WO2023240225A1 - Constitutively active polymeric sting mimics for antitumor immunity - Google Patents
Constitutively active polymeric sting mimics for antitumor immunity Download PDFInfo
- Publication number
- WO2023240225A1 WO2023240225A1 PCT/US2023/068182 US2023068182W WO2023240225A1 WO 2023240225 A1 WO2023240225 A1 WO 2023240225A1 US 2023068182 W US2023068182 W US 2023068182W WO 2023240225 A1 WO2023240225 A1 WO 2023240225A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cancer
- unisting
- mrna
- cells
- polynucleotide
- Prior art date
Links
- 230000005809 anti-tumor immunity Effects 0.000 title description 12
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 185
- 239000000203 mixture Substances 0.000 claims abstract description 86
- 239000002157 polynucleotide Substances 0.000 claims abstract description 70
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 70
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 70
- 201000011510 cancer Diseases 0.000 claims abstract description 65
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 39
- 229960005486 vaccine Drugs 0.000 claims abstract description 37
- 201000010099 disease Diseases 0.000 claims abstract description 22
- 230000028993 immune response Effects 0.000 claims abstract description 19
- 208000035475 disorder Diseases 0.000 claims abstract description 17
- 108020004999 messenger RNA Proteins 0.000 claims description 114
- 101000643024 Homo sapiens Stimulator of interferon genes protein Proteins 0.000 claims description 109
- 102100035533 Stimulator of interferon genes protein Human genes 0.000 claims description 105
- 108090000623 proteins and genes Proteins 0.000 claims description 74
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 69
- 238000000034 method Methods 0.000 claims description 67
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 59
- 229920001184 polypeptide Polymers 0.000 claims description 56
- -1 cationic lipid Chemical class 0.000 claims description 52
- 102000004169 proteins and genes Human genes 0.000 claims description 48
- 239000003814 drug Substances 0.000 claims description 38
- 230000001086 cytosolic effect Effects 0.000 claims description 32
- 238000012384 transportation and delivery Methods 0.000 claims description 30
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 29
- 210000004899 c-terminal region Anatomy 0.000 claims description 28
- 150000002632 lipids Chemical class 0.000 claims description 28
- 230000002601 intratumoral effect Effects 0.000 claims description 26
- 208000026310 Breast neoplasm Diseases 0.000 claims description 23
- 206010006187 Breast cancer Diseases 0.000 claims description 22
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 21
- 125000003729 nucleotide group Chemical group 0.000 claims description 19
- 108020004414 DNA Proteins 0.000 claims description 14
- 239000002671 adjuvant Substances 0.000 claims description 14
- 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 claims description 14
- 239000002105 nanoparticle Substances 0.000 claims description 13
- 239000002981 blocking agent Substances 0.000 claims description 12
- 229920001223 polyethylene glycol Polymers 0.000 claims description 11
- 239000002202 Polyethylene glycol Substances 0.000 claims description 9
- 208000014018 liver neoplasm Diseases 0.000 claims description 9
- 230000001939 inductive effect Effects 0.000 claims description 8
- 230000002452 interceptive effect Effects 0.000 claims description 8
- 201000007270 liver cancer Diseases 0.000 claims description 8
- 208000020816 lung neoplasm Diseases 0.000 claims description 8
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 8
- 150000003904 phospholipids Chemical class 0.000 claims description 8
- 238000007910 systemic administration Methods 0.000 claims description 8
- 229930182558 Sterol Natural products 0.000 claims description 7
- 235000012000 cholesterol Nutrition 0.000 claims description 7
- 230000003053 immunization Effects 0.000 claims description 7
- 238000002649 immunization Methods 0.000 claims description 7
- 230000004048 modification Effects 0.000 claims description 7
- 238000012986 modification Methods 0.000 claims description 7
- 235000003702 sterols Nutrition 0.000 claims description 7
- 108020005345 3' Untranslated Regions Proteins 0.000 claims description 6
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 6
- 208000000453 Skin Neoplasms Diseases 0.000 claims description 6
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical class O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 6
- 201000005202 lung cancer Diseases 0.000 claims description 6
- 230000007935 neutral effect Effects 0.000 claims description 6
- 201000000849 skin cancer Diseases 0.000 claims description 6
- 150000003432 sterols Chemical class 0.000 claims description 6
- 108091026898 Leader sequence (mRNA) Proteins 0.000 claims description 5
- 108091023045 Untranslated Region Proteins 0.000 claims description 5
- 108060003951 Immunoglobulin Proteins 0.000 claims description 3
- 150000002148 esters Chemical class 0.000 claims description 3
- 102000018358 immunoglobulin Human genes 0.000 claims description 3
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 claims description 2
- 230000004936 stimulating effect Effects 0.000 abstract description 3
- 101710196623 Stimulator of interferon genes protein Proteins 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 135
- 101000941029 Homo sapiens Endoplasmic reticulum junction formation protein lunapark Proteins 0.000 description 88
- 101000991410 Homo sapiens Nucleolar and spindle-associated protein 1 Proteins 0.000 description 88
- 102100030991 Nucleolar and spindle-associated protein 1 Human genes 0.000 description 88
- 238000011282 treatment Methods 0.000 description 79
- 230000014509 gene expression Effects 0.000 description 65
- 210000004881 tumor cell Anatomy 0.000 description 59
- 241000699670 Mus sp. Species 0.000 description 55
- 235000001014 amino acid Nutrition 0.000 description 55
- 229940024606 amino acid Drugs 0.000 description 54
- 108091070501 miRNA Proteins 0.000 description 53
- 239000002679 microRNA Substances 0.000 description 53
- 150000001413 amino acids Chemical class 0.000 description 48
- 235000018102 proteins Nutrition 0.000 description 44
- 230000004083 survival effect Effects 0.000 description 34
- XRILCFTWUCUKJR-INFSMZHSSA-N 2'-3'-cGAMP Chemical compound C([C@H]([C@H]1O)O2)OP(O)(=O)O[C@H]3[C@@H](O)[C@H](N4C5=NC=NC(N)=C5N=C4)O[C@@H]3COP(O)(=O)O[C@H]1[C@@H]2N1C=NC2=C1NC(N)=NC2=O XRILCFTWUCUKJR-INFSMZHSSA-N 0.000 description 31
- 239000002953 phosphate buffered saline Substances 0.000 description 30
- 210000001744 T-lymphocyte Anatomy 0.000 description 29
- 230000011664 signaling Effects 0.000 description 29
- 230000004913 activation Effects 0.000 description 28
- 238000009472 formulation Methods 0.000 description 28
- 238000004458 analytical method Methods 0.000 description 26
- 101001011382 Homo sapiens Interferon regulatory factor 3 Proteins 0.000 description 24
- 102100029843 Interferon regulatory factor 3 Human genes 0.000 description 24
- 238000002560 therapeutic procedure Methods 0.000 description 23
- 150000007523 nucleic acids Chemical class 0.000 description 21
- 230000004614 tumor growth Effects 0.000 description 20
- 210000001519 tissue Anatomy 0.000 description 19
- 230000000694 effects Effects 0.000 description 17
- 102000039446 nucleic acids Human genes 0.000 description 17
- 108020004707 nucleic acids Proteins 0.000 description 17
- 102100025248 C-X-C motif chemokine 10 Human genes 0.000 description 15
- 101000858088 Homo sapiens C-X-C motif chemokine 10 Proteins 0.000 description 15
- 238000000684 flow cytometry Methods 0.000 description 15
- 125000005647 linker group Chemical group 0.000 description 15
- 239000002773 nucleotide Substances 0.000 description 15
- 230000004044 response Effects 0.000 description 15
- 238000001890 transfection Methods 0.000 description 15
- 230000000259 anti-tumor effect Effects 0.000 description 14
- 239000000872 buffer Substances 0.000 description 14
- 239000007924 injection Substances 0.000 description 14
- 238000002347 injection Methods 0.000 description 14
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 13
- 239000003112 inhibitor Substances 0.000 description 13
- 108091040751 miR-130a stem-loop Proteins 0.000 description 13
- 108091041344 miR-130a-1 stem-loop Proteins 0.000 description 13
- 108091091365 miR-130a-2 stem-loop Proteins 0.000 description 13
- 108091028466 miR-130b stem-loop Proteins 0.000 description 13
- 108091026034 miR-130b-1 stem-loop Proteins 0.000 description 13
- 108091025972 miR-130b-2 stem-loop Proteins 0.000 description 13
- 239000004055 small Interfering RNA Substances 0.000 description 13
- 229940044665 STING agonist Drugs 0.000 description 12
- 239000012091 fetal bovine serum Substances 0.000 description 12
- 230000006870 function Effects 0.000 description 12
- 238000001727 in vivo Methods 0.000 description 12
- 238000011201 multiple comparisons test Methods 0.000 description 12
- 238000001543 one-way ANOVA Methods 0.000 description 12
- 230000037361 pathway Effects 0.000 description 12
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 11
- 241001529936 Murinae Species 0.000 description 11
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 11
- 108020004459 Small interfering RNA Proteins 0.000 description 11
- 239000002246 antineoplastic agent Substances 0.000 description 11
- 239000002585 base Substances 0.000 description 11
- 239000003636 conditioned culture medium Substances 0.000 description 11
- 230000001404 mediated effect Effects 0.000 description 11
- 239000012528 membrane Substances 0.000 description 11
- 230000003278 mimic effect Effects 0.000 description 11
- 230000001225 therapeutic effect Effects 0.000 description 11
- 108010050904 Interferons Proteins 0.000 description 10
- 102000014150 Interferons Human genes 0.000 description 10
- 208000006552 Lewis Lung Carcinoma Diseases 0.000 description 10
- 238000003559 RNA-seq method Methods 0.000 description 10
- 239000003795 chemical substances by application Substances 0.000 description 10
- 238000009169 immunotherapy Methods 0.000 description 10
- 210000004185 liver Anatomy 0.000 description 10
- 206010061289 metastatic neoplasm Diseases 0.000 description 10
- 238000006467 substitution reaction Methods 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 239000013598 vector Substances 0.000 description 10
- 102100032912 CD44 antigen Human genes 0.000 description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 9
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 9
- 101000665442 Homo sapiens Serine/threonine-protein kinase TBK1 Proteins 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 description 9
- 102100038192 Serine/threonine-protein kinase TBK1 Human genes 0.000 description 9
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 9
- 230000003247 decreasing effect Effects 0.000 description 9
- 229940079322 interferon Drugs 0.000 description 9
- 108091050874 miR-19a stem-loop Proteins 0.000 description 9
- 241000239290 Araneae Species 0.000 description 8
- 108091006146 Channels Proteins 0.000 description 8
- 102100031256 Cyclic GMP-AMP synthase Human genes 0.000 description 8
- 108030002637 Cyclic GMP-AMP synthases Proteins 0.000 description 8
- 102000004127 Cytokines Human genes 0.000 description 8
- 108090000695 Cytokines Proteins 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 108091007767 MALAT1 Proteins 0.000 description 8
- 230000027455 binding Effects 0.000 description 8
- 229940127089 cytotoxic agent Drugs 0.000 description 8
- 230000001419 dependent effect Effects 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 230000015654 memory Effects 0.000 description 8
- 239000013642 negative control Substances 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 7
- 239000012980 RPMI-1640 medium Substances 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 125000000539 amino acid group Chemical group 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 238000007385 chemical modification Methods 0.000 description 7
- 230000003828 downregulation Effects 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 238000010199 gene set enrichment analysis Methods 0.000 description 7
- 238000003384 imaging method Methods 0.000 description 7
- 238000001990 intravenous administration Methods 0.000 description 7
- 108091068975 miR-19a-3 stem-loop Proteins 0.000 description 7
- 230000002829 reductive effect Effects 0.000 description 7
- 238000007920 subcutaneous administration Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 6
- 108020004705 Codon Proteins 0.000 description 6
- 102000001398 Granzyme Human genes 0.000 description 6
- 108060005986 Granzyme Proteins 0.000 description 6
- 101001018097 Homo sapiens L-selectin Proteins 0.000 description 6
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 6
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 6
- 102100033467 L-selectin Human genes 0.000 description 6
- 108060001084 Luciferase Proteins 0.000 description 6
- 239000005089 Luciferase Substances 0.000 description 6
- 101000643023 Mus musculus Stimulator of interferon genes protein Proteins 0.000 description 6
- 108010058846 Ovalbumin Proteins 0.000 description 6
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 6
- 238000007792 addition Methods 0.000 description 6
- 239000012298 atmosphere Substances 0.000 description 6
- 230000029918 bioluminescence Effects 0.000 description 6
- 238000005415 bioluminescence Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 210000000481 breast Anatomy 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 230000000875 corresponding effect Effects 0.000 description 6
- 231100000433 cytotoxic Toxicity 0.000 description 6
- 230000001472 cytotoxic effect Effects 0.000 description 6
- 239000013604 expression vector Substances 0.000 description 6
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 238000007918 intramuscular administration Methods 0.000 description 6
- 238000007912 intraperitoneal administration Methods 0.000 description 6
- 108091027943 miR-16 stem-loop Proteins 0.000 description 6
- 239000002777 nucleoside Substances 0.000 description 6
- 229940092253 ovalbumin Drugs 0.000 description 6
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 6
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 6
- 239000011591 potassium Substances 0.000 description 6
- 229910052700 potassium Inorganic materials 0.000 description 6
- 230000019491 signal transduction Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 210000000952 spleen Anatomy 0.000 description 6
- 230000009885 systemic effect Effects 0.000 description 6
- 238000013519 translation Methods 0.000 description 6
- 238000007492 two-way ANOVA Methods 0.000 description 6
- 238000005199 ultracentrifugation Methods 0.000 description 6
- 239000003981 vehicle Substances 0.000 description 6
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 5
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 5
- 239000004475 Arginine Substances 0.000 description 5
- 102100027207 CD27 antigen Human genes 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 5
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 5
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical group C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 5
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 5
- 102000003814 Interleukin-10 Human genes 0.000 description 5
- 108090000174 Interleukin-10 Proteins 0.000 description 5
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 5
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 5
- 239000004472 Lysine Substances 0.000 description 5
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 description 5
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 5
- 230000006023 anti-tumor response Effects 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 5
- 125000002091 cationic group Chemical group 0.000 description 5
- 230000000973 chemotherapeutic effect Effects 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- 238000003119 immunoblot Methods 0.000 description 5
- 230000003308 immunostimulating effect Effects 0.000 description 5
- 230000001976 improved effect Effects 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 230000026731 phosphorylation Effects 0.000 description 5
- 238000006366 phosphorylation reaction Methods 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 230000000770 proinflammatory effect Effects 0.000 description 5
- 230000002035 prolonged effect Effects 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 208000022679 triple-negative breast carcinoma Diseases 0.000 description 5
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 4
- 108020003589 5' Untranslated Regions Proteins 0.000 description 4
- 229940126253 ADU-S100 Drugs 0.000 description 4
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 4
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 4
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 4
- 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 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 102100022057 Hepatocyte nuclear factor 1-alpha Human genes 0.000 description 4
- 101001045751 Homo sapiens Hepatocyte nuclear factor 1-alpha Proteins 0.000 description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 4
- 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 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 4
- 102100040247 Tumor necrosis factor Human genes 0.000 description 4
- 102000013814 Wnt Human genes 0.000 description 4
- 108050003627 Wnt Proteins 0.000 description 4
- 239000012190 activator Substances 0.000 description 4
- 229960005305 adenosine Drugs 0.000 description 4
- 229960003767 alanine Drugs 0.000 description 4
- 229960002932 anastrozole Drugs 0.000 description 4
- YBBLVLTVTVSKRW-UHFFFAOYSA-N anastrozole Chemical compound N#CC(C)(C)C1=CC(C(C)(C#N)C)=CC(CN2N=CN=C2)=C1 YBBLVLTVTVSKRW-UHFFFAOYSA-N 0.000 description 4
- 235000009582 asparagine Nutrition 0.000 description 4
- 229960001230 asparagine Drugs 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 239000013592 cell lysate Substances 0.000 description 4
- 238000000749 co-immunoprecipitation Methods 0.000 description 4
- 210000001072 colon Anatomy 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 239000000562 conjugate Substances 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 210000001808 exosome Anatomy 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- 102000037865 fusion proteins Human genes 0.000 description 4
- 230000030279 gene silencing Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 4
- 102000050022 human STING1 Human genes 0.000 description 4
- 230000001506 immunosuppresive effect Effects 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 201000001441 melanoma Diseases 0.000 description 4
- 210000003071 memory t lymphocyte Anatomy 0.000 description 4
- 229960000485 methotrexate Drugs 0.000 description 4
- 108091031326 miR-15b stem-loop Proteins 0.000 description 4
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 4
- 125000003835 nucleoside group Chemical group 0.000 description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 4
- 210000002307 prostate Anatomy 0.000 description 4
- 229940096913 pseudoisocytidine Drugs 0.000 description 4
- 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 4
- 229920005989 resin Polymers 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 150000003384 small molecules Chemical class 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 229960005026 toremifene Drugs 0.000 description 4
- XFCLJVABOIYOMF-QPLCGJKRSA-N toremifene Chemical compound C1=CC(OCCN(C)C)=CC=C1C(\C=1C=CC=CC=1)=C(\CCCl)C1=CC=CC=C1 XFCLJVABOIYOMF-QPLCGJKRSA-N 0.000 description 4
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 3
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 3
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 3
- 229930024421 Adenine Natural products 0.000 description 3
- OIRDTQYFTABQOQ-KQYNXXCUSA-N Adenosine Natural products C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 3
- 102100036170 C-X-C motif chemokine 9 Human genes 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- 102100027221 CD81 antigen Human genes 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- 150000008574 D-amino acids Chemical class 0.000 description 3
- 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 3
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 3
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 241000206602 Eukaryota Species 0.000 description 3
- 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 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 3
- 108020005004 Guide RNA Proteins 0.000 description 3
- 101000947172 Homo sapiens C-X-C motif chemokine 9 Proteins 0.000 description 3
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 3
- 101001010600 Homo sapiens Interleukin-12 subunit alpha Proteins 0.000 description 3
- 101000813777 Homo sapiens Splicing factor ESS-2 homolog Proteins 0.000 description 3
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 102100030698 Interleukin-12 subunit alpha Human genes 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 102100025354 Macrophage mannose receptor 1 Human genes 0.000 description 3
- 108010031099 Mannose Receptor Proteins 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 229930012538 Paclitaxel Natural products 0.000 description 3
- 108091093037 Peptide nucleic acid Proteins 0.000 description 3
- 108091036407 Polyadenylation Proteins 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 3
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 3
- 102000006382 Ribonucleases Human genes 0.000 description 3
- 108010083644 Ribonucleases Proteins 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- 108091027967 Small hairpin RNA Proteins 0.000 description 3
- 108091081024 Start codon Proteins 0.000 description 3
- 230000005867 T cell response Effects 0.000 description 3
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 3
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000013543 active substance Substances 0.000 description 3
- 229960000643 adenine Drugs 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 125000001931 aliphatic group Chemical group 0.000 description 3
- QWCKQJZIFLGMSD-UHFFFAOYSA-N alpha-aminobutyric acid Chemical compound CCC(N)C(O)=O QWCKQJZIFLGMSD-UHFFFAOYSA-N 0.000 description 3
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 3
- 150000001412 amines Chemical class 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 239000000074 antisense oligonucleotide Substances 0.000 description 3
- 238000012230 antisense oligonucleotides Methods 0.000 description 3
- 238000003491 array Methods 0.000 description 3
- 125000003118 aryl group Chemical group 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000004422 calculation algorithm Methods 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 238000000942 confocal micrograph Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000016396 cytokine production Effects 0.000 description 3
- 210000000172 cytosol Anatomy 0.000 description 3
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 3
- 210000004443 dendritic cell Anatomy 0.000 description 3
- 239000002552 dosage form Substances 0.000 description 3
- 230000007783 downstream signaling Effects 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 150000004665 fatty acids Chemical group 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 229960003180 glutathione Drugs 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 229960001438 immunostimulant agent Drugs 0.000 description 3
- 239000003022 immunostimulating agent Substances 0.000 description 3
- 230000005917 in vivo anti-tumor Effects 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 108091074057 miR-16-1 stem-loop Proteins 0.000 description 3
- 108091056204 miR-16-2 stem-loop Proteins 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 150000003833 nucleoside derivatives Chemical class 0.000 description 3
- 229960001592 paclitaxel Drugs 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- 238000010837 poor prognosis Methods 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 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 3
- 230000000699 topical effect Effects 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 230000002103 transcriptional effect Effects 0.000 description 3
- 239000012096 transfection reagent Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 3
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 3
- 229940045145 uridine Drugs 0.000 description 3
- STUWGJZDJHPWGZ-LBPRGKRZSA-N (2S)-N1-[4-methyl-5-[2-(1,1,1-trifluoro-2-methylpropan-2-yl)-4-pyridinyl]-2-thiazolyl]pyrrolidine-1,2-dicarboxamide Chemical compound S1C(C=2C=C(N=CC=2)C(C)(C)C(F)(F)F)=C(C)N=C1NC(=O)N1CCC[C@H]1C(N)=O STUWGJZDJHPWGZ-LBPRGKRZSA-N 0.000 description 2
- MWWSFMDVAYGXBV-FGBSZODSSA-N (7s,9s)-7-[(2r,4s,5r,6s)-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;hydron;chloride 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)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 MWWSFMDVAYGXBV-FGBSZODSSA-N 0.000 description 2
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 2
- CITHEXJVPOWHKC-UUWRZZSWSA-N 1,2-di-O-myristoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCC CITHEXJVPOWHKC-UUWRZZSWSA-N 0.000 description 2
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 2
- UVBYMVOUBXYSFV-XUTVFYLZSA-N 1-methylpseudouridine Chemical compound O=C1NC(=O)N(C)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 UVBYMVOUBXYSFV-XUTVFYLZSA-N 0.000 description 2
- UVBYMVOUBXYSFV-UHFFFAOYSA-N 1-methylpseudouridine Natural products O=C1NC(=O)N(C)C=C1C1C(O)C(O)C(CO)O1 UVBYMVOUBXYSFV-UHFFFAOYSA-N 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- SDEAXTCZPQIFQM-UHFFFAOYSA-N 6-n-(4,4-dimethyl-5h-1,3-oxazol-2-yl)-4-n-[3-methyl-4-([1,2,4]triazolo[1,5-a]pyridin-7-yloxy)phenyl]quinazoline-4,6-diamine Chemical compound C=1C=C(OC2=CC3=NC=NN3C=C2)C(C)=CC=1NC(C1=C2)=NC=NC1=CC=C2NC1=NC(C)(C)CO1 SDEAXTCZPQIFQM-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
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 2
- RHXHGRAEPCAFML-UHFFFAOYSA-N 7-cyclopentyl-n,n-dimethyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide Chemical compound N1=C2N(C3CCCC3)C(C(=O)N(C)C)=CC2=CN=C1NC(N=C1)=CC=C1N1CCNCC1 RHXHGRAEPCAFML-UHFFFAOYSA-N 0.000 description 2
- PEHVGBZKEYRQSX-UHFFFAOYSA-N 7-deaza-adenine Chemical compound NC1=NC=NC2=C1C=CN2 PEHVGBZKEYRQSX-UHFFFAOYSA-N 0.000 description 2
- HCGHYQLFMPXSDU-UHFFFAOYSA-N 7-methyladenine Chemical compound C1=NC(N)=C2N(C)C=NC2=N1 HCGHYQLFMPXSDU-UHFFFAOYSA-N 0.000 description 2
- OGHAROSJZRTIOK-KQYNXXCUSA-O 7-methylguanosine Chemical compound C1=2N=C(N)NC(=O)C=2[N+](C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OGHAROSJZRTIOK-KQYNXXCUSA-O 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108091023037 Aptamer Proteins 0.000 description 2
- 101100107610 Arabidopsis thaliana ABCF4 gene Proteins 0.000 description 2
- BFYIZQONLCFLEV-DAELLWKTSA-N Aromasine Chemical compound O=C1C=C[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC(=C)C2=C1 BFYIZQONLCFLEV-DAELLWKTSA-N 0.000 description 2
- 241000193755 Bacillus cereus Species 0.000 description 2
- 108010006654 Bleomycin Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 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
- 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 2
- 201000009030 Carcinoma Diseases 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 241000701489 Cauliflower mosaic virus Species 0.000 description 2
- 108010049048 Cholera Toxin Proteins 0.000 description 2
- 102000009016 Cholera Toxin Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 2
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 2
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 2
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 2
- 241000194033 Enterococcus Species 0.000 description 2
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 2
- VWUXBMIQPBEWFH-WCCTWKNTSA-N Fulvestrant Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3[C@H](CCCCCCCCCS(=O)CCCC(F)(F)C(F)(F)F)CC2=C1 VWUXBMIQPBEWFH-WCCTWKNTSA-N 0.000 description 2
- 241000223195 Fusarium graminearum Species 0.000 description 2
- 241000237858 Gastropoda Species 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- BLCLNMBMMGCOAS-URPVMXJPSA-N Goserelin Chemical compound C([C@@H](C(=O)N[C@H](COC(C)(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(=O)NNC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 BLCLNMBMMGCOAS-URPVMXJPSA-N 0.000 description 2
- 108010069236 Goserelin Proteins 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 101000801359 Homo sapiens Acetylcholinesterase Proteins 0.000 description 2
- 101000943274 Homo sapiens Cholinesterase Proteins 0.000 description 2
- 101000959820 Homo sapiens Interferon alpha-1/13 Proteins 0.000 description 2
- 101100462513 Homo sapiens TP53 gene Proteins 0.000 description 2
- 101000750302 Homo sapiens Vasodilator-stimulated phosphoprotein Proteins 0.000 description 2
- 101900134826 Human metapneumovirus Phosphoprotein Proteins 0.000 description 2
- 102000026633 IL6 Human genes 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical class [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 108010011185 KCNQ1 Potassium Channel Proteins 0.000 description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 2
- 150000008575 L-amino acids Chemical class 0.000 description 2
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- 239000002136 L01XE07 - Lapatinib Substances 0.000 description 2
- 206010025323 Lymphomas Diseases 0.000 description 2
- 108010090665 Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase Proteins 0.000 description 2
- 101900303553 Measles virus Phosphoprotein Proteins 0.000 description 2
- 206010027406 Mesothelioma Diseases 0.000 description 2
- 208000034578 Multiple myelomas Diseases 0.000 description 2
- 241000711408 Murine respirovirus Species 0.000 description 2
- 101100043703 Mus musculus Sting1 gene Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- 102100030397 N-acetylmuramoyl-L-alanine amidase Human genes 0.000 description 2
- 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 2
- 206010061309 Neoplasm progression Diseases 0.000 description 2
- 101900205472 Newcastle disease virus Hemagglutinin-neuraminidase Proteins 0.000 description 2
- 101001002820 Nipah virus Phosphoprotein Proteins 0.000 description 2
- 108010089430 Phosphoproteins Proteins 0.000 description 2
- 102000007982 Phosphoproteins Human genes 0.000 description 2
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 102100037444 Potassium voltage-gated channel subfamily KQT member 1 Human genes 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 230000026279 RNA modification Effects 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 102000004389 Ribonucleoproteins Human genes 0.000 description 2
- 108010081734 Ribonucleoproteins Proteins 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 101100068078 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GCN4 gene Proteins 0.000 description 2
- 206010039491 Sarcoma Diseases 0.000 description 2
- 201000010208 Seminoma Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 241000205077 Staphylothermus marinus Species 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 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 2
- 108091046869 Telomeric non-coding RNA Proteins 0.000 description 2
- 108020005038 Terminator Codon Proteins 0.000 description 2
- FOCVUCIESVLUNU-UHFFFAOYSA-N Thiotepa Chemical compound C1CN1P(N1CC1)(=S)N1CC1 FOCVUCIