and without limitation where each linkage between nucleotide residues can consist of a standard phosphodiester linkage, and in addition, may contain one or more modified linkages, for example and without limitation, substitution of the non-bridging oxygen atom with a nitrogen atom (i.e., a phosphoramidate linkage, a sulfur atom (i.e., a phosphorothioate linkage), or an alkyl or aryl group (i.e., alkyl or aryl phosphonates), substitution of the bridging oxygen atom with a sulfur atom (i.e., phosphorothiolate), substitution of the phosphodiester bond with a peptide bond (i.e., peptide nucleic acid or PNA), or formation of one or more additional covalent bonds (i.e., locked nucleic acid or LNA), which has an
- Nucleic acid(s) that are “complementary” or “complement(s)” are those that are capable of base-pairing according to the standard Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules.
- the term “complementary” or “complement(s)” may refer to nucleic acid(s) that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above.
- substantially complementary may refer to a nucleic acid comprising at least one sequence of consecutive nucleobases, or semiconsecutive nucleobases if one or more nucleobase moieties are not present in the molecule, are capable of hybridizing to at least one nucleic acid strand or duplex even if less than all nucleobases do not base pair with a counterpart nucleobase.
- a “substantially complementary” nucleic acid contains at least one sequence in which about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, to about 100%, and any range therein, of the nucleobase sequence is capable of base-pairing with at least one single or double-stranded nucleic acid molecule during hybridization.
- the term “substantially complementary” refers to at least one nucleic acid that may hybridize to at least one nucleic acid strand or duplex in stringent conditions.
- a “partially complementary” nucleic acid comprises at least one sequence that may hybridize in low stringency conditions to at least one single or double-stranded nucleic acid, or contains at least one sequence in which less than about 70% of the nucleobase sequence is capable of base-pairing with at least one single or double-stranded nucleic acid molecule during hybridization.
- non-complementary refers to nucleic acid sequence that lacks the ability to form at least one Watson-Crick base pair through specific hydrogen bonds.
- blunt end refers to the end of a dsDNA molecule having 5′ and 3′ ends, wherein the 5′ and 3′ ends terminate at the same nucleotide position. Thus, the blunt end comprises no 5′ or 3′ overhang.
- overhang refers to the end of a dsDNA molecule having 5′ and 3′ ends, wherein the 5′ and 3′ ends terminate at different nucleotide positions, leaving at least one nucleotide on the end of either the 5′ and 3′ end which has no nucleotide to hydrogen bond with.
- cap is a guanine nucleoside that is joined via its 5′-carbon to a triphosphate group that is, in turn, joined to the 5′-carbon of the most 5′-nucleotide of the primary mRNA transcript, and in most eukaryotes, the nitrogen at the 7 position of guanine in the cap nucleotide is methylated. Most eukaryotic cellular mRNA transcripts and most eukaryotic viral mRNA transcripts are blocked or “capped” at their 5′ terminus. In addition to mRNA, some other forms of eukaryotic RNA, such as but not limited to, small nuclear RNA (“snRNA”) and pre-micro RNA (i.e.
- pre-miRNA the primary transcripts that are processed to miRNA
- the cap is required to varying degrees for processing and maturation of an RNA transcripts in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of the mRNA to protein.
- sample means a material obtained or isolated from a fresh or preserved biological sample or synthetically-created source that contains nucleic acids of interest.
- a sample is the biological material that contains the variable immune region(s) for which data or information are sought.
- Samples can include at least one cell, fetal cell, cell culture, tissue specimen, blood, serum, plasma, saliva, urine, tear, vaginal secretion, sweat, lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascites fluid, fecal matter, body exudates, umbilical cord blood, chorionic villi, amniotic fluid, embryonic tissue, multicellular embryo, lysate, extract, solution, or reaction mixture suspected of containing immune nucleic acids of interest. Samples can also include non-human sources, such as non-human primates, rodents and other mammals, other animals, plants, fungi, bacteria, and viruses.
- substantially known refers to having sufficient sequence information in order to permit preparation of a nucleic acid molecule, including its amplification. This will typically be about 100%, although in some embodiments some portion of an adaptor sequence is random or degenerate. Thus, in specific embodiments, substantially known refers to about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.
- Nucleic acids of the present disclosure can comprise amplification, reverse transcription, and/or in vitro transcription primers and oligonucleotides, tumor derived nucleic acids and/or recombinant nucleic acids encoding one or more proteins or peptides, such as those which may induce an immune response that is effective against a tumor or tumor cell.
- generation of nucleic acids which may induce an immune response comprises reverse transcription of target mRNA to generate cDNA.
- Methods of reverse transcription are well known in the art (U.S. Pat. Nos. 5,962,271 and 6,518,019, incorporated herein by reference). Reverse transcription is performed by a RNA dependent DNA polymerase, also known as a reverse transcriptase.
- the reverse transcriptase binds to a primer hybridized to the 3′ end of a target mRNA, and synthesizes DNA complementary to the mRNA.
- the reverse transcriptase may add several non-templated nucleotides to the 3′ end of the newly synthesized cDNA, such as a short poly(C) tail.
- a second primer may be hybridized to the 3′ end of the nascent cDNA, and the reverse transcriptase may continue synthesizing DNA complementary to the primer, using its template switch activity to use the second primer as its template.
- the primers or oligonucleotides used in reverse transcription or amplification may allow for the addition of exogenous DNA or RNA sequence to the 5′ and 3′ ends of the target sequences during complementary strand synthesis.
- the primers or oligonucleotides used for priming the reverse transcription or amplification reactions may incorporate a variety of features including, but not limited to transcriptional promoters, ribosomal binding sites, or restriction endonuclease cleavage sites.
- a primer may include a transcriptional promoter sequence.
- cDNA prepared by reverse transcription can be amplified or transcribed to RNA.
- cDNAs may be amplified by PCR, to increase the concentration of the cDNA.
- the primers for reverse transcription may be used as the primers for PCR, or separate primers may be added.
- the PCR reaction may utilize the reverse transcriptase in order to amplify the cDNA, or a DNA dependent DNA polymerase may be added to increase the efficiency of DNA amplification.
- the primers for reverse transcription or DNA amplification may include transcription promoter sequences, such as the T7 promoter sequence.
- cDNA or amplified cDNA may be transcribed in an in vitro transcription process to generate large quantities of the initial target RNA using any commercially available RNA polymerase or in vitro transcription kit, such as AMPLISCRIBETM T7-FLASHTM (LUCIGEN® Cat. No. ASF3257).
- a nucleic acid composition contains both a tumor derived nucleic acid population and a recombinant nucleic acid component. This combination nucleic acid composition increases the prevalence of certain known tumor antigens or other nucleic acids encoding proteins or peptides that enhance the effectiveness of the methods and compositions described herein.
- a “tumor-derived” nucleic acid refers to a nucleic acid that has its origin in a tumor cell, and which includes RNA corresponding to a tumor antigen(s). Included is RNA that encodes all or a portion of a tumor antigen or a previously identified tumor antigen. Such nucleic acid can be “in vitro transcribed,” e.g., reverse transcribed to produce cDNA that can be amplified by PCR and subsequently be transcribed in vitro, with or without cloning the cDNA. Also included is RNA that is provided as a fractionated preparation of tumor cell.
- RNA preparation e.g., total RNA or total poly A RNA
- the preparation is fractionated with respect to a non-RNA component(s) of the cell in order to decrease the concentration of a non-RNA component, such as protein, lipid, and/or DNA and enrich the preparation for RNA.
- a non-RNA component such as protein, lipid, and/or DNA
- the preparation can be further fractionated with respect to the RNA (e.g., by subtractive hybridization) such that “tumor-specific” or “pathogen-specific” RNA is produced.
- tumor-specific RNA is meant an RNA sample that, relative to unfractionated tumor-derived RNA, has a high content of RNA that is preferentially present in a tumor cell compared with a non-tumor cell.
- tumor-specific RNA includes RNA that is present in a tumor cell, but not present in a non-tumor cell.
- an RNA sample that includes RNA that is present both in tumor and non-tumor cells, but is present at a higher level in tumor cells than in non-tumor cells.
- RNA that encodes a previously identified tumor antigen and which is produced in vitro e.g., from a plasmid or by PCR.
- tumor-specific RNA can be prepared by fractionating an RNA sample such that the percentage of RNA corresponding to a tumor antigen is increased, relative to unfractionated tumor-derived RNA.
- tumor-specific RNA can be prepared by fractionating tumor-derived RNA using conventional subtractive hybridization techniques against RNA from non-tumor cells.
- RNA sample i.e., the fractionated tumor preparation
- the RNA sample is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% RNA (wt/vol).
- RNA in 500 ⁇ l of Opti-MEM can be mixed with a cationic lipid at a concentration of 10 to 100 ⁇ g, and incubated at room temperature for 20 to 30 minutes.
- suitable lipids include LIPOFECTINTM (1:1 (w/w) DOTMA:DOPE), LIPOFECTAMINETM (3:1 (w/w) DOSPA:DOPE), DODAC:DOPE (1:1), CHOL:DOPE (1:1), DMEDA, CHOL, DDAB, DMEDA, DODAC, DOPE, DORI, DORIE, DOSPA, DOTAP, and DOTMA.
- RNA-lipid complex is then added to 1-3 ⁇ 10 6 cells, preferably 2 ⁇ 10 6 , antigen-presenting cells in a total volume of approximately 2 ml (e.g., in Opti-MEM), and incubated at 37° C. for 2 to 4 hours.
- the RNA can be introduced into the antigen presenting cells by employing conventional techniques, such as electroporation or calcium phosphate transfection with 1-5 ⁇ 10 6 cells and 5 to 50 ⁇ g of RNA. Typically, 5-20 ⁇ g of poly A RNA or 25-50 ⁇ g of total RNA are typically used.
- the preparation When the RNA is provided as a tumor preparation, the preparation typically is fractionated or otherwise treated to decrease the concentration of proteins, lipids, and/or DNA in the preparation, and enrich the preparation for RNA.
- art-known RNA purification methods can be used to at least partially purify the RNA from the tumor cell or pathogen. It is also acceptable to treat the RNA preparation with proteases or RNase-free DNases.
- dendritic cells for use according to the embodiments are isolated from a subject that is to be treated by a method of the embodiments.
- dendritic cells may be from a different subject, such as an HLA-matched donor.
- the dendritic cells are from a bank of dendritic cells having a defined HLA typing.
- primed dendritic cells for use according to the embodiments are homologously-loaded with antigen as detailed herein and in U.S. Pat. No. 8,728,806.
- dendritic cell precursors and immature dendritic cells can be isolated by collecting heparinized blood, by apheresis or leukapheresis, by preparation of buffy coats, rosetting, centrifugation, density gradient centrifugation (e.g., using Ficoll (such as FICOLL-PAQUE®), PERCOLL® (colloidal silica particles (15-30 mm diameter) coated with non-dialyzable polyvinylpyrrolidone (PVP)), sucrose, and the like), differential lysis of cells, filtration, and the like.
- Ficoll such as FICOLL-PAQUE®
- PERCOLL® colloidal silica particles (15-30 mm diameter) coated with non-dialyzable polyvinylpyrrolidone (PVP)
- a leukocyte population can be prepared, such as, for example, by collecting blood from a subject, defribrinating to remove the platelets and lysing the red blood cells.
- Dendritic cell precursors and immature dendritic cells can optionally be enriched for monocytic dendritic cell precursors by, for example, centrifugation through a PERCOLL® gradient.
- dendritic cell precursors can be selected using CD14 selection of G-CSF mobilized peripheral blood.
- Dendritic cell precursors and immature dendritic cells optionally can be prepared in a closed, aseptic system.
- the terms “closed, aseptic system” or “closed system” refer to a system in which exposure to non-sterilize, ambient, or circulating air or other non-sterile conditions is minimized or eliminated.
- Closed systems for isolating dendritic cell precursors and immature dendritic cells generally exclude density gradient centrifugation in open top tubes, open air transfer of cells, culture of cells in tissue culture plates or unsealed flasks, and the like.
- the closed system allows aseptic transfer of the dendritic cell precursors and immature dendritic cells from an initial collection vessel to a sealable tissue culture vessel without exposure to non-sterile air.
- monocytic dendritic cell precursors are isolated by adherence to a monocyte-binding substrate.
- a population of leukocytes e.g., isolated by leukapheresis
- a monocytic dendritic cell precursor adhering substrate e.g., isolated by leukapheresis
- the monocytic dendritic cell precursors in the leukocyte population preferentially adhere to the substrate.
- Other leukocytes including other potential dendritic cell precursors
- exhibit reduced binding affinity to the substrate thereby allowing the monocytic dendritic cell precursors to be preferentially enriched on the surface of the substrate.
- Suitable substrates include, for example, those having a large surface area to volume ratio. Such substrates can be, for example, a particulate or fibrous substrate. Suitable particulate substrates include, for example, glass particles, plastic particles, glass-coated plastic particles, glass-coated polystyrene particles, and other beads suitable for protein absorption. Suitable fibrous substrates include microcapillary tubes and microvillous membrane. The particulate or fibrous substrate usually allows the adhered monocytic dendritic cell precursors to be eluted without substantially reducing the viability of the adhered cells. A particulate or fibrous substrate can be substantially non-porous to facilitate elution of monocytic dendritic cell precursors or dendritic cells from the substrate. A “substantially non-porous” substrate is a substrate in which at least a majority of pores present in the substrate are smaller than the cells to minimize entrapping cells in the substrate.
- Adherence of the monocytic dendritic cell precursors to the substrate can optionally be enhanced by addition of binding media.
- Suitable binding media include monocytic dendritic cell precursor culture media (e.g., AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, and the like) supplemented, individually or in any combination, with for example, cytokines (e.g., Granulocyte/Macrophage Colony Stimulating Factor (GM-CSF), Interleukin 4 (IL-4), or Interleukin 13 (IL-13)), blood plasma, serum (e.g., human serum, such as autologous or allogenic sera), purified proteins, such as serum albumin, divalent cations (e.g., calcium and/or magnesium ions) and other molecules that aid in the specific adherence of monocytic dendritic cell precursors to the substrate, or that prevent adherence of non-monocytic dendritic cell precursors to the substrate.
- the non-adhering leukocytes are separated from the monocytic dendritic cell precursor/substrate complexes.
- Any suitable means can be used to separate the non-adhering cells from the complexes.
- the mixture of the non-adhering leukocytes and the complexes can be allowed to settle, and the non-adhering leukocytes and media decanted or drained.
- the mixture can be centrifuged, and the supernatant containing the non-adhering leukocytes decanted or drained from the pelleted complexes.
- Isolated dendritic cell precursors can be cultured ex vivo for differentiation, maturation and/or expansion.
- isolated immature dendritic cells, dendritic cell precursors, T cells, and other cells refers to cells that, by human hand, exists apart from their native environment, and are therefore not a product of nature. Isolated cells can exist in purified form, in semi-purified form, or in a non-native environment.
- ex vivo differentiation typically involves culturing dendritic cell precursors, or populations of cells having dendritic cell precursors, in the presence of one or more differentiation agents.
- Suitable differentiating agents can be, for example, cellular growth factors (e.g., cytokines such as (GM-CSF), Interleukin 4 (IL-4), Interleukin 13 (IL-13), and/or combinations thereof).
- cytokines such as (GM-CSF), Interleukin 4 (IL-4), Interleukin 13 (IL-13), and/or combinations thereof.
- the monocytic dendritic cells precursors are differentiated to form monocyte-derived immature dendritic cells.
- the dendritic cell precursors can be cultured and differentiated in suitable culture conditions.
- suitable tissue culture media include AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, and the like.
- the tissue culture media can be supplemented with serum, amino acids, vitamins, cytokines, such as GM-CSF and/or IL-4, divalent cations, and the like, to promote differentiation of the cells.
- the dendritic cell precursors can be cultured in the serum-free media.
- Such culture conditions can optionally exclude any animal-derived products.
- a typical cytokine combination in a typical dendritic cell culture medium is about 500 units/ml each of GM-CSF (50 ng/ml) and IL-4 (10 ng/ml).
- Dendritic cell precursors when differentiated to form immature dendritic cells, are phenotypically similar to skin Langerhans cells. Immature dendritic cells typically are CD14 ⁇ and CD11c+, express low levels of CD86 and CD83, and are able to capture soluble antigens via specialized endocytosis. The immature DC expressed very high levels of CD86. Also, the population was mixed in terms of CD14 and CD11C. Though the majority were CD11c+, there were distinct subpopulations that were CD11c ⁇ and CD 14+.
- the immature dendritic cells are matured to form mature dendritic cells.
- Mature DC lose the ability to take up antigen and display up-regulated expression of costimulatory cell surface molecules and various cytokines.
- mature DC express higher levels of MHC class I and II antigens than immature dendritic cells, and mature dendritic cells are generally identified as being CD80+, CD83+, CD86+, and CD14 ⁇ .
- Greater MHC expression leads to an increase in antigen density on the DC surface, while up regulation of costimulatory molecules CD80 and CD86 strengthens the T cell activation signal through the counterparts of the costimulatory molecules, such as CD28 on the T cells.
- Mature dendritic cells of the present invention can be prepared (i.e., matured) by contacting the immature dendritic cells with effective amounts or concentrations of a nucleic acid composition and a tumor antigen composition.
- Effective amounts of nucleic acid composition typically range from at most, at least, or about 0.01, 0.1, 1, 5, 10, to 10, 15, 20, 50, 100 ng or mg of nucleic acid per culture dish or per cell, including all values and ranges there between.
- Effective amounts of tumor antigen composition typically range from at most, at least, or about 0.01, 0.1, 1, 5, 10, to 10, 15, 20, 50, 100 ng or mg of protein per culture dish or per cell.
- tumor antigen/cell 0.001 ng of tumor antigen/cell to 1 ⁇ g of tumor antigen/million cells
- the tumor antigen composition can optionally be heat inactivated or treated (e.g., exposed to protease) prior to contact with dendritic cells. Maturing the immature dendritic cells with a nucleic acid composition and a tumor antigen composition primes the mature dendritic cells for a type 1 (Th-1) response.
- the immature DC are typically contacted with effective amounts of a nucleic acid composition and a tumor antigen composition for at most, at least, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 minutes, hours, or days.
- the immature dendritic cells can be cultured and matured in suitable maturation culture conditions. Suitable tissue culture media include AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, and the like.
- the tissue culture media can be supplemented with amino acids, vitamins, cytokines, such as GM-CSF and/or IL-4, divalent cations, and the like, to promote maturation of the cells.
- Dendritic cell precursors, immature dendritic cells, and mature dendritic cells, either primed or unprimed, with antigens can be cryopreserved for use at a later date. Methods for cryopreservation are well-known in the art. For example, U.S. Pat. No. 5,788,963, which is incorporated herein by reference in its entirety.
- a nucleic acid or nucleic acid primed dendritic cell is a dendritic cell that was incubated or transfected with RNA, e.g., RNA derived from a tumor or tumor cell.
- RNA can be transfected using conventional nucleic acid transfection methods, such as lipid-mediated transfection, electroporation, and calcium phosphate transfection.
- RNA can be introduced into a DC by incubating the DC with the RNA (or extract) for 1 to 24 hours (e.g., 2 hours) at 37° C.
- the nucleic acid-loaded antigen-presenting cells of the present disclosure can be used to stimulate CTL proliferation in vivo or ex vivo.
- the ability of the nucleic acid-loaded dendritic cells to stimulate a CTL response can be measured by assaying the ability of the effector cells to lyse target cells.
- the commonly-used europium release assay can be used.
- 5-10 ⁇ 106 target cells are labeled with europium diethylenetriamine pentaacetate for 20 minutes at 4° C.
- the genetic modification comprises introduction of an exogenous transgene in the cells, such as an inhibitory nucleic acid.
- the transgene may be a suicide gene, such as a gene encoding thymidine kinase, under the control of an inducible promoter.
- administered dendritic cells can be killed-off by induction of the promoter controlling expression of the suicide gene.
- the genetic modification comprises a genomic deletion or insertion in the cell population.
- one or more HLA gene may be disrupted to render the dendritic cells as an effective HLA match for a subject to be treated.
- the embodiments concern dendritic cells that have been genetically modified, such as to reduce the expression of CTLA-4.
- the genetic modification comprises introduction of an exogenous inhibitory nucleic acid specific to CTLA-4.
- the inhibitory nucleic acid is a RNA, such as a RNA that is expressed from a DNA vector in the dendritic cells.
- the inhibitory nucleic acid may be a siRNAs, dsRNA, miRNA or shRNA that is introduced in the dendritic cells. A detailed disclosure of such RNAs is provided above.
- the genetic modification comprises a genomic deletion or insertion in the cell population that reduces CTLA-4.
- the dendritic cells comprises a hemizygous or homozygous deletion within the CTLA-4 gene.
- one or both copies of the CTLA-4 gene of a dendritic cell can be completely or partially deleted, such that expression the CTLA-4 polypeptide is inhibited.
- modification the cells so that they do not express one or more CTLA-4 gene may comprise introducing into the cells an artificial nuclease that specifically targets the CTLA-4 locus.
- the artificial nuclease may be a zinc finger nuclease, TALEN, or CRISPR/Cas9.
- introducing into the cells an artificial nuclease may comprise introducing mRNA encoding the artificial nuclease into the cells.
- dendritic cell therapies which include dendritic cells transformed with nucleic acids generated by the embodiments, it may be desirable to combine these compositions with other agents effective in the treatment of the disease of interest.
- the treatment of cancer may be implemented with a primed dendritic cell composition of the present embodiments along with other anti-cancer agents.
- An “anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell.
- This process may involve contacting the cells with the anti-cancer peptide or nanoparticle complex and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the dendritic cell composition and the other includes the second agent(s).
- Treatment with a dendritic cell composition may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
- the other agent and dendritic cell composition are applied separately to the subject, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and the dendritic cell composition would still be able to exert an advantageously combined effect on the cell.
- dendritic cell therapy is “A” and the secondary agent, such as radiotherapy, chemotherapy or anti-inflammatory agent, is “B”:
- administration of dendritic cell therapy of the present embodiments to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of the vector. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described hyperproliferative cell therapy.
- Cancer therapies also include a variety of combination therapies.
- a dendritic cell composition of the embodiments is administered (or formulated) in conjunction with a chemotherapeutic agent.
- the chemotherapeutic agent is a protein kinase inhibitor such as a EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF inhibitors.
- Nonlimiting examples of protein kinase inhibitors include Afatinib, Axitinib, Bevacizumab, Bosutinib, Cetuximab, Crizotinib, Dasatinib, Erlotinib, Fostamatinib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib, Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, Miltefosine, Perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus,
- alkylating agents such as thiotepa and cyclosphosphamide
- alkyl sulfonates such as busulfan, improsulfan and piposulfan
- aziridines such as benzodopa, carboquone, meturedopa, and uredopa
- ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine
- acetogenins especially bullatacin and bullatacinone
- a camptothecin including the synthetic analogue topotecan
- bryostatin callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duo
- compositions provided herein may be used in combination with gefitinib.
- present embodiments may be practiced in combination with Gleevac (e.g., from about 400 to about 800 mg/day of Gleevac may be administered to a patient).
- one or more chemotherapeutic may be used in combination with the compositions provided herein.
- Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
- Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
- contacted and “exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
- both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
- the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the therapeutic composition.
- Viral vectors for the expression of a gene product are well known in the art, and include such eukaryotic expression systems as adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, lentiviruses, poxviruses including vaccinia viruses, and papiloma viruses, including SV40.
- the administration of expression constructs can be accomplished with lipid based vectors such as liposomes or DOTAP:cholesterol vesicles. All of these methods are well known in the art (see, e.g. Sambrook et al., 1989; Ausubel et al., 1998; Ausubel, 1996).
- Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatments provided herein, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
- Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed.
- Tumor resection refers to physical removal of at least part of a tumor.
- treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present embodiments may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
- a dendritic cell composition of the embodiments is administered to lymphoid tissue that drained the previous site for the tumor.
- kits for generating cDNA from mRNA in a sample and the in vitro transcription of said cDNA.
- a “kit” refers to a combination of physical elements.
- a kit may include, for example, one or more components including without limitation specific primers, enzymes, reaction buffers, an instruction sheet, and other elements useful to practice the technology described herein. These physical elements can be arranged in any way suitable for carrying out the invention.
- the kit may further comprise a DNA polymerase, including for example, ⁇ 29 polymerase, Bst polymerase, Taq polymerase, Vent polymerase, DNA polymerase I, the Klenow fragment of DNA polymerase I, 9° Nm Polymerase, T4 polymerase, T7 DNA polymerase, Pfu DNA polymerase, Q5® polymerase (New England Biolabs), or a RNA polymerase, and a reverse transcriptase.
- a DNA polymerase including for example, ⁇ 29 polymerase, Bst polymerase, Taq polymerase, Vent polymerase, DNA polymerase I, the Klenow fragment of DNA polymerase I, 9° Nm Polymerase, T4 polymerase, T7 DNA polymerase, Pfu DNA polymerase, Q5® polymerase (New England Biolabs), or a RNA polymerase, and a reverse transcriptase.
- a mutant form of T7 phage DNA polymerase that lacks 3′-5′
- the kit may further comprise a RNA polymerase for in vitro transcription, including for example, E. coli RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, T7 RNA polymerase, or mutants thereof.
- a RNA polymerase for in vitro transcription including for example, E. coli RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, T7 RNA polymerase, or mutants thereof.
- kits may be packaged either in aqueous media or in lyophilized form.
- the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted (e.g., aliquoted into the wells of a microtiter plate). Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a single vial.
- the kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
- kits will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented. It is contemplated that such reagents are embodiments of kits of the invention. Such kits, however, are not limited to the particular items identified above and may include any reagent used for the manipulation or characterization of the methylation of a gene.
- the container means of the kits will generally include at least one vial, test tube, flask, bottle, or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain additional containers into which the additional components may be separately placed. However, various combinations of components may be comprised in a container.
- the kits of the present invention also will typically include a means for packaging the component containers in close confinement for commercial sale. Such packaging may include injection or blow-molded plastic containers into which the desired component containers are retained.
- RNA stabilization solution RNA stabilization solution
- the cryovial containing the RNALATERTM and dissected tumor is then left at 4° C. overnight to prevent RNase activity.
- the cryovial containing the tissue in RNALATERTM solution was centrifuged at 10,000 RPM for 1 min at 4° C. After centrifugation, the supernatant was removed by pipetting, and the dissected pieces of tissues were weighed. 10% of the tissue was used for subsequent preparation of total RNA, while the remaining 90% was used for subsequent preparation of antigenic lysates.
- Preparation of antigenic lysates Dissected tumor tissue was added to a 15 mL conical tube and dilute to 100 mg/mL in sterile PBS. A homogenous suspension of tissue in PBS was then made using a handheld tissue homogenizer. The tube containing the homogenous suspension was then immersed in liquid nitrogen or a dry ice/ethanol bath to freeze the suspension. After freezing, the cell suspension was then incubated in a 55° C. water bath until it was completely thawed. The freeze thaw cycle was repeated two further times. Following the final thaw, to ensure sterility and complete cell death, the cell suspension was Gamma irradiated with >2500 Gy of radiation via a cesium irradiator. Lysates were then stored at ⁇ 20° C. until further use.
- Total RNA was isolated from tissue stabilized with RNALATERTM reagent using the RNEASY® mini kit (Qiagen cat #74104), which utilizes spin columns, or the TRIPURE® (Roche) method, which utilizes a guanidinium thiocyanate and phenol mixture, each according to the manufacturer's instructions.
- RNeasy Total RNA preparation 600 ⁇ L of Buffer RLT was added to 30 mg of tissue and the resulting mixture was homogenized using a handheld tissue homogenizer. 600 ⁇ L of 70% ethanol was added to the lysate, and mixed by pipetting. 700 ⁇ L of the sample was transferred to a RNeasy spin column, and placed in a 2 mL collection tube. The sample was centrifuged for 1 min at 8,000 ⁇ g. Following centrifugation, the flow-through was discarded. The remaining lysate was added to the spin column and was centrifuged again. Following centrifugation, the flow-through was discarded. 350 ⁇ L of buffer RW1 was added to the column and centrifuged.
- DNase I solution was prepared by adding 10 ⁇ L of DNase I stock solution to 70 ⁇ L of buffer RDD, followed by gentle mixing. 80 ⁇ L of DNase I solution was then added to the column and incubated at room temperature for 15 min. Following DNase I incubation, 350 ⁇ L of buffer RW1 was added to the column and the column was centrifuged for 1 min at 8000 ⁇ g. Following centrifugation the flow-through was discarded. The membrane was then washed by the addition of another 700 ⁇ L of buffer RW1, and another 1 min centrifugation at 8000 ⁇ g, followed by two 500 ⁇ l washes with buffer RPE, with two centrifugations at 8000 ⁇ g. The membrane was then dried by centrifugation for 2 min.
- RNA was eluted into a new column by direct addition of 50 ⁇ l of pre-warmed nuclease free water to the membrane in the column, 1 minute of incubation, and centrifugation at 8000 ⁇ g for 1 minute. In order to concentrate the RNA, the elution was performed a second time using the eluent. Total RNA was then quantified. Total RNA was stored at ⁇ 80° C. for long term storage.
- Guanidinium Thiocyanate/Phenol/Chloroform RNA Isolation 1 mL of guanidinium thiocyanate and phenol reagent (TRIpureTM reagent from Roche, Cat. NO. 11 667 157 001) was added for every 50 mg of tumor tissue and the resulting mixture was homogenized using a handheld tissue homogenizer. The homogenized mixture was then centrifuged at 13,000 RPM for 5 min at 4° C. to clear the homogenate. The supernatant from the homogenate was transferred to a new RNase free tube. To clear the supernatant, 400 uL of chloroform was added, and the mixture was mixed thoroughly by inversion.
- TripureTM reagent TripureTM reagent from Roche, Cat. NO. 11 667 157 001
- the supernatant/chloroform mixture was then incubated at room temperature for 5-10 min.
- the mixture was then centrifuged at 4500 RPM for 45 min or 12,000 ⁇ g for 15 min at 4° C. in order to clear it.
- the resulting mixture is present in 3 phases: the organic phase, the interface, and the aqueous phase.
- the aqueous phase is collected by pipetting and transferred directly to a pre-chilled tube.
- 0.5 mL of isopropanol were added for every 1 mL of starting guanidinium thiocyanate and phenol reagent.
- the isopropanol/supernatant mixture was mixed gently and incubated at room temperature for 10 min.
- RNA was pelleted by centrifugation at 4500 RPM for 45 min or 12000 ⁇ G for 15 min at 4° C.
- the pelleted RNA was washed with 1 mL of 70% ethanol and centrifuged again. The supernatant was removed by pipetting and the pellet was allowed to air dry for 5-10 minutes on ice. The pellet was then dissolved in 100 uL of RNase free water, and incubated at 55° C. for 5 min. The concentration of total RNA was then measured. Total RNA was stored at ⁇ 80° C. for long term storage.
- RNA isolated using the guanidinium thiocyanate method was DNase treated by the addition of 1 uL of amplification grade DNase (Invitrogen Cat. No. 18068-015) to 4 ug of total RNA in 10 ⁇ buffer diluted to 1 ⁇ . DNase reaction was allowed to proceed for 15 minutes, and stopped by the addition of 1 uL of EDTA.
- RNA/primer annealing mix was prepared in a sterile 0.2 mL PCR tube: 4 uL of a 10 uM solution of SEQ ID NO: 1 (VKWD oligo), 4 uL of a 10 uM solution of SEQ ID NO: 2 (CDS 64T+Oligo), 4 ug of sample RNA, and water to a total reaction volume of 19 uL.
- the solution was mixed and spun briefly in a microfuge to condense the solution to the bottom of the tube. The solution was then incubated at 72° C. for 2 min in a preheated thermocycler, and then cooled to 4° C. The sample was then stored on ice while the first strand synthesis master mix was prepared.
- First strand synthesis master mixes were prepared to account for each reaction plus one no template control, and prepared from reagents in the Superscript® First Strand Synthesis Kit (Invitrogen Cat. No. 11904-018).
- the master mix contains, per sample: 4 uL of 10 ⁇ First-Strand Buffer, 4 uL DTT (100 mM), 1 uL of RNase inhibitor (RNase OutTM) 4 uL MgCl 2 (25 mM), 4 uL dNTP Mix (10 mM each of dATP, dGTP, dCTP, and dTTP), and 4 uL of Superscript® II Reverse Transcriptase, for a total volume of 21 uL per sample.
- first strand synthesis mastermix 21 uL of first strand synthesis mastermix was added to each tube containing 19 ul of RNA/primer annealing mix. Each reaction, including the NTC, was incubated at 42° C. for 1 hour to synthesize the first strand of the cDNA. The first strand synthesis reaction was then collected by a brief centrifugation in a microcentrifuge and chilled on ice prior to second strand cDNA synthesis.
- the second strand synthesis master mix was prepared by adding 100 uL FastStartTM high fidelity reaction buffer with MgCl2, 40 uL of 10 ⁇ mol/L CDS 64T+Oligo (SEQ ID NO: 2), 20 uL of 20 ⁇ mol/L Powerswitch T7 primer (SEQ ID NO: 3), 20 uL dNTP mix (10 mM each, dATP, dGTP, dCTP, dTTP), 20 uL of FastStart high fidelity enzyme, and 780 uL of water, for a total of 980 uL per sample. 20 uL of the first strand synthesis reaction was added to the second strand synthesis master mix. The resulting mixture was then split into equal volumes of 100 uL each in 0.2 mL PCR tubes. Each of these reactions was then subjected to thermal cycling using the following program:
- Double stranded cDNA was then purified using the Salt-ethabol precipitation method.
- Sodium acetate (3M) was added to the sample at a 1:10 vol:vol ratio, followed by a 2:1 vol:vol ratio of chilled 100% ethanol.
- the sample was mixed gently and then incubated at ⁇ 20° C. for 1 hour.
- the mixture was then centrifuged at 13,000 RPM for 20 min to pellet the DNA.
- the supernatant was carefully pipetted off the pelleted DNA.
- the resulting pellet was then washed by adding 100 uL of chilled 70% ethanol and resuspended.
- the solution was then centrifuged for 10 min at 13,000 RPM, and the supernatant was pipetted off.
- the pellet was then air dried for 10 minutes before dissolving in 20 uL of nuclease free water.
- the concentration of the cDNA was measured by nanodrop.
- In vitro transcription was performed using the AMPLISCRIBETM T7-FLASHTM in vitro amplification kit (LUCIGENTM Cat. No. ASF3257). 20 uL reaction volumes were prepared by adding, in the following order: X uL of RNase free water (X being the amount of water required to achieve 20 uL reaction volume); 1 ug of cDNA template; 2 uL of AIVIPLISCRIBETM T7-FLASHTM 10 ⁇ reaction buffer; 1.8 uL each of 100 mM ATP, CTP, GTP and UTP; 2 uL DTT; 0.5 uL of RIBOGUARDTM RNase inhibitor; and 2 uL of AMPLISCRIBETM T7-FLASHTM Enzyme solution.
- the reaction was then incubated for 1 hr at 42° C. in a thermocycler. Following incubation, 1 uL of RNase-free DNase I was added, and the reaction was incubated at 37° C. for 15 min.
- the DNase treated in vitro transcription reaction was brought to 50 uL by addition of 29 uL of RNase free water.
- the in vitro transcription reaction was then purified by salt and ethanol precipitation and purification. 50 uL of 5 M ammonium acetate and 100 uL of ice cold 100% ethanol were added to the in vitro transcription reaction and mixed. The resulting mixture was then incubated for 1 hr at ⁇ 20° C. After incubation, the mixture was centrifuged at 10,000 ⁇ g for 15 min at 4° C.
- the supernatant was removed from the pellet by pipetting.
- the pellet was then washed with 100 uL of cold 70% ethanol and centrifuged again at 10,000 ⁇ g for 15 min at 4° C.
- the supernatant was removed by pipetting and the pellet was allowed to air dry before resuspending in 50 uL of RNase free water.
- a capping master mix was prepared by adding 10 uL of 10 ⁇ SCRIPTCAPTM Capping buffer, 10 uL of 10 mM GTP, 2.5 uL of 20 mM S-adenosyl methionine, 2.5 uL SCRIPTGUARDTM RNase inhibitor, and 4 uL SCRIPTCAPTM 2′-O-methyltransferase, per sample.
- the capping reaction was assembled by adding 4 uL of the SCRIPTCAPTM capping enzyme to 29 uL of the capping master mix, and adding that mix to the heat denatured RNA. The capping reaction was allowed to proceed at 37° C. for 30 min.
- Capped RNA was purified by salt and ethanol precipitation and purification as above, and resuspended in 50 uL of RNase free water. To assess the quality of the capped mRNA, 1 ug of capped mRNA was run on a 50 mL, 1% agarose non-denaturing gel. The capped mRNA was stored at ⁇ 80° C.
- Antigenic lysate preparation To generate MHC class II determinants, 1 mL of PBS per 100 mg of tissue was added to the isolated tissue fractions, and the tissues were disrupted using a POLYTRON® PT1200E tissue homogenizer (Kinematica, Inc., Bohemia, New York). The homogenized tissue fractions were then subjected to three repetitive freeze-thaw cycles, cycling the homogenized mixtures between liquid nitrogen and a 55 degree C. water bath, and stored at ⁇ 20° C.
- IFN ⁇ production by T cells co cultured with dendritic cells loaded with capped mRNA were treated for 5 hours with 10 ⁇ g/ml brefeldin A (eBioscience). Cells were stained for surface markers with anti-CD3, anti-CD8, and anti-CD25 antibodies and then fixed/permeabilized for intracellular staining of IFN- ⁇ using the Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer's instructions.
- Homologous antigenic loading leads to enhanced production of AIMp1 and retention of CTLA-4.
- Enhanced production of AIMp1 was demonstrated by Western blot.
- Enhanced retention of CTLA-4 was demonstrated by CTLA-4 western blot of cell culture supernatants (to determine release) and intracellular flow cytometry to determine retained CTLA-4 content. Histogram analysis of intracellular CTLA-4 is gated on the CD11c+CD80+CD83+CD86+ cell population.
- Electrophoreses and blotting proteins samples were separated by SDS-gel electrophoreses (Invitrogen) with subsequent transfer to a 0.45 ⁇ m nitrocellulose membrane (Bio-Rad) for antibody probing. All blocking and antibody staining steps were carried out in 5% BSA (RPI, Grainger) in 1 ⁇ TB ST buffer (0.05% Tween-20). Western blotting chemiluminescent signal was detected with SUPERSIGNAL® West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) using a CHEMIDOC® XRS digital imaging system supported by Image Lab software version 2.0.1 (Bio-Rad). Densitometry was performed using Image Lab software.
- RNA isolation from fresh tissue yields high quality RNA—To determine whether fresh or frozen tissue yielded higher quality RNA for downstream processing, total RNA was isolated from 30 mg of 3 different frozen tumor tissues or a single fresh tumor. As can be seen in FIG. 1 , the RNA isolated from the three frozen tissues exhibits significant degradation, visible as a large smear in the lanes. Lane 4 has two clearly defined bands, indicating that the integrity of the RNA is high.
- Total RNA was isolated from 30 mg of tumor tissue using either the RNEASY® mini kit (Qiagen) or using the TRIPURE® RNA isolation reagent (Roche), which is a guanidinium thiocyanate/phenol/chloroform based method. Both methods yielded high quality RNA as visible in FIG. 2 , though the TRIPURE® method yielded 4.4-fold more RNA than the RNEASY® kit from the same input amount.
- FIG. 3 depicts a schematic, including the primers, for the method used for reverse transcription of mRNA isolated from tumor cells, and subsequent in vitro transcription of the cDNA according to the present methods (see Example 1).
- Two different reverse transcription and in vitro transcription methods were compared ( FIG. 4 ).
- Lanes 1 and 2 depict either uncapped RNA or capped RNA as prepared by the Slagter-Jager method (Slagter-Jager et al., 2013).
- Lanes 3 and 4 show either uncapped RNA or RNA prepared by the methods described herein. Previous methods show an enrichment of very small transcripts, while the methods described herein yielded a smear of RNA at larger molecular weights.
- FIG. 5 depicts an agarose gel of the capped RNA generated by each method. Lane 1 shows RNA post-transcriptionally capped, while Lane 2 shows RNA which was co-transcriptionally capped. Clearly, the transcript smear is much more evident in lane 2, in which the RNA was produced by the present methods and co-transcriptionally capped. Tables 1 and 2 show the concentrations, volume, and yields of products generated by the Slagter-Jager method (Slagter-Jager et al., 2013). Konduri/Decker Hybrid preparation is according to the present methods.
- RNA prepared from TRIPURE TM isolated total RNA.
- TRIPURE ® method Concentration Volume Yield RNA yield 2 ng/ul 40 ul 80 ug Argos method ds-cDNA 20 ng/ul 120 ul 2.4 ug uncapped mRNA 2.5 ug/ul 100 ul 250 ug capped mRNA 2.4 ug/ul 30 ul 72 ug co-transcribed capped 2.7 ng/ul 30 ul 81 ug mRNA Konduri/Decker Hybrid ds-cDNA 21 ng/ul 120 ul 2.5 ug uncapped mRNA 2.6 ug/ul 100 ul 260 ug capped mRNA 2.0 ug/ul 40 ul 80 ug co-transcribed capped 3.3 ug/ul 60 ul 198 ug mRNA
- IFN ⁇ production is enhanced by co-culture with loaded DCs—Dendritic cells were homologously loaded with antigenic lysate and mRNA prepared by the methods described herein, or heterologously loaded with prostate mRNA and pancreatic tumor lysate (mismatched).
- FIG. 6 shows relative IFN ⁇ levels produced by T cells cocultured with the indicated dendritic cells. Dendritic cells loaded with capped RNA and antigenic lysate clearly stimulate IFN ⁇ production, and does so better than dendritic cells loaded with uncapped RNA, or immature or unloaded dendritic cells.
- FIG. 7 shows the results of flow cytometric analysis of CD8 + CD25 + cells sorted for IFN ⁇ levels, after co-culture with dendritic cells homologously loaded with either uncapped or capped mRNA produced by the methods described herein and lysate, or loaded with prostate mRNA and pancreatic tumor lysate (mismatched). Homologously loading lysate and mRNA produced by the methods described herein significantly enhances the levels of antigen specific CD8 + CD25 + T cells.
- FIG. 8 shows a western blot for AIMp1 and CTLA-4 in the lysates with (3-actin provided as a control.
- Homologous antigenic loading with capped mRNA made by the methods provided herein have increased amounts of AIMp1 and a high amount of intracellular CTLA-4, as can be seen in lane 5.
- flow cytometry was performed on dendritic cells either unloaded, loaded with just mRNA, just lysate, both, or mismatched antigens.
- CTLA4 retention is significantly enhanced in CD11c+CD80+CD83+CD86+ cells loaded with both the mRNA and homologous antigenic lysate. Further confirming enhanced CTLA4 retention by cells homologously loaded with antigenic lysate and mRNA generated by the methods provided herein, dendritic cell lysates and supernatant were probed for CLTA4 by western blot ( FIG. 10 ). Loading with antigenic lysate and mRNA prepared by either method decreased the amount of CTLA4 present in the supernatant, indicating enhanced retention.
- Homologous antigenic loading increases IL-12 transcripts in dendritic cells—Dendritic cells were evaluated for IL-12a and IL-12b production by RT-qPCR.
- antigenic loading with both lysate and mRNA generated by the methods provided herein increases both IL-12 transcripts better than loading with just lysate, mRNA, or heterologous loading, as well as increasing IL-12 transcript production better than loading with mRNA prepared by previous methods (Column-IVT).
- results shown in FIG. 13 A show monocyte-derived human DC that were loaded with a) native poly A tumor mRNA and homologous/heterologous lysate or 2) capped IVT-amplified mRNA and homologous/heterologous lysate.
- DC were then cocultured with T cells and the percent of activated CD8+CD25+Ifng+ cells were analyzed by flow. T cells cocultured with unloaded DC or DC loaded with uncapped mRNA served as controls.
- FIG. 13 B shows results with DC loaded with various combinations of poly A mRNA or IVT-amplified mRNA and homologous or heterologous cell lysates were matured for 48 hours, and intracellular CTLA4 levels were analyzed by flow cytometry. Singly loaded and unloaded DCs serve as controls. Upregulation of intracellular CTLA-4 is indicative of increased retention and therefore reduced secretion.
- Tissue Total Template ds- Template Capped Tumor Type Experiment in mg RNA Total RNA cDNA ds-cDNA IVT Primary PDAC 1 15 mg 1.4 ug 1.4 ug 41.0 ug 1 ug 45 ug Primary PDAC 2 10 mg 3.6 ug 3.0 ug 32.6 ug 1 ug 94 ug Primary 3 20 mg 1.4 ug 1.3 ug 22.6 ug 2 ⁇ 1 ug 300 ug Prostate Primary PDAC 4 10 mg 3.0 ug 3.0 ug 45.0 ug 1 ug 70 ug Dermal 5 80 mg 66.7 ug 5 ug 22.0 ug 2 ⁇ 2 ug 360 ug metastasis from HR-positive breast carcinoma Pleural cavity 6 60 mg 30.0 ug 4 ug 60.0 ug 2 ⁇ 5 ug 270 ug metastsis from poorly
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Plant Pathology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Bioinformatics & Computational Biology (AREA)
- Oncology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Provided herein are compositions and methods for the synthesis, amplification, and in vitro transcription of full-length cDNA, or cDNA fragments. Methods are provided for reverse transcription of RNA and amplification for in vitro transcription. Further provided are method for loading of dendritic cells with the RNA and homologous lysate for immune stimulation.
Description
- This application is a divisional application of U.S. patent application Ser. No. 16/772,682, filed Jun. 12, 2020, which is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/US2018/066051, filed Dec. 17, 2018, which claims the benefit of U.S. Provisional Patent Application No. 62/599,472, filed Dec. 15, 2017, the entirety of each of which is incorporated herein by reference.
- The sequence listing that is contained in the file named “BACMP0006USD1 Sequence Listing.xml”, which is 5 KB (as measured in Microsoft Windows®) and was created on May 26, 2023, is filed herewith by electronic submission and is incorporated by reference herein.
- The present invention relates generally to the field of molecular biology. More particularly, it concerns reverse transcription of mRNA and amplification of those products.
- An important tool in molecular biology is the ability replicate DNA and RNA. DNA can be replicated and amplified in vitro by the polymerase chain reaction, in which primers are hybridized to opposite ends, and opposite strands of a target nucleic acids, and the primers are extended by a DNA dependent DNA polymerase. RNA, which generally exists transiently in vivo, can be reverse transcribed by a RNA dependent DNA polymerase (reverse transcriptase) in order to generate a DNA copy, which is far more stable than RNA. The DNA which is generated from reverse transcription, referred to as complementary DNA or cDNA, can then be amplified, sequenced, transcribed back into RNA, incorporated into a vector for cloning, or any combination thereof.
- A variety of methods and enzymes currently exist to reverse transcribe RNA into DNA. Frequently, when designing primers for cDNA synthesis from mRNA templates, researchers take advantage of the poly(A) tail which exists on the 3′ end of mRNA. This allows researchers to use primers with a poly(T) sequence at the 3′ end of the primer, and makes capturing 3′ sequence information from mRNA very simple. The second primer, corresponding to the 5′ end of the target sequence, comprises either random or targeted sequences, as in standard PCR. A third option for the 5′ primer is to take advantage of the inherent template switching activity of some reverse transcriptase enzymes and hybridize the second primer to a polynucleotide overhang generated by the reverse transcriptase, and continue to synthesize the strand complementary to the second primer (see, for example, U.S. Pat. No. 5,962,271). The complementary DNA strand can then be copied using a standard PCR reaction to amplify the sequence. The primers used in cDNA synthesis and amplification may include a promoter so that the cDNA can be transcribed using a process of in vitro transcription to generate large quantities of target mRNA. RNA generated by in vitro transcription can then be used to induce antigen presentation on dendritic cells. The methods for reverse transcription and in vitro transcription, however, suffer from high bias during the reverse transcription process, and inefficient in vitro transcription, demonstrating a need for improved methods and compositions to enhance these processes.
- In a first embodiment, there is provided an oligonucleotide comprising the sequence of SEQ ID NO: 1. In some aspects, the oligonucleotide is further defined as a primer. In certain aspects, the oligonucleotide comprises less than 40 nucleotides. In a specific aspect, the oligonucleotide consists of SEQ ID NO: 1.
- In a further embodiment, there is provided a composition comprising a first primer and a second primer, wherein the first primer comprises a nucleic acid sequence of SEQ ID NO: 1, and wherein the second primer comprises a nucleic acid sequence of SEQ ID NO: 2. In several aspects, the composition is further defined as a nucleic acid amplification reaction mixture. In other aspects, the composition further comprises a third primer, wherein the third primer comprises a nucleic acid sequence of SEQ ID NO: 3.
- In yet a further embodiment, there is provided a method of synthesizing a first cDNA strand from a template mRNA comprising the steps of: a) hybridizing a first primer to the template mRNA, wherein the first primer comprises a nucleic acid sequence of SEQ ID NO: 2; b) extending the first primer with a reverse transcriptase that has terminal transferase and template switching activity to generate a partial first cDNA strand with an oligo(C) overhang; c) hybridizing a second primer to the oligo(C) overhang of the partial first cDNA strand, wherein the second primer comprises a nucleic acid sequence of SEQ ID NO: 1; and d) extending the partial first cDNA strand from the oligo(C) overhang using the second primer as the template, thereby generating a first cDNA strand. In some aspects, the method further comprises steps e) separating the template mRNA and first cDNA strand; f) hybridizing a third primer to the first cDNA strand; and g) extending the third primer to generate a second cDNA strand, thereby generating a double stranded cDNA. In certain aspects, the second primer comprises a nucleic acid sequence of SEQ ID NO: 1. In several aspects, the third primer comprises a nucleic acid sequence of SEQ ID NO: 3. In further aspects, the template mRNA is obtained from a sample. In some specific aspects, the sample is a tumor sample. In certain specific aspects, the tumor sample is stored in an RNA stabilization solution after removal from a subject. In still further aspects, the RNA stabilization solution is RNALATER®.
- In additional aspects, the method further comprises synthesizing RNA from the double stranded cDNA. In some aspects, the synthesizing RNA from the double stranded cDNA comprises in vitro transcription. In more specific aspects, the in vitro transcription comprises adding a fourth primer, a fifth primer, and an RNA polymerase, and synthesizing RNA from the double stranded cDNA with the RNA polymerase. In a particular aspect, the fourth primer comprises a nucleic acid sequence of SEQ ID NO: 1. In a further aspect, the fourth primer comprises a nucleic acid sequence of SEQ ID NO: 3. In another aspect, the method further comprises capping the RNA.
- In certain aspects, the method further comprises amplifying the double stranded cDNA. In some aspects, the second primer comprises the nucleic acid sequence of SEQ ID NO: 1. In other aspects, the third primer comprises the nucleic acid sequence of SEQ ID NO: 3. In particular aspects, amplifying the double stranded cDNA comprises adding a DNA dependent DNA polymerase, a fourth primer, and a fifth primer and amplifying the cDNA by polymerase chain reaction. In some specific aspects, the fourth primer comprises the nucleic acid sequence of SEQ ID NO: 1. In another aspect, the fifth primer comprises the nucleic acid sequence of SEQ ID NO: 3.
- In further aspects, the method additionally comprises in vitro transcribing the amplified cDNA to generate sense-strand amplified mRNA. In several aspects, the second primer comprises the nucleic acid sequence of SEQ ID NO: 1. In certain aspects, the third primer comprises the nucleic acid sequence of SEQ ID NO: 3. In some additional aspects, the in vitro transcribing of the amplified cDNA comprises adding primers having the sequence of SEQ ID NO: 1 and SEQ ID NO: 3 to the amplified cDNA, and hybridizing said primers to the amplified cDNA. In a particular aspect, the in vitro transcribing of the amplified cDNA comprises adding an RNA polymerase to the amplified cDNA, and extending the hybridized primers to generate RNA. In another aspect, the method further comprises capping the amplified RNA.
- A further embodiment of the invention provides, a method for transducing a dendritic cell population comprising contacting the dendritic cell population with a nucleic acid encoding one or more antigens, wherein the nucleic acid comprises RNA generated by any of the methods and aspects described above. In some aspects, the method further comprises contacting the dendritic cell population with a tumor cell lysate. In another aspect, the tumor cell lysate comprises a tumor antigen with an epitope having a sequence that overlaps a minimum of 5 amino acids with the sequence of the nucleic acid encoding one or more antigens.
- In yet a further embodiment, there is provided a method for providing an immune response in a subject having a diseased cell population comprising: a) obtaining a primed dendritic cell population produced by the method according to claim 36 or 37; and b) administering an effective amount of the primed dendritic cell population to the subject. In certain aspects, the antigen-primed dendritic cell has been primed with an antigen associated with a cancer, an autoimmune disease or an infectious disease. In several aspects, the antigen-primed dendritic cell has been primed with at least one tumor antigen. In additional aspects, the method further comprises administering an immune checkpoint inhibitor. In another aspect, the immune checkpoint inhibitor is a CTLA-4 antagonist. In certain aspects, the immune checkpoint inhibitor is ipilimumab, pembrolizumab or nivolumab.
- Still yet a further embodiment of the invention provides a kit comprising an oligonucleotide primer of
claim 1, an oligonucleotide primer of SEQ ID NO:2 and/or an oligonucleotide primer of SEQ ID NO:3. In some aspects, the kit may additionally comprise nucleic acids isolated from cancer cells, a DNA polymerase, and/or an RNA stabilization solution. In a particular aspect, the RNA stabilization solution is RNALATER®. In another aspect, the kit is further defined as a cDNA synthesis kit. In additional aspects, the kit further comprises dNTPs, MgCl2, reverse transcriptase, and/or RNase inhibitor. - As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
- As used herein in the specification and claims, “a” or “an” may mean one or more. As used herein in the specification and claims, when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein, in the specification and claim, “another” or “a further” may mean at least a second or more.
- As used herein in the specification and claims, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
- Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating certain embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
- The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
-
FIG. 1 : Freshly harvested tumor samples yield better quality RNA than frozen archived samples. Pictured, RNA isolated from either frozen or fresh tumor tissue was run on a non-denaturing 1% agarose gel. From left to right: L: ladder; 1: frozen tumor sample RNA; 2 frozen tumor sample RNA; 3: Frozen tumor sample RNA; 4: Fresh tumor sample RNA. -
FIG. 2 : Comparison of two different methods of RNA isolation. Pictured, RNA samples were run on a non-denaturing 1% agarose gel. Lane 1: RNA isolated from 30 mg of tumor tissue by column purification. Lane 2: RNA isolated from 30 mg of tumor tissue by guanidinium thiocyanate/phenol/chloroform purification. -
FIG. 3 : Schematic showing the process of reverse transcription used in the present invention, indicating the primers used. VKWD primer has the nucleotide sequence of SEQ ID NO: 1. CDS64T+oligo primer has the nucleotide sequence of SEQ ID NO: 2. T7 power switch primer has the nucleotide sequence of SEQ ID NO: 3. -
FIG. 4 : Comparison of methods of reverse transcription followed by in vitro transcription. Pictured, 4 different protocols were used to generate in vitro transcribed RNA from identical starting material.Lane 1, uncapped transcripts generated by the Slagter-Jager method (Slagter-Jager et al., 2013).Lane 2, capped transcripts generated by the Slagter-Jager method (Slagter-Jager et al., 2013).Lane 3, uncapped RNA generated by the methods described herein.Lane 4, capped RNA generated by the methods described herein. -
FIG. 5 : Comparison of mRNA generated and capped by two different methods. 5 ug of in vitro transcribed mRNA was run on a 1% agarose, non-denaturing gel.Lane 1, mRNA generated by previous method (Slagter-Jager et al., 2013) and capped post-transcription.Lane 2, mRNA generated by the present methods and capped co-transcriptionally. -
FIG. 6 : IFNγ production is significantly enhanced by T cells cocultured with dendritic cells loaded with capped mRNA and lysates. Pictured, relative amounts of IFN-γ secretion as measured by ELISA for cells loaded with uncapped or capped mRNA generated by the previous method (Argos (Slagter-Jager)) or the present methods (VKWD), and lysates, or with mismatched mRNA and lysates. -
FIG. 7 : Antigen specific CD8+CD25+ T cell production is enhanced when co-cultured with dendritic cells homologously loaded with capped mRNA and lysate. Pictured, flow cytometric analysis of T cells for antigen specific activation markers following re-stimulation with dendritic cells loaded with the mRNA shown below the data. -
FIG. 8 : Western blot analysis of dendritic cell lysates. Lane 1: unloaded dendritic cells. Lane 2: dendritic cells loaded with uncapped mRNA generated using the Slagter-Jager method. Lane 3: dendritic cells loaded with capped mRNA generated using the Slagter-Jager method. Lane 4: dendritic cells loaded with uncapped mRNA generated using the methods described herein. Lane 5: dendritic cells loaded with capped mRNA generated using the methods described herein. Lane 6: dendritic cells loaded with mismatched mRNA and lysate. Lane 7: dendritic cells loaded with mRNA generated using the methods described herein and capped co-transcriptionally. -
FIG. 9 : Homologous antigenic loading with in vitro transcribed and amplified mRNA leads to enhanced retention of CTLA4. Pictured are flow cytometric analyses of dendritic cells either unloaded, or loaded with the indicated: mRNA, Lysate, 2× (mRNA and Lysate), and MM (mismatched mRNA and lysate). -
FIG. 10 : Enhanced retention and reduced release of CTLA4 from the homologously loaded dendritic cells. Pictured are western blots detecting CTLA4 in the supernatant or the lysate of dendritic cell cultures. Dendritic cells were either unloaded (UL) or loaded with mRNA, lysate, both, or mismatched mRNA and lysate. mRNA was isolated by either a column based method or guanidinium thiocyanate/phenol/chloroform method. -
FIG. 11 : IL-12 transcript levels are elevated in dendritic cells homologously loaded with lysates and in vitro transcribed mRNA. Shown are IL-12a and IL-12b levels as detected by ELISA for dendritic cells loaded with the indicated RNA and/or lysate. The left axis indicates arbitrary transcriptional units normalized to unloaded control DC and set at 1.0. -
FIG. 12 : Flow chart describing the preparation of a dendritic cell vaccine. -
FIG. 13A-B : Amplified mRNA generates TH1 immune responses in vitro with the same efficiency as native poly-A mRNA when loaded into DC with homologous lysate. (A) Monocyte-derived human DC were loaded with a) native poly A tumor mRNA and homologous/heterologous lysate or 2) capped IVT-amplified mRNA and homologous/heterologous lysate. DC were then cocultured with T cells and the percent of activated CD8+CD25+Ifng+ cells were analyzed by flow. T cells cocultured with unloaded DC or DC loaded with uncapped mRNA served as controls. (B) DC loaded with various combinations of poly A mRNA or IVT-amplified mRNA and homologous or heterologous cell lysates were matured for 48 hours, and intracellular CTLA4 levels were analyzed by flow cytometry. Singly loaded and unloaded DCs serve as controls. Upregulation of intracellular CTLA-4 is indicative of increased retention and therefore reduced secretion. UL-unloaded DC, uncapped mRNA-DC loaded with uncapped IVT mRNA, mRNA-DC loaded with poly A mRNA, lysate-DC loaded with lysate, 2× w/uncapped-DC loaded with uncapped IVT mRNA and homologous lysate, 2× capped-DC loaded with capped IVT mRNA and homologous lysate, MM uncapped-DC loaded with uncapped IVT mRNA and heterologous lysate, MM capped-DC loaded with capped IVT mRNA and heterologous lysate. - Reverse transcription is a process in which a RNA dependent DNA polymerase synthesizes DNA using RNA as a template, and has become increasingly important as a tool in molecular biology for understanding gene expression and sequencing cellular RNA. Using a process of in vitro transcription and amplification following reverse transcription allows for the production of large quantities of RNA from a template RNA. However, there is a need for methods to decrease the bias and increase the efficiency of the reverse transcription process.
- Accordingly, in certain embodiments, the present disclosure provides methods for decreasing the bias of the reverse transcription process, and provides methods for increasing amplification efficiency of the reverse transcribed molecules. In particular, the present studies found the nucleotide composition and sequence of the primers to be important to the reverse transcription process, as well as the incorporation of a promoter sequence in the primers, for efficient in vitro transcription.
- Specifically, some embodiments provide compositions and methods for the synthesis, amplification, and in vitro transcription of full-length cDNA, or cDNA fragments. The method may comprise contacting RNA with a primer which can anneal to the poly(A) tail of mRNA, a suitable enzyme which possesses reverse transcriptase activity, and a template switching oligonucleotide under conditions sufficient to permit the template-dependent extension of the primer a cDNA complementary to the mRNA template. The template switching oligonucleotide can hybridize to a reverse transcriptase generated overhang at the 3′ end of the newly generated cDNA, and allow for the reverse transcriptase to continue to synthesize the complement of the template switching oligonucleotide on the 3′ end of the cDNA. Subsequent amplification can introduce a promoter sequence for in vitro transcription. In vitro transcribed RNA can then be loaded into dendritic cells with homologous lysate for immune stimulation. Thus, further embodiments provide methods for loading of dendritic cells and use of the dendritic cell vaccine for the treatment of diseases, such as cancer.
- “Amplification,” as used herein, refers to an in vitro process for increasing the number of copies of a nucleotide sequence or sequences. Nucleic acid amplification results in the incorporation of nucleotides into DNA or RNA. As used herein, one amplification reaction may consist of many rounds of DNA replication. For example, one PCR reaction may consist of 30-100 “cycles” of denaturation and replication.
- “Polymerase chain reaction,” or “PCR,” means a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors well-known to those of ordinary skill in the art, e.g., exemplified by the references: McPherson et al., editors, PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively).
- “Primer” means an oligonucleotide, either natural or synthetic that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3′ end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Usually primers are extended by a DNA polymerase. Primers are generally of a length compatible with its use in synthesis of primer extension products, and are usually are in the range of between 8 to 100 nucleotides in length, such as 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to 40, and so on, more typically in the range of between 18-40, 20-35, 21-30 nucleotides long, and any length between the stated ranges. Typical primers can be in the range of between 10-50 nucleotides long, such as 15-45, 18-40, 20-30, 21-25 and so on, and any length between the stated ranges. In some embodiments, the primers are usually not more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.
- “Incorporating,” as used herein, means becoming part of a nucleic acid polymer.
- The term “in the absence of exogenous manipulation” as used herein refers to there being modification of a nucleic acid molecule without changing the solution in which the nucleic acid molecule is being modified. In specific embodiments, it occurs in the absence of the hand of man or in the absence of a machine that changes solution conditions, which may also be referred to as buffer conditions. However, changes in temperature may occur during the modification.
- A “nucleoside” is a base-sugar combination, i.e., a nucleotide lacking a phosphate. It is recognized in the art that there is a certain inter-changeability in usage of the terms nucleoside and nucleotide. For example, the nucleotide deoxyuridine triphosphate, dUTP, is a deoxyribonucleoside triphosphate. After incorporation into DNA, it serves as a DNA monomer, formally being deoxyuridylate, i.e., dUMP or deoxyuridine monophosphate. One may say that one incorporates dUTP into DNA even though there is no dUTP moiety in the resultant DNA. Similarly, one may say that one incorporates deoxyuridine into DNA even though that is only a part of the substrate molecule.
- “Nucleotide,” as used herein, is a term of art that refers to a base-sugar-phosphate combination. Nucleotides are the monomeric units of nucleic acid polymers, i.e., of DNA and RNA. The term includes ribonucleotide triphosphates, such as rATP, rCTP, rGTP, or rUTP, and deoxyribonucleotide triphosphates, such as dATP, dCTP, dUTP, dGTP, or dTTP.
- The term “nucleic acid” or “polynucleotide” will generally refer to at least one molecule or strand of DNA, RNA, DNA-RNA chimera or a derivative or analog thereof, comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine “A,” guanine “G,” thymine “T” and cytosine “C”) or RNA (e.g., A, G, uracil “U” and C). The term “nucleic acid” encompasses the terms “oligonucleotide” and “polynucleotide.” “Oligonucleotide,” as used herein, refers collectively and interchangeably to two terms of art, “oligonucleotide” and “polynucleotide.” Note that although oligonucleotide and polynucleotide are distinct terms of art, there is no exact dividing line between them and they are used interchangeably herein. The term “adaptor” may also be used interchangeably with the terms “oligonucleotide” and “polynucleotide.” In addition, the term “adaptor” can indicate a linear adaptor (either single stranded or double stranded) or a stem-loop adaptor. These definitions generally refer to at least one single-stranded molecule, but in specific embodiments will also encompass at least one additional strand that is partially, substantially, or fully complementary to at least one single-stranded molecule. Thus, a nucleic acid may encompass at least one double-stranded molecule or at least one triple-stranded molecule that comprises one or more complementary strand(s) or “complement(s)” of a particular sequence comprising a strand of the molecule. As used herein, a single stranded nucleic acid may be denoted by the prefix “ss,” and a double-stranded nucleic acid by the prefix “ds.”
- “Oligonucleotide,” as used herein, refers collectively and interchangeably to two terms of art, “oligonucleotide” and “polynucleotide.” Note that although oligonucleotide and polynucleotide are distinct terms of art, there is no exact dividing line between them and they are used interchangeably herein. The term “adaptor” may also be used interchangeably with the terms “oligonucleotide” and “polynucleotide.”
- A “nucleic acid molecule” or “nucleic acid target molecule” refers to any single-stranded or double-stranded nucleic acid molecule including standard canonical bases, hypermodified bases, non-natural bases, or any combination of the bases thereof. For example and without limitation, the nucleic acid molecule contains the four canonical DNA bases—adenine, cytosine, guanine, and thymine, and/or the four canonical RNA bases—adenine, cytosine, guanine, and uracil. Uracil can be substituted for thymine when the nucleoside contains a 2′-deoxyribose group. The nucleic acid molecule can be transformed from RNA into DNA and from DNA into RNA. For example, and without limitation, mRNA can be created into complementary DNA (cDNA) using reverse transcriptase and DNA can be created into RNA using RNA polymerase. A nucleic acid molecule can be of biological or synthetic origin. Examples of nucleic acid molecules include genomic DNA, cDNA, RNA, a DNA/RNA hybrid, amplified DNA, a pre-existing nucleic acid library, etc. A nucleic acid may be obtained from a human sample, such as blood, serum, plasma, cerebrospinal fluid, cheek scrapings, biopsy, semen, urine, feces, saliva, sweat, etc. A nucleic acid molecule may be subjected to various treatments, such as repair treatments and fragmenting treatments. Fragmenting treatments include mechanical, sonic, and hydrodynamic shearing. Repair treatments include nick repair via extension and/or ligation, polishing to create blunt ends, removal of damaged bases, such as deaminated, derivatized, abasic, or crosslinked nucleotides, etc. A nucleic acid molecule of interest may also be subjected to chemical modification (e.g., bisulfite conversion, methylation/demethylation), extension, amplification (e.g., PCR, isothermal, etc.), etc.
- “Analogous” forms of purines and pyrimidines are well known in the art, and include, but are not limited to aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid, and 2,6-diaminopurine. The nucleic acid molecule can also contain one or more hypermodified bases, for example and without limitation, 5-hydroxymethyluracil, 5-hydroxyuracil, α-putrescinylthymine, 5-hydroxymethylcytosine, 5-hydroxycytosine, 5-methylcytosine, N4-methyl cytosine, 2-aminoadenine, α-carbamoylmethyladenine, N6-methyladenine, inosine, xanthine, hypoxanthine, 2,6-diaminpurine, and N7-methylguanine. The nucleic acid molecule can also contain one or more non-natural bases, for example and without limitation, 7-deaza-7-hydroxymethyladenine, 7-deaza-7-hydroxymethylguanine, isocytosine (isoC), 5-methylisocytosine, and isoguanine (isoG). The nucleic acid molecule containing only canonical, hypermodified, non-natural bases, or any combinations the bases thereof, can also contain, for example and without limitation where each linkage between nucleotide residues can consist of a standard phosphodiester linkage, and in addition, may contain one or more modified linkages, for example and without limitation, substitution of the non-bridging oxygen atom with a nitrogen atom (i.e., a phosphoramidate linkage, a sulfur atom (i.e., a phosphorothioate linkage), or an alkyl or aryl group (i.e., alkyl or aryl phosphonates), substitution of the bridging oxygen atom with a sulfur atom (i.e., phosphorothiolate), substitution of the phosphodiester bond with a peptide bond (i.e., peptide nucleic acid or PNA), or formation of one or more additional covalent bonds (i.e., locked nucleic acid or LNA), which has an additional bond between the 2′-oxygen and the 4′-carbon of the ribose sugar.
- Nucleic acid(s) that are “complementary” or “complement(s)” are those that are capable of base-pairing according to the standard Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules. As used herein, the term “complementary” or “complement(s)” may refer to nucleic acid(s) that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above. The term “substantially complementary” may refer to a nucleic acid comprising at least one sequence of consecutive nucleobases, or semiconsecutive nucleobases if one or more nucleobase moieties are not present in the molecule, are capable of hybridizing to at least one nucleic acid strand or duplex even if less than all nucleobases do not base pair with a counterpart nucleobase. In certain embodiments, a “substantially complementary” nucleic acid contains at least one sequence in which about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, to about 100%, and any range therein, of the nucleobase sequence is capable of base-pairing with at least one single or double-stranded nucleic acid molecule during hybridization. In certain embodiments, the term “substantially complementary” refers to at least one nucleic acid that may hybridize to at least one nucleic acid strand or duplex in stringent conditions. In certain embodiments, a “partially complementary” nucleic acid comprises at least one sequence that may hybridize in low stringency conditions to at least one single or double-stranded nucleic acid, or contains at least one sequence in which less than about 70% of the nucleobase sequence is capable of base-pairing with at least one single or double-stranded nucleic acid molecule during hybridization.
- The term “non-complementary” refers to nucleic acid sequence that lacks the ability to form at least one Watson-Crick base pair through specific hydrogen bonds.
- The term “blunt end” as used herein refers to the end of a dsDNA molecule having 5′ and 3′ ends, wherein the 5′ and 3′ ends terminate at the same nucleotide position. Thus, the blunt end comprises no 5′ or 3′ overhang.
- The term “overhang” as used herein refers to the end of a dsDNA molecule having 5′ and 3′ ends, wherein the 5′ and 3′ ends terminate at different nucleotide positions, leaving at least one nucleotide on the end of either the 5′ and 3′ end which has no nucleotide to hydrogen bond with.
- The term “cap” as used herein is a guanine nucleoside that is joined via its 5′-carbon to a triphosphate group that is, in turn, joined to the 5′-carbon of the most 5′-nucleotide of the primary mRNA transcript, and in most eukaryotes, the nitrogen at the 7 position of guanine in the cap nucleotide is methylated. Most eukaryotic cellular mRNA transcripts and most eukaryotic viral mRNA transcripts are blocked or “capped” at their 5′ terminus. In addition to mRNA, some other forms of eukaryotic RNA, such as but not limited to, small nuclear RNA (“snRNA”) and pre-micro RNA (i.e. “pre-miRNA”, the primary transcripts that are processed to miRNA) are also capped. The 5′ caps of eukaryotic cellular and viral mRNAs (and some other forms of RNA) play important roles in RNA stability and processing. For example, the cap is required to varying degrees for processing and maturation of an RNA transcripts in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of the mRNA to protein.
- “Sample” means a material obtained or isolated from a fresh or preserved biological sample or synthetically-created source that contains nucleic acids of interest. In certain embodiments, a sample is the biological material that contains the variable immune region(s) for which data or information are sought. Samples can include at least one cell, fetal cell, cell culture, tissue specimen, blood, serum, plasma, saliva, urine, tear, vaginal secretion, sweat, lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascites fluid, fecal matter, body exudates, umbilical cord blood, chorionic villi, amniotic fluid, embryonic tissue, multicellular embryo, lysate, extract, solution, or reaction mixture suspected of containing immune nucleic acids of interest. Samples can also include non-human sources, such as non-human primates, rodents and other mammals, other animals, plants, fungi, bacteria, and viruses.
- As used herein in relation to a nucleotide sequence, “substantially known” refers to having sufficient sequence information in order to permit preparation of a nucleic acid molecule, including its amplification. This will typically be about 100%, although in some embodiments some portion of an adaptor sequence is random or degenerate. Thus, in specific embodiments, substantially known refers to about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.
- Nucleic acids of the present disclosure can comprise amplification, reverse transcription, and/or in vitro transcription primers and oligonucleotides, tumor derived nucleic acids and/or recombinant nucleic acids encoding one or more proteins or peptides, such as those which may induce an immune response that is effective against a tumor or tumor cell. In some embodiments, generation of nucleic acids which may induce an immune response comprises reverse transcription of target mRNA to generate cDNA. Methods of reverse transcription are well known in the art (U.S. Pat. Nos. 5,962,271 and 6,518,019, incorporated herein by reference). Reverse transcription is performed by a RNA dependent DNA polymerase, also known as a reverse transcriptase. The reverse transcriptase binds to a primer hybridized to the 3′ end of a target mRNA, and synthesizes DNA complementary to the mRNA. The reverse transcriptase may add several non-templated nucleotides to the 3′ end of the newly synthesized cDNA, such as a short poly(C) tail. A second primer may be hybridized to the 3′ end of the nascent cDNA, and the reverse transcriptase may continue synthesizing DNA complementary to the primer, using its template switch activity to use the second primer as its template.
- The primers or oligonucleotides used in reverse transcription or amplification may allow for the addition of exogenous DNA or RNA sequence to the 5′ and 3′ ends of the target sequences during complementary strand synthesis. The primers or oligonucleotides used for priming the reverse transcription or amplification reactions may incorporate a variety of features including, but not limited to transcriptional promoters, ribosomal binding sites, or restriction endonuclease cleavage sites. In some embodiments, a primer may include a transcriptional promoter sequence.
- cDNA prepared by reverse transcription can be amplified or transcribed to RNA. cDNAs may be amplified by PCR, to increase the concentration of the cDNA. The primers for reverse transcription may be used as the primers for PCR, or separate primers may be added. The PCR reaction may utilize the reverse transcriptase in order to amplify the cDNA, or a DNA dependent DNA polymerase may be added to increase the efficiency of DNA amplification. The primers for reverse transcription or DNA amplification may include transcription promoter sequences, such as the T7 promoter sequence. cDNA or amplified cDNA may be transcribed in an in vitro transcription process to generate large quantities of the initial target RNA using any commercially available RNA polymerase or in vitro transcription kit, such as AMPLISCRIBE™ T7-FLASH™ (LUCIGEN® Cat. No. ASF3257).
- In certain embodiments a nucleic acid composition contains both a tumor derived nucleic acid population and a recombinant nucleic acid component. This combination nucleic acid composition increases the prevalence of certain known tumor antigens or other nucleic acids encoding proteins or peptides that enhance the effectiveness of the methods and compositions described herein.
- A “tumor-derived” nucleic acid refers to a nucleic acid that has its origin in a tumor cell, and which includes RNA corresponding to a tumor antigen(s). Included is RNA that encodes all or a portion of a tumor antigen or a previously identified tumor antigen. Such nucleic acid can be “in vitro transcribed,” e.g., reverse transcribed to produce cDNA that can be amplified by PCR and subsequently be transcribed in vitro, with or without cloning the cDNA. Also included is RNA that is provided as a fractionated preparation of tumor cell. Because even unfractionated RNA preparation (e.g., total RNA or total poly A RNA) can be used, it is not necessary that a tumor antigen be identified. In one embodiment, the preparation is fractionated with respect to a non-RNA component(s) of the cell in order to decrease the concentration of a non-RNA component, such as protein, lipid, and/or DNA and enrich the preparation for RNA. If desired, the preparation can be further fractionated with respect to the RNA (e.g., by subtractive hybridization) such that “tumor-specific” or “pathogen-specific” RNA is produced.
- By “tumor-specific” RNA is meant an RNA sample that, relative to unfractionated tumor-derived RNA, has a high content of RNA that is preferentially present in a tumor cell compared with a non-tumor cell. For example, tumor-specific RNA includes RNA that is present in a tumor cell, but not present in a non-tumor cell. Also encompassed in this definition is an RNA sample that includes RNA that is present both in tumor and non-tumor cells, but is present at a higher level in tumor cells than in non-tumor cells. Also included within this definition is RNA that encodes a previously identified tumor antigen and which is produced in vitro, e.g., from a plasmid or by PCR. Alternatively, tumor-specific RNA can be prepared by fractionating an RNA sample such that the percentage of RNA corresponding to a tumor antigen is increased, relative to unfractionated tumor-derived RNA. For example, tumor-specific RNA can be prepared by fractionating tumor-derived RNA using conventional subtractive hybridization techniques against RNA from non-tumor cells.
- Methods suitable for producing tumor-derived nucleic acid or RNA are provided herein. These nucleic acids can be used for priming dendritic cells, and in the preparation of mature dendritic cells. It is not necessary that the nucleic acid be provided to the DC in a purified form. Preferably, the RNA sample (i.e., the fractionated tumor preparation) is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% RNA (wt/vol).
- Art-known transfection methods are suitable for introducing the tumor-derived nucleic acid into a dendritic cell. For example, 5-50 μg of RNA in 500 μl of Opti-MEM can be mixed with a cationic lipid at a concentration of 10 to 100 μg, and incubated at room temperature for 20 to 30 minutes. Other suitable lipids include LIPOFECTIN™ (1:1 (w/w) DOTMA:DOPE), LIPOFECTAMINE™ (3:1 (w/w) DOSPA:DOPE), DODAC:DOPE (1:1), CHOL:DOPE (1:1), DMEDA, CHOL, DDAB, DMEDA, DODAC, DOPE, DORI, DORIE, DOSPA, DOTAP, and DOTMA. The resulting RNA-lipid complex is then added to 1-3×106 cells, preferably 2×106, antigen-presenting cells in a total volume of approximately 2 ml (e.g., in Opti-MEM), and incubated at 37° C. for 2 to 4 hours. Alternatively, the RNA can be introduced into the antigen presenting cells by employing conventional techniques, such as electroporation or calcium phosphate transfection with 1-5×106 cells and 5 to 50 μg of RNA. Typically, 5-20 μg of poly A RNA or 25-50 μg of total RNA are typically used.
- When the RNA is provided as a tumor preparation, the preparation typically is fractionated or otherwise treated to decrease the concentration of proteins, lipids, and/or DNA in the preparation, and enrich the preparation for RNA. For example, art-known RNA purification methods can be used to at least partially purify the RNA from the tumor cell or pathogen. It is also acceptable to treat the RNA preparation with proteases or RNase-free DNases.
- Methods for isolating culturing and priming dendritic cells are well known in the art. For example, U.S. Pat. No. 8,728,806, which is incorporated herein by reference in its entirety, provides detailed methods for providing antigen primed dendritic cells that may be used in the compositions and methods of the embodiments. In certain aspects, dendritic cells for use according to the embodiments are isolated from a subject that is to be treated by a method of the embodiments. In other aspects, dendritic cells may be from a different subject, such as an HLA-matched donor. In certain aspects, the dendritic cells are from a bank of dendritic cells having a defined HLA typing. In preferred aspects, primed dendritic cells for use according to the embodiments are homologously-loaded with antigen as detailed herein and in U.S. Pat. No. 8,728,806.
- Methods for isolating cell populations enriched for dendritic cell precursors and immature dendritic cells from various sources, including blood and bone marrow, are known in the art. For example, dendritic cell precursors and immature dendritic cells can be isolated by collecting heparinized blood, by apheresis or leukapheresis, by preparation of buffy coats, rosetting, centrifugation, density gradient centrifugation (e.g., using Ficoll (such as FICOLL-PAQUE®), PERCOLL® (colloidal silica particles (15-30 mm diameter) coated with non-dialyzable polyvinylpyrrolidone (PVP)), sucrose, and the like), differential lysis of cells, filtration, and the like. In certain embodiments, a leukocyte population can be prepared, such as, for example, by collecting blood from a subject, defribrinating to remove the platelets and lysing the red blood cells. Dendritic cell precursors and immature dendritic cells can optionally be enriched for monocytic dendritic cell precursors by, for example, centrifugation through a PERCOLL® gradient. In other aspects, dendritic cell precursors can be selected using CD14 selection of G-CSF mobilized peripheral blood.
- Dendritic cell precursors and immature dendritic cells optionally can be prepared in a closed, aseptic system. As used herein, the terms “closed, aseptic system” or “closed system” refer to a system in which exposure to non-sterilize, ambient, or circulating air or other non-sterile conditions is minimized or eliminated. Closed systems for isolating dendritic cell precursors and immature dendritic cells generally exclude density gradient centrifugation in open top tubes, open air transfer of cells, culture of cells in tissue culture plates or unsealed flasks, and the like. In a typical embodiment, the closed system allows aseptic transfer of the dendritic cell precursors and immature dendritic cells from an initial collection vessel to a sealable tissue culture vessel without exposure to non-sterile air.
- In certain embodiments, monocytic dendritic cell precursors are isolated by adherence to a monocyte-binding substrate. For example, a population of leukocytes (e.g., isolated by leukapheresis) can be contacted with a monocytic dendritic cell precursor adhering substrate. When the population of leukocytes is contacted with the substrate, the monocytic dendritic cell precursors in the leukocyte population preferentially adhere to the substrate. Other leukocytes (including other potential dendritic cell precursors) exhibit reduced binding affinity to the substrate, thereby allowing the monocytic dendritic cell precursors to be preferentially enriched on the surface of the substrate.
- Suitable substrates include, for example, those having a large surface area to volume ratio. Such substrates can be, for example, a particulate or fibrous substrate. Suitable particulate substrates include, for example, glass particles, plastic particles, glass-coated plastic particles, glass-coated polystyrene particles, and other beads suitable for protein absorption. Suitable fibrous substrates include microcapillary tubes and microvillous membrane. The particulate or fibrous substrate usually allows the adhered monocytic dendritic cell precursors to be eluted without substantially reducing the viability of the adhered cells. A particulate or fibrous substrate can be substantially non-porous to facilitate elution of monocytic dendritic cell precursors or dendritic cells from the substrate. A “substantially non-porous” substrate is a substrate in which at least a majority of pores present in the substrate are smaller than the cells to minimize entrapping cells in the substrate.
- Adherence of the monocytic dendritic cell precursors to the substrate can optionally be enhanced by addition of binding media. Suitable binding media include monocytic dendritic cell precursor culture media (e.g., AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, and the like) supplemented, individually or in any combination, with for example, cytokines (e.g., Granulocyte/Macrophage Colony Stimulating Factor (GM-CSF), Interleukin 4 (IL-4), or Interleukin 13 (IL-13)), blood plasma, serum (e.g., human serum, such as autologous or allogenic sera), purified proteins, such as serum albumin, divalent cations (e.g., calcium and/or magnesium ions) and other molecules that aid in the specific adherence of monocytic dendritic cell precursors to the substrate, or that prevent adherence of non-monocytic dendritic cell precursors to the substrate. In certain embodiments, the blood plasma or serum can be heated-inactivated. The heat-inactivated plasma can be autologous or heterologous to the leukocytes.
- Following adherence of monocytic dendritic cell precursors to the substrate, the non-adhering leukocytes are separated from the monocytic dendritic cell precursor/substrate complexes. Any suitable means can be used to separate the non-adhering cells from the complexes. For example, the mixture of the non-adhering leukocytes and the complexes can be allowed to settle, and the non-adhering leukocytes and media decanted or drained. Alternatively, the mixture can be centrifuged, and the supernatant containing the non-adhering leukocytes decanted or drained from the pelleted complexes.
- Isolated dendritic cell precursors can be cultured ex vivo for differentiation, maturation and/or expansion. (As used herein, isolated immature dendritic cells, dendritic cell precursors, T cells, and other cells, refers to cells that, by human hand, exists apart from their native environment, and are therefore not a product of nature. Isolated cells can exist in purified form, in semi-purified form, or in a non-native environment.) Briefly, ex vivo differentiation typically involves culturing dendritic cell precursors, or populations of cells having dendritic cell precursors, in the presence of one or more differentiation agents. Suitable differentiating agents can be, for example, cellular growth factors (e.g., cytokines such as (GM-CSF), Interleukin 4 (IL-4), Interleukin 13 (IL-13), and/or combinations thereof). In certain embodiments, the monocytic dendritic cells precursors are differentiated to form monocyte-derived immature dendritic cells.
- The dendritic cell precursors can be cultured and differentiated in suitable culture conditions. Suitable tissue culture media include AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, and the like. The tissue culture media can be supplemented with serum, amino acids, vitamins, cytokines, such as GM-CSF and/or IL-4, divalent cations, and the like, to promote differentiation of the cells. In certain embodiments, the dendritic cell precursors can be cultured in the serum-free media. Such culture conditions can optionally exclude any animal-derived products. A typical cytokine combination in a typical dendritic cell culture medium is about 500 units/ml each of GM-CSF (50 ng/ml) and IL-4 (10 ng/ml). Dendritic cell precursors, when differentiated to form immature dendritic cells, are phenotypically similar to skin Langerhans cells. Immature dendritic cells typically are CD14− and CD11c+, express low levels of CD86 and CD83, and are able to capture soluble antigens via specialized endocytosis. The immature DC expressed very high levels of CD86. Also, the population was mixed in terms of CD14 and CD11C. Though the majority were CD11c+, there were distinct subpopulations that were CD11c− and
CD 14+. - The immature dendritic cells are matured to form mature dendritic cells. Mature DC lose the ability to take up antigen and display up-regulated expression of costimulatory cell surface molecules and various cytokines. Specifically, mature DC express higher levels of MHC class I and II antigens than immature dendritic cells, and mature dendritic cells are generally identified as being CD80+, CD83+, CD86+, and CD14−. Greater MHC expression leads to an increase in antigen density on the DC surface, while up regulation of costimulatory molecules CD80 and CD86 strengthens the T cell activation signal through the counterparts of the costimulatory molecules, such as CD28 on the T cells.
- Mature dendritic cells of the present invention can be prepared (i.e., matured) by contacting the immature dendritic cells with effective amounts or concentrations of a nucleic acid composition and a tumor antigen composition. Effective amounts of nucleic acid composition typically range from at most, at least, or about 0.01, 0.1, 1, 5, 10, to 10, 15, 20, 50, 100 ng or mg of nucleic acid per culture dish or per cell, including all values and ranges there between. Effective amounts of tumor antigen composition typically range from at most, at least, or about 0.01, 0.1, 1, 5, 10, to 10, 15, 20, 50, 100 ng or mg of protein per culture dish or per cell. In certain aspects 0.001 ng of tumor antigen/cell to 1 μg of tumor antigen/million cells) can be used. The tumor antigen composition can optionally be heat inactivated or treated (e.g., exposed to protease) prior to contact with dendritic cells. Maturing the immature dendritic cells with a nucleic acid composition and a tumor antigen composition primes the mature dendritic cells for a type 1 (Th-1) response.
- The immature DC are typically contacted with effective amounts of a nucleic acid composition and a tumor antigen composition for at most, at least, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 minutes, hours, or days. The immature dendritic cells can be cultured and matured in suitable maturation culture conditions. Suitable tissue culture media include AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, and the like. The tissue culture media can be supplemented with amino acids, vitamins, cytokines, such as GM-CSF and/or IL-4, divalent cations, and the like, to promote maturation of the cells.
- Maturation of dendritic cells can be monitored by methods known in the art. Cell surface markers can be detected in assays familiar to the art, such as flow cytometry, immunohistochemistry, and the like. The cells can also be monitored for cytokine production (e.g., by ELISA, FACS, or other immune assay). Dendritic cell precursors, immature dendritic cells, and mature dendritic cells, either primed or unprimed, with antigens can be cryopreserved for use at a later date. Methods for cryopreservation are well-known in the art. For example, U.S. Pat. No. 5,788,963, which is incorporated herein by reference in its entirety.
- A nucleic acid or nucleic acid primed dendritic cell is a dendritic cell that was incubated or transfected with RNA, e.g., RNA derived from a tumor or tumor cell. Such RNA can be transfected using conventional nucleic acid transfection methods, such as lipid-mediated transfection, electroporation, and calcium phosphate transfection. For example, RNA can be introduced into a DC by incubating the DC with the RNA (or extract) for 1 to 24 hours (e.g., 2 hours) at 37° C.
- The nucleic acid-loaded antigen-presenting cells of the present disclosure can be used to stimulate CTL proliferation in vivo or ex vivo. The ability of the nucleic acid-loaded dendritic cells to stimulate a CTL response can be measured by assaying the ability of the effector cells to lyse target cells. For example, the commonly-used europium release assay can be used. Typically, 5-10×106 target cells are labeled with europium diethylenetriamine pentaacetate for 20 minutes at 4° C. After
several washes 104 europium-labeled target cells and serial dilutions of effector cells at an effector:target ratio ranging from 50:1 to 6.25:1 are incubated in 200 μl RPMI 1640 with 10% heat-inactivated fetal calf serum in 96-well plates. The plates are centrifuged at 500×g for 3 minutes and the incubated at 37° C. in 5% CO2 for 4 hours. A 50 μl aliquot of the supernatant is collected, and europium release is measured by time resolved fluorescence (Volgmann et al., J. Immunol. Methods 119:45-51, 1989). - A. Genetically Modified Dendritic Cells
- Certain aspects of the embodiments concern dendritic cells that have been genetically modified. In some aspects, the genetic modification comprises introduction of an exogenous transgene in the cells, such as an inhibitory nucleic acid. In further aspects, the transgene may be a suicide gene, such as a gene encoding thymidine kinase, under the control of an inducible promoter. Thus, in some aspects, after stimulating an immune response, administered dendritic cells can be killed-off by induction of the promoter controlling expression of the suicide gene.
- In further aspects, the genetic modification comprises a genomic deletion or insertion in the cell population. For example, one or more HLA gene may be disrupted to render the dendritic cells as an effective HLA match for a subject to be treated.
- Further aspects of the embodiments concern dendritic cells that have been genetically modified, such as to reduce the expression of CTLA-4. In some aspects, the genetic modification comprises introduction of an exogenous inhibitory nucleic acid specific to CTLA-4. In certain aspects, the inhibitory nucleic acid is a RNA, such as a RNA that is expressed from a DNA vector in the dendritic cells. In further aspects, the inhibitory nucleic acid may be a siRNAs, dsRNA, miRNA or shRNA that is introduced in the dendritic cells. A detailed disclosure of such RNAs is provided above.
- In further aspects, the genetic modification comprises a genomic deletion or insertion in the cell population that reduces CTLA-4. In other aspects, the dendritic cells comprises a hemizygous or homozygous deletion within the CTLA-4 gene. For example, in some aspects, one or both copies of the CTLA-4 gene of a dendritic cell can be completely or partially deleted, such that expression the CTLA-4 polypeptide is inhibited. In some aspects, modification the cells so that they do not express one or more CTLA-4 gene may comprise introducing into the cells an artificial nuclease that specifically targets the CTLA-4 locus. In various aspects, the artificial nuclease may be a zinc finger nuclease, TALEN, or CRISPR/Cas9. In various aspects, introducing into the cells an artificial nuclease may comprise introducing mRNA encoding the artificial nuclease into the cells.
- In order to increase the effectiveness of dendritic cell therapies which include dendritic cells transformed with nucleic acids generated by the embodiments, it may be desirable to combine these compositions with other agents effective in the treatment of the disease of interest.
- As a non-limiting example, the treatment of cancer may be implemented with a primed dendritic cell composition of the present embodiments along with other anti-cancer agents. An “anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the anti-cancer peptide or nanoparticle complex and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the dendritic cell composition and the other includes the second agent(s).
- Treatment with a dendritic cell composition may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and dendritic cell composition are applied separately to the subject, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and the dendritic cell composition would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly where several days (e.g., 2, 3, 4, 5, 6 or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 weeks) lapse between the respective administrations.
- Various combinations may be employed, where dendritic cell therapy is “A” and the secondary agent, such as radiotherapy, chemotherapy or anti-inflammatory agent, is “B”:
-
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
In certain embodiments, administration of dendritic cell therapy of the present embodiments to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of the vector. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described hyperproliferative cell therapy. - A. Chemotherapy
- Cancer therapies also include a variety of combination therapies. In some aspects a dendritic cell composition of the embodiments is administered (or formulated) in conjunction with a chemotherapeutic agent. For example, in some aspects the chemotherapeutic agent is a protein kinase inhibitor such as a EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF inhibitors. Nonlimiting examples of protein kinase inhibitors include Afatinib, Axitinib, Bevacizumab, Bosutinib, Cetuximab, Crizotinib, Dasatinib, Erlotinib, Fostamatinib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib, Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, Miltefosine, Perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, Alvocidib, Geni stein, Selumetinib, AZD-6244, Vatalanib, P1446A-AG-024322, ZD1839, P276-00, GW572016 or a mixture thereof.
- Yet further combination chemotherapies include, for example, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammalI and calicheamicin omegaIl; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the compositions provided herein may be used in combination with gefitinib. In other embodiments, the present embodiments may be practiced in combination with Gleevac (e.g., from about 400 to about 800 mg/day of Gleevac may be administered to a patient). In certain embodiments, one or more chemotherapeutic may be used in combination with the compositions provided herein.
- B. Radiotherapy
- Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
- The terms “contacted” and “exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
- C. Gene Therapy
- In yet another embodiment, the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the therapeutic composition. Viral vectors for the expression of a gene product are well known in the art, and include such eukaryotic expression systems as adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, lentiviruses, poxviruses including vaccinia viruses, and papiloma viruses, including SV40. Alternatively, the administration of expression constructs can be accomplished with lipid based vectors such as liposomes or DOTAP:cholesterol vesicles. All of these methods are well known in the art (see, e.g. Sambrook et al., 1989; Ausubel et al., 1998; Ausubel, 1996).
- D. Surgery
- Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatments provided herein, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
- Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present embodiments may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue. In some aspects, following tumor resection a dendritic cell composition of the embodiments is administered to lymphoid tissue that drained the previous site for the tumor.
- The technology herein includes kits for generating cDNA from mRNA in a sample, and the in vitro transcription of said cDNA. A “kit” refers to a combination of physical elements. For example, a kit may include, for example, one or more components including without limitation specific primers, enzymes, reaction buffers, an instruction sheet, and other elements useful to practice the technology described herein. These physical elements can be arranged in any way suitable for carrying out the invention.
- The kit may further comprise a DNA polymerase, including for example, Φ29 polymerase, Bst polymerase, Taq polymerase, Vent polymerase, DNA polymerase I, the Klenow fragment of DNA polymerase I, 9° Nm Polymerase, T4 polymerase, T7 DNA polymerase, Pfu DNA polymerase, Q5® polymerase (New England Biolabs), or a RNA polymerase, and a reverse transcriptase. A mutant form of T7 phage DNA polymerase that lacks 3′-5′ exonuclease activity, or a mixture thereof.
- The kit may further comprise a RNA polymerase for in vitro transcription, including for example, E. coli RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, T7 RNA polymerase, or mutants thereof.
- The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted (e.g., aliquoted into the wells of a microtiter plate). Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a single vial. The kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
- A kit will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented. It is contemplated that such reagents are embodiments of kits of the invention. Such kits, however, are not limited to the particular items identified above and may include any reagent used for the manipulation or characterization of the methylation of a gene.
- The container means of the kits will generally include at least one vial, test tube, flask, bottle, or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain additional containers into which the additional components may be separately placed. However, various combinations of components may be comprised in a container. The kits of the present invention also will typically include a means for packaging the component containers in close confinement for commercial sale. Such packaging may include injection or blow-molded plastic containers into which the desired component containers are retained.
- The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
- Fresh tissue processing—Following surgical removal of the tumor, tumor tissue was weighed and sliced into small pieces (about 20 mg/piece or smaller). All pieces were placed in a 1.5 mL cryovial with 1 mL of an RNA stabilization solution (RNALATER™, Invitrogen). The cryovial containing the RNALATER™ and dissected tumor is then left at 4° C. overnight to prevent RNase activity. Following overnight incubation at 4° C., the cryovial containing the tissue in RNALATER™ solution was centrifuged at 10,000 RPM for 1 min at 4° C. After centrifugation, the supernatant was removed by pipetting, and the dissected pieces of tissues were weighed. 10% of the tissue was used for subsequent preparation of total RNA, while the remaining 90% was used for subsequent preparation of antigenic lysates.
- Preparation of antigenic lysates—Dissected tumor tissue was added to a 15 mL conical tube and dilute to 100 mg/mL in sterile PBS. A homogenous suspension of tissue in PBS was then made using a handheld tissue homogenizer. The tube containing the homogenous suspension was then immersed in liquid nitrogen or a dry ice/ethanol bath to freeze the suspension. After freezing, the cell suspension was then incubated in a 55° C. water bath until it was completely thawed. The freeze thaw cycle was repeated two further times. Following the final thaw, to ensure sterility and complete cell death, the cell suspension was Gamma irradiated with >2500 Gy of radiation via a cesium irradiator. Lysates were then stored at −20° C. until further use.
- Preparation of antigenic mRNA—Total RNA was isolated from tissue stabilized with RNALATER™ reagent using the RNEASY® mini kit (Qiagen cat #74104), which utilizes spin columns, or the TRIPURE® (Roche) method, which utilizes a guanidinium thiocyanate and phenol mixture, each according to the manufacturer's instructions.
- RNeasy Total RNA preparation—600 μL of Buffer RLT was added to 30 mg of tissue and the resulting mixture was homogenized using a handheld tissue homogenizer. 600 μL of 70% ethanol was added to the lysate, and mixed by pipetting. 700 μL of the sample was transferred to a RNeasy spin column, and placed in a 2 mL collection tube. The sample was centrifuged for 1 min at 8,000×g. Following centrifugation, the flow-through was discarded. The remaining lysate was added to the spin column and was centrifuged again. Following centrifugation, the flow-through was discarded. 350 μL of buffer RW1 was added to the column and centrifuged. DNase I solution was prepared by adding 10 μL of DNase I stock solution to 70 μL of buffer RDD, followed by gentle mixing. 80 μL of DNase I solution was then added to the column and incubated at room temperature for 15 min. Following DNase I incubation, 350 μL of buffer RW1 was added to the column and the column was centrifuged for 1 min at 8000×g. Following centrifugation the flow-through was discarded. The membrane was then washed by the addition of another 700 μL of buffer RW1, and another 1 min centrifugation at 8000×g, followed by two 500 μl washes with buffer RPE, with two centrifugations at 8000×g. The membrane was then dried by centrifugation for 2 min. RNA was eluted into a new column by direct addition of 50 μl of pre-warmed nuclease free water to the membrane in the column, 1 minute of incubation, and centrifugation at 8000×g for 1 minute. In order to concentrate the RNA, the elution was performed a second time using the eluent. Total RNA was then quantified. Total RNA was stored at −80° C. for long term storage.
- Guanidinium Thiocyanate/Phenol/Chloroform RNA Isolation—1 mL of guanidinium thiocyanate and phenol reagent (TRIpure™ reagent from Roche, Cat. NO. 11 667 157 001) was added for every 50 mg of tumor tissue and the resulting mixture was homogenized using a handheld tissue homogenizer. The homogenized mixture was then centrifuged at 13,000 RPM for 5 min at 4° C. to clear the homogenate. The supernatant from the homogenate was transferred to a new RNase free tube. To clear the supernatant, 400 uL of chloroform was added, and the mixture was mixed thoroughly by inversion. The supernatant/chloroform mixture was then incubated at room temperature for 5-10 min. The mixture was then centrifuged at 4500 RPM for 45 min or 12,000×g for 15 min at 4° C. in order to clear it. Following centrifugation, the resulting mixture is present in 3 phases: the organic phase, the interface, and the aqueous phase. The aqueous phase is collected by pipetting and transferred directly to a pre-chilled tube. 0.5 mL of isopropanol were added for every 1 mL of starting guanidinium thiocyanate and phenol reagent. The isopropanol/supernatant mixture was mixed gently and incubated at room temperature for 10 min. Following incubation, the RNA was pelleted by centrifugation at 4500 RPM for 45 min or 12000×G for 15 min at 4° C. The pelleted RNA was washed with 1 mL of 70% ethanol and centrifuged again. The supernatant was removed by pipetting and the pellet was allowed to air dry for 5-10 minutes on ice. The pellet was then dissolved in 100 uL of RNase free water, and incubated at 55° C. for 5 min. The concentration of total RNA was then measured. Total RNA was stored at −80° C. for long term storage. Prior to first strand synthesis, total RNA isolated using the guanidinium thiocyanate method was DNase treated by the addition of 1 uL of amplification grade DNase (Invitrogen Cat. No. 18068-015) to 4 ug of total RNA in 10× buffer diluted to 1×. DNase reaction was allowed to proceed for 15 minutes, and stopped by the addition of 1 uL of EDTA.
- cDNA synthesis—For each sample and control, the following RNA/primer annealing mix was prepared in a sterile 0.2 mL PCR tube: 4 uL of a 10 uM solution of SEQ ID NO: 1 (VKWD oligo), 4 uL of a 10 uM solution of SEQ ID NO: 2 (CDS 64T+Oligo), 4 ug of sample RNA, and water to a total reaction volume of 19 uL. The solution was mixed and spun briefly in a microfuge to condense the solution to the bottom of the tube. The solution was then incubated at 72° C. for 2 min in a preheated thermocycler, and then cooled to 4° C. The sample was then stored on ice while the first strand synthesis master mix was prepared.
- First strand synthesis master mixes were prepared to account for each reaction plus one no template control, and prepared from reagents in the Superscript® First Strand Synthesis Kit (Invitrogen Cat. No. 11904-018). The master mix contains, per sample: 4 uL of 10× First-Strand Buffer, 4 uL DTT (100 mM), 1 uL of RNase inhibitor (RNase Out™) 4 uL MgCl2 (25 mM), 4 uL dNTP Mix (10 mM each of dATP, dGTP, dCTP, and dTTP), and 4 uL of Superscript® II Reverse Transcriptase, for a total volume of 21 uL per sample.
- 21 uL of first strand synthesis mastermix was added to each tube containing 19 ul of RNA/primer annealing mix. Each reaction, including the NTC, was incubated at 42° C. for 1 hour to synthesize the first strand of the cDNA. The first strand synthesis reaction was then collected by a brief centrifugation in a microcentrifuge and chilled on ice prior to second strand cDNA synthesis.
- The second strand synthesis master mix was prepared by adding 100 uL FastStart™ high fidelity reaction buffer with MgCl2, 40 uL of 10 μmol/L CDS 64T+Oligo (SEQ ID NO: 2), 20 uL of 20 μmol/L Powerswitch T7 primer (SEQ ID NO: 3), 20 uL dNTP mix (10 mM each, dATP, dGTP, dCTP, dTTP), 20 uL of FastStart high fidelity enzyme, and 780 uL of water, for a total of 980 uL per sample. 20 uL of the first strand synthesis reaction was added to the second strand synthesis master mix. The resulting mixture was then split into equal volumes of 100 uL each in 0.2 mL PCR tubes. Each of these reactions was then subjected to thermal cycling using the following program:
-
1x 95° C. for 5 min 11x 95° C. for 30 s 65° C. for 30 s 67° C. for 6 min 9x 95° C. for 35 s 65° C. for 35 s 67° C. for 6 min 5 secondsFor each additional cycle, the 67° C. extension increases by an additional 5 seconds, e.g. cycle 1 = 6:05, cycle 2 = 6:10,cycle 3 = 6:15, etc.1x 67° C. for 7 min Hold 4° C. - Double stranded cDNA was then purified using the Salt-ethabol precipitation method. Sodium acetate (3M) was added to the sample at a 1:10 vol:vol ratio, followed by a 2:1 vol:vol ratio of chilled 100% ethanol. The sample was mixed gently and then incubated at −20° C. for 1 hour. The mixture was then centrifuged at 13,000 RPM for 20 min to pellet the DNA. The supernatant was carefully pipetted off the pelleted DNA. The resulting pellet was then washed by adding 100 uL of chilled 70% ethanol and resuspended. The solution was then centrifuged for 10 min at 13,000 RPM, and the supernatant was pipetted off. The pellet was then air dried for 10 minutes before dissolving in 20 uL of nuclease free water. The concentration of the cDNA was measured by nanodrop.
- In vitro transcription—In vitro transcription was performed using the AMPLISCRIBE™ T7-FLASH™ in vitro amplification kit (LUCIGEN™ Cat. No. ASF3257). 20 uL reaction volumes were prepared by adding, in the following order: X uL of RNase free water (X being the amount of water required to achieve 20 uL reaction volume); 1 ug of cDNA template; 2 uL of AIVIPLISCRIBE™ T7-FLASH™ 10× reaction buffer; 1.8 uL each of 100 mM ATP, CTP, GTP and UTP; 2 uL DTT; 0.5 uL of RIBOGUARD™ RNase inhibitor; and 2 uL of AMPLISCRIBE™ T7-FLASH™ Enzyme solution. The reaction was then incubated for 1 hr at 42° C. in a thermocycler. Following incubation, 1 uL of RNase-free DNase I was added, and the reaction was incubated at 37° C. for 15 min. The DNase treated in vitro transcription reaction was brought to 50 uL by addition of 29 uL of RNase free water. The in vitro transcription reaction was then purified by salt and ethanol precipitation and purification. 50 uL of 5 M ammonium acetate and 100 uL of ice cold 100% ethanol were added to the in vitro transcription reaction and mixed. The resulting mixture was then incubated for 1 hr at −20° C. After incubation, the mixture was centrifuged at 10,000×g for 15 min at 4° C. The supernatant was removed from the pellet by pipetting. The pellet was then washed with 100 uL of cold 70% ethanol and centrifuged again at 10,000×g for 15 min at 4° C. The supernatant was removed by pipetting and the pellet was allowed to air dry before resuspending in 50 uL of RNase free water.
- Capping of uncapped in vitro transcription generated RNA—Capping of the RNA generated by in vitro transcription generated RNA was performed using the SCRIPTCAP™ m7G Capping System and
SCRIPTCAP™ 2′-O-Methyltransferase Kit (CELL SCRIPT™ Cat. Nos. C-SCCE0625 and C-SCMT0625) according to the manufacturer's instructions. Briefly, 50-60 ug in vitro transcribed uncapped RNA was added to RNase free water to a total volume of 67 uL. The diluted RNA was then incubated at 65° C. for 10 min to denature, and subsequently stored on ice. A capping master mix was prepared by adding 10 uL of 10× SCRIPTCAP™ Capping buffer, 10 uL of 10 mM GTP, 2.5 uL of 20 mM S-adenosyl methionine, 2.5 uL SCRIPTGUARD™ RNase inhibitor, and 4uL SCRIPTCAP™ 2′-O-methyltransferase, per sample. The capping reaction was assembled by adding 4 uL of the SCRIPTCAP™ capping enzyme to 29 uL of the capping master mix, and adding that mix to the heat denatured RNA. The capping reaction was allowed to proceed at 37° C. for 30 min. Capped RNA was purified by salt and ethanol precipitation and purification as above, and resuspended in 50 uL of RNase free water. To assess the quality of the capped mRNA, 1 ug of capped mRNA was run on a 50 mL, 1% agarose non-denaturing gel. The capped mRNA was stored at −80° C. - Antigenic lysate preparation—To generate MHC class II determinants, 1 mL of PBS per 100 mg of tissue was added to the isolated tissue fractions, and the tissues were disrupted using a POLYTRON® PT1200E tissue homogenizer (Kinematica, Inc., Bohemia, New York). The homogenized tissue fractions were then subjected to three repetitive freeze-thaw cycles, cycling the homogenized mixtures between liquid nitrogen and a 55 degree C. water bath, and stored at −20° C.
- Human (Decker et al., 2006; Decker et al., 2009) and wild type mouse (Konduri et al., 2013) dendritic cells were prepared, loaded, and matured as described. In vitro co-cultures were performed as described previously (Decker et al., 2006; Decker et al., 2009; Konduri et al., 2013).
- IFNγ production by T cells co cultured with dendritic cells loaded with capped mRNA. Total PBMC in the co-culture were treated for 5 hours with 10 μg/ml brefeldin A (eBioscience). Cells were stained for surface markers with anti-CD3, anti-CD8, and anti-CD25 antibodies and then fixed/permeabilized for intracellular staining of IFN-γ using the Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer's instructions.
- Homologous antigenic loading leads to enhanced production of AIMp1 and retention of CTLA-4. Enhanced production of AIMp1 was demonstrated by Western blot. Enhanced retention of CTLA-4 was demonstrated by CTLA-4 western blot of cell culture supernatants (to determine release) and intracellular flow cytometry to determine retained CTLA-4 content. Histogram analysis of intracellular CTLA-4 is gated on the CD11c+CD80+CD83+CD86+ cell population.
- Western blot analysis. Preparation of whole cell lysate: cells were lysed in 1% NP-40 lysis buffer containing protease inhibitor cocktail,
phosphatase inhibitor cocktail 2 and phosphatase inhibitor cocktail 3 (all purchased from Sigma-Aldrich) on ice with vortexing every 10-15 minutes. Cell lysate was centrifuged at 14,000×g for 15 minutes and the cleared lysate was denatured with laemmli buffer (Bio-Rad) containing 5% β-mercaptoethanol (Bio-Rad) for 10 minutes. Denatured whole cell lysate samples were stored at −20° C. for further analysis. Electrophoreses and blotting: proteins samples were separated by SDS-gel electrophoreses (Invitrogen) with subsequent transfer to a 0.45 μm nitrocellulose membrane (Bio-Rad) for antibody probing. All blocking and antibody staining steps were carried out in 5% BSA (RPI, Grainger) in 1×TB ST buffer (0.05% Tween-20). Western blotting chemiluminescent signal was detected with SUPERSIGNAL® West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) using a CHEMIDOC® XRS digital imaging system supported by Image Lab software version 2.0.1 (Bio-Rad). Densitometry was performed using Image Lab software. - RNA isolation from fresh tissue yields high quality RNA—To determine whether fresh or frozen tissue yielded higher quality RNA for downstream processing, total RNA was isolated from 30 mg of 3 different frozen tumor tissues or a single fresh tumor. As can be seen in
FIG. 1 , the RNA isolated from the three frozen tissues exhibits significant degradation, visible as a large smear in the lanes.Lane 4 has two clearly defined bands, indicating that the integrity of the RNA is high. - Comparison of commercial RNA isolation methods—Total RNA was isolated from 30 mg of tumor tissue using either the RNEASY® mini kit (Qiagen) or using the TRIPURE® RNA isolation reagent (Roche), which is a guanidinium thiocyanate/phenol/chloroform based method. Both methods yielded high quality RNA as visible in
FIG. 2 , though the TRIPURE® method yielded 4.4-fold more RNA than the RNEASY® kit from the same input amount. - In Vitro Transcribed RNA from mRNA isolated from tumor cells—
FIG. 3 depicts a schematic, including the primers, for the method used for reverse transcription of mRNA isolated from tumor cells, and subsequent in vitro transcription of the cDNA according to the present methods (see Example 1). Two different reverse transcription and in vitro transcription methods were compared (FIG. 4 ).Lanes Lanes - Enrichment of small transcripts by co-transcriptional capping. In order to insure accurate representation of all transcripts, two methods were tested for capping of in vitro transcribed mRNA.
FIG. 5 depicts an agarose gel of the capped RNA generated by each method.Lane 1 shows RNA post-transcriptionally capped, whileLane 2 shows RNA which was co-transcriptionally capped. Clearly, the transcript smear is much more evident inlane 2, in which the RNA was produced by the present methods and co-transcriptionally capped. Tables 1 and 2 show the concentrations, volume, and yields of products generated by the Slagter-Jager method (Slagter-Jager et al., 2013). Konduri/Decker Hybrid preparation is according to the present methods. -
TABLE 1 In vitro transcribed RNA prepared from RNEASY ® isolated total RNA. RNEASY ® column Concentration Volume Yield RNA yield 200 ng/ul 90 ul 18 ug Argos method ds-cDNA 20 ng/ ul 100 ul 2 ug uncapped mRNA 2.8 ug/ ul 100 ul 280 ug capped mRNA 2.0 ug/ul 20 ul 40 ug co-transcribed capped 90 ng/ul 25 ul 2.2 ug mRNA Konduri/Decker Hybrid ds-cDNA 47 ng/ ul 100 ul 4.7 ug uncapped mRNA 3.0 ug/ ul 100 ul 300 ug capped mRNA 2.5 ug/ul 20 ul 50 ug co-transcribed capped 2.0 ug/ul 35 ul 70 ug mRNA -
TABLE 2 In vitro transcribed RNA prepared from TRIPURE ™ isolated total RNA. TRIPURE ® method Concentration Volume Yield RNA yield 2 ng/ul 40 ul 80 ug Argos method ds-cDNA 20 ng/ul 120 ul 2.4 ug uncapped mRNA 2.5 ug/ ul 100 ul 250 ug capped mRNA 2.4 ug/ul 30 ul 72 ug co-transcribed capped 2.7 ng/ul 30 ul 81 ug mRNA Konduri/Decker Hybrid ds-cDNA 21 ng/ul 120 ul 2.5 ug uncapped mRNA 2.6 ug/ ul 100 ul 260 ug capped mRNA 2.0 ug/ul 40 ul 80 ug co-transcribed capped 3.3 ug/ul 60 ul 198 ug mRNA - IFNγ production is enhanced by co-culture with loaded DCs—Dendritic cells were homologously loaded with antigenic lysate and mRNA prepared by the methods described herein, or heterologously loaded with prostate mRNA and pancreatic tumor lysate (mismatched).
FIG. 6 shows relative IFNγ levels produced by T cells cocultured with the indicated dendritic cells. Dendritic cells loaded with capped RNA and antigenic lysate clearly stimulate IFNγ production, and does so better than dendritic cells loaded with uncapped RNA, or immature or unloaded dendritic cells. - Co-culture with homologously loaded dendritic cells enhances antigen specific CD8+CD25+ T cells—
FIG. 7 shows the results of flow cytometric analysis of CD8+CD25+ cells sorted for IFNγ levels, after co-culture with dendritic cells homologously loaded with either uncapped or capped mRNA produced by the methods described herein and lysate, or loaded with prostate mRNA and pancreatic tumor lysate (mismatched). Homologously loading lysate and mRNA produced by the methods described herein significantly enhances the levels of antigen specific CD8+CD25+ T cells. - Homologous antigenic loading enhances production of AIMp1 and retention of CTLA4—Dendritic cells were evaluated for AIMp1 production and CTLA4 retention after either homologous antigenic loading or heterologous antigenic loading (mismatch).
FIG. 8 shows a western blot for AIMp1 and CTLA-4 in the lysates with (3-actin provided as a control. Homologous antigenic loading with capped mRNA made by the methods provided herein have increased amounts of AIMp1 and a high amount of intracellular CTLA-4, as can be seen inlane 5. To confirm this, flow cytometry was performed on dendritic cells either unloaded, loaded with just mRNA, just lysate, both, or mismatched antigens.FIG. 9 shows that CTLA4 retention is significantly enhanced in CD11c+CD80+CD83+CD86+ cells loaded with both the mRNA and homologous antigenic lysate. Further confirming enhanced CTLA4 retention by cells homologously loaded with antigenic lysate and mRNA generated by the methods provided herein, dendritic cell lysates and supernatant were probed for CLTA4 by western blot (FIG. 10 ). Loading with antigenic lysate and mRNA prepared by either method decreased the amount of CTLA4 present in the supernatant, indicating enhanced retention. - Homologous antigenic loading increases IL-12 transcripts in dendritic cells—Dendritic cells were evaluated for IL-12a and IL-12b production by RT-qPCR. As can be seen in
FIG. 11 , antigenic loading with both lysate and mRNA generated by the methods provided herein (TRIpure-IVT) increases both IL-12 transcripts better than loading with just lysate, mRNA, or heterologous loading, as well as increasing IL-12 transcript production better than loading with mRNA prepared by previous methods (Column-IVT). - Additional studies were performed demonstrating that amplified mRNA generates TH1 immune responses in vitro with the same efficiency as native poly-A mRNA when loaded into DC with homologous lysate. For example, results shown in
FIG. 13A show monocyte-derived human DC that were loaded with a) native poly A tumor mRNA and homologous/heterologous lysate or 2) capped IVT-amplified mRNA and homologous/heterologous lysate. DC were then cocultured with T cells and the percent of activated CD8+CD25+Ifng+ cells were analyzed by flow. T cells cocultured with unloaded DC or DC loaded with uncapped mRNA served as controls.FIG. 13B shows results with DC loaded with various combinations of poly A mRNA or IVT-amplified mRNA and homologous or heterologous cell lysates were matured for 48 hours, and intracellular CTLA4 levels were analyzed by flow cytometry. Singly loaded and unloaded DCs serve as controls. Upregulation of intracellular CTLA-4 is indicative of increased retention and therefore reduced secretion. UL-unloaded DC, uncapped mRNA-DC loaded with uncapped IVT mRNA, mRNA-DC loaded with poly A mRNA, lysate-DC loaded with lysate, 2× w/uncapped-DC loaded with uncapped IVT mRNA and homologous lysate, 2× capped-DC loaded with capped IVT mRNA and homologous lysate, MM uncapped-DC loaded with uncapped IVT mRNA and heterologous lysate, MM capped-DC loaded with capped IVT mRNA and heterologous lysate. - Methods of the embodiments were able to amplify sufficient amounts of mRNA for use in stimulating an immune response from very small amounts of starting tissue. Results presented in Table 3 below shows the amount to RNA that could be generated from a variety of starting samples.
-
TABLE 3 Additional preparation of nucleic acids from tumor tissues by the methods of the embodiments. Tissue Total Template ds- Template Capped Tumor Type Experiment in mg RNA Total RNA cDNA ds-cDNA IVT Primary PDAC 1 15 mg 1.4 ug 1.4 ug 41.0 ug 1 ug 45 ug Primary PDAC 2 10 mg 3.6 ug 3.0 ug 32.6 ug 1 ug 94 ug Primary 3 20 mg 1.4 ug 1.3 ug 22.6 ug 2 × 1 ug 300 ug Prostate Primary PDAC 4 10 mg 3.0 ug 3.0 ug 45.0 ug 1 ug 70 ug Dermal 5 80 mg 66.7 ug 5 ug 22.0 ug 2 × 2 ug 360 ug metastasis from HR-positive breast carcinoma Pleural cavity 6 60 mg 30.0 ug 4 ug 60.0 ug 2 × 5 ug 270 ug metastsis from poorly- differentiated head and neck sarcoma Bone metastasis 7 20 mg 4.0 ug 4 ug 21.3 ug 2 × 2.5 ug 240 ug from esophageal adenocarcinoma - All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
- The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
- Slagter-Jäger et al., “Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response,” Mol Ther Nucleic Acids, 2(5): e91, 2013.
Claims (28)
1. An oligonucleotide comprising the sequence of SEQ ID NO: 1.
2-9. (canceled)
10. A method of synthesizing a first cDNA strand from a template mRNA comprising the steps of:
a) hybridizing a first primer to the template mRNA, wherein the first primer comprises a nucleic acid sequence of SEQ ID NO: 2;
b) extending the first primer with a reverse transcriptase that has terminal transferase and template switching activity to generate a partial first cDNA strand with an oligo(C) overhang;
c) hybridizing a second primer to the oligo(C) overhang of the partial first cDNA strand, wherein the second primer comprises a nucleic acid sequence of SEQ ID NO: 1; and
d) extending the partial first cDNA strand from the oligo(C) overhang using the second primer as the template, thereby generating a first cDNA strand.
11. The method of claim 10 , further comprising:
e) separating the template mRNA and first cDNA strand;
f) hybridizing a third primer to the first cDNA strand; and
g) extending the third primer to generate a second cDNA strand, thereby generating a double stranded cDNA.
12. The method of claim 11 , wherein the second primer comprises a nucleic acid sequence of SEQ ID NO: 1.
13. The method of claim 11 , wherein the third primer comprises a nucleic acid sequence of SEQ ID NO: 3.
14. The method of claim 11 , wherein the template mRNA is obtained from a sample.
15. The method of claim 14 , wherein the sample is a tumor sample.
16-17. (canceled)
18. A method of preparing an RNA by transcribing a cDNA synthesized from an mRNA, the method comprising the steps of:
a) in an in vitro reaction mixture, hybridizing a first primer to the mRNA, wherein the first primer comprises both SEQ ID NO: 2 and a poly(T) sequence whereby the first primer anneals to the mRNA;
b) extending the first primer with a reverse transcriptase that has terminal transferase and template switching activity to generate a partial first cDNA strand with an oligo(C) overhang;
c) hybridizing a second primer comprising SEQ ID NO: 1 to the oligo(C) overhang of the partial first cDNA strand;
d) extending the partial first cDNA strand in the 3′ direction from the oligo(C) overhang using the second primer as a template, thereby generating a first cDNA strand;
e) hybridizing a third primer that comprises an RNA promoter to the first cDNA strand;
f) extending the third primer to generate a second cDNA strand, thereby generating a double stranded cDNA; and
g) synthesizing RNA from the double stranded cDNA.
19. The method of claim 18 , wherein the synthesizing RNA from the double stranded cDNA comprises in vitro transcription.
20-22. (canceled)
23. The method of claim 18 , wherein the method further comprises capping the RNA.
24. The method of claim 18 , further comprising amplifying the double stranded cDNA before step g).
25-26. (canceled)
27. The method of claim 24 , wherein amplifying the double stranded cDNA comprises adding a DNA dependent DNA polymerase, a fourth primer, and a fifth primer and amplifying the cDNA by polymerase chain reaction.
28. The method of claim 27 , wherein the fourth primer comprises the nucleic acid sequence of SEQ ID NO: 2.
29. The method of claim 27 , wherein the fifth primer comprises the nucleic acid sequence of SEQ ID NO: 3.
30. The method of claim 24 , further comprising in vitro transcribing the amplified cDNA to generate sense-strand amplified mRNA.
31-34. (canceled)
35. The method of claim 30 , further comprising capping the amplified RNA.
36. A method for transducing a dendritic cell population comprising contacting the dendritic cell population with a nucleic acid encoding one or more antigens, wherein the nucleic acid comprises RNA generated by the method of claim 18 .
37. The method of claim 36 , further comprising contacting the dendritic cell population with a tumor cell lysate.
38. (canceled)
39. A method for providing an immune response in a subject having a diseased cell population comprising:
a) obtaining a primed dendritic cell population produced by the method according to claim 37 ; and
b) administering an effective amount of the primed dendritic cell population to the subject.
40-44. (canceled)
45. A kit comprising an oligonucleotide primer of claim 1 , an oligonucleotide primer of SEQ ID NO:2 and/or an oligonucleotide primer of SEQ ID NO:3.
46-51. (canceled)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18/324,633 US20240026436A1 (en) | 2017-12-15 | 2023-05-26 | Methods and compositions for the amplification of mrna |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201762599472P | 2017-12-15 | 2017-12-15 | |
PCT/US2018/066051 WO2019118978A1 (en) | 2017-12-15 | 2018-12-17 | Methods and compositions for the amplification of mrna |
US202016772682A | 2020-06-12 | 2020-06-12 | |
US18/324,633 US20240026436A1 (en) | 2017-12-15 | 2023-05-26 | Methods and compositions for the amplification of mrna |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2018/066051 Division WO2019118978A1 (en) | 2017-12-15 | 2018-12-17 | Methods and compositions for the amplification of mrna |
US16/772,682 Division US11781178B2 (en) | 2017-12-15 | 2018-12-17 | Methods and compositions for the amplification of mRNA |
Publications (1)
Publication Number | Publication Date |
---|---|
US20240026436A1 true US20240026436A1 (en) | 2024-01-25 |
Family
ID=66819714
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/772,682 Active 2039-04-18 US11781178B2 (en) | 2017-12-15 | 2018-12-17 | Methods and compositions for the amplification of mRNA |
US18/324,633 Pending US20240026436A1 (en) | 2017-12-15 | 2023-05-26 | Methods and compositions for the amplification of mrna |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/772,682 Active 2039-04-18 US11781178B2 (en) | 2017-12-15 | 2018-12-17 | Methods and compositions for the amplification of mRNA |
Country Status (7)
Country | Link |
---|---|
US (2) | US11781178B2 (en) |
EP (1) | EP3724331A4 (en) |
JP (1) | JP2021508244A (en) |
CN (1) | CN111712572A (en) |
AU (1) | AU2018386331A1 (en) |
CA (1) | CA3085486A1 (en) |
WO (1) | WO2019118978A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11781178B2 (en) | 2017-12-15 | 2023-10-10 | Baylor College Of Medicine | Methods and compositions for the amplification of mRNA |
Family Cites Families (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5962271A (en) | 1996-01-03 | 1999-10-05 | Cloutech Laboratories, Inc. | Methods and compositions for generating full-length cDNA having arbitrary nucleotide sequence at the 3'-end |
EP1005481B1 (en) | 1997-04-22 | 2009-10-14 | Life Technologies Corporation | Methods for the production of aslv reverse transcriptases composed of multiple subunits |
US20050069938A1 (en) * | 2003-09-26 | 2005-03-31 | Youxiang Wang | Amplification of polynucleotides by rolling circle amplification |
WO2005052128A2 (en) * | 2003-11-25 | 2005-06-09 | Argos Therapeutics, Inc. | Mrna transfected antigen presenting cells |
EP2222344A4 (en) | 2007-11-30 | 2012-11-07 | Baylor College Medicine | Dendritic cell vaccine compositions and uses of same |
CN101377021A (en) | 2008-09-25 | 2009-03-04 | 海南大学 | Method for constructing cDNA library |
WO2010065876A2 (en) | 2008-12-06 | 2010-06-10 | The Board Of Regents Of The University Of Texas System | Methods and compositions related to th-1 dendritic cells |
CA3172119A1 (en) * | 2009-12-28 | 2011-07-07 | Evogene Ltd. | Isolated polynucleotides and polypeptides and methods of using same for increasing plant yield, biomass, growth rate, vigor, oil content, abiotic stress tolerance of plants and nitrogen use efficiency |
EP2566518A4 (en) | 2010-05-07 | 2013-12-25 | Baylor Res Inst | Dendritic cell immunoreceptors (dcir)-mediated crosspriming of human cd8+ t cells |
CA2799453A1 (en) | 2010-06-04 | 2011-12-08 | Pioneer Hi-Bred International, Inc. | Compositions and methods for insecticidal control of stinkbugs |
WO2013029076A1 (en) * | 2011-08-26 | 2013-03-07 | Universität Graz | Enzymatic alkene cleavage |
LT2756098T (en) * | 2011-09-16 | 2018-09-10 | Lexogen Gmbh | Method for making library of nucleic acid molecules |
AU2016259020B2 (en) * | 2015-05-07 | 2021-12-09 | Baylor College Of Medicine | Dendritic cell immunotherapy |
IL299926A (en) * | 2016-03-31 | 2023-03-01 | Biontech Us Inc | Neoantigens and methods of their use |
US11781178B2 (en) | 2017-12-15 | 2023-10-10 | Baylor College Of Medicine | Methods and compositions for the amplification of mRNA |
-
2018
- 2018-12-17 US US16/772,682 patent/US11781178B2/en active Active
- 2018-12-17 EP EP18888097.5A patent/EP3724331A4/en active Pending
- 2018-12-17 CN CN201880087136.7A patent/CN111712572A/en active Pending
- 2018-12-17 CA CA3085486A patent/CA3085486A1/en active Pending
- 2018-12-17 JP JP2020532802A patent/JP2021508244A/en active Pending
- 2018-12-17 WO PCT/US2018/066051 patent/WO2019118978A1/en unknown
- 2018-12-17 AU AU2018386331A patent/AU2018386331A1/en active Pending
-
2023
- 2023-05-26 US US18/324,633 patent/US20240026436A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
AU2018386331A1 (en) | 2020-07-02 |
CA3085486A1 (en) | 2019-06-20 |
US20200308638A1 (en) | 2020-10-01 |
JP2021508244A (en) | 2021-03-04 |
EP3724331A4 (en) | 2021-09-01 |
EP3724331A1 (en) | 2020-10-21 |
WO2019118978A1 (en) | 2019-06-20 |
US11781178B2 (en) | 2023-10-10 |
CN111712572A (en) | 2020-09-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220213440A1 (en) | Dendritic cell immunotherapy | |
WO2018112032A1 (en) | Methods and compositions for targeting tumor-infiltrating tregs using inhibitors of ccr8 and tnfrsf8 | |
US20210179687A1 (en) | Targeting lilrb4 with car-t or car-nk cells in the treatment of cancer | |
US20240026436A1 (en) | Methods and compositions for the amplification of mrna | |
JP2024510505A (en) | Methods for tumor-infiltrating lymphocyte (TIL) expansion and gene knockout in TILs associated with CD39/CD69 selection | |
CA3215633A1 (en) | Chimeric antigen receptor-modified granulocyte-macrophage progenitors for cancer immunotherapy | |
WO2018013589A1 (en) | Methods and compositions for thymic transplantation | |
WO2023092097A1 (en) | Fragment consensus methods for ultrasensitive detection of aberrant methylation | |
CN115461461A (en) | MiRNA-193a for promoting immunogenic cell death | |
EP3965831A2 (en) | Targeting otub1 in immunotherapy | |
US20220392638A1 (en) | Precision enrichment of pathology specimens | |
NL2019548B1 (en) | T-cell based immunotherapy | |
WO2018013585A1 (en) | Methods and compositions for modulating thymic function | |
JP5210303B2 (en) | How to manipulate stem cells | |
US20220017900A1 (en) | Methods of cancer treatment | |
CA3234457A1 (en) | Natural killer cells and methods of use thereof | |
WO2023211921A1 (en) | Use of spop mutations as a predictive biomarker | |
TW202128196A (en) | Methods of treatment using a genetically modified autologous t cell immunotherapy | |
Mertens | Chemical modifications of DNA activate the cGAS/STING-signaling pathway even in the presence of the cytosolic exonuclease TREX1 | |
Dorrian et al. | Graft-versus-host Disease Presenting Along Blaschko Lines: Cutaneous Mosaicism | |
WO2023245041A2 (en) | Enhancing the activity of cellular therapies in the tumor microenvironment | |
WO2023137447A1 (en) | Alk gene fusions and uses thereof | |
Nishimura et al. | Down-regulation by a new anti-inflammatory compound, FR167653, of differentiation and maturation of human monocytes and bone marrow CD34+ cells to dendritic cells |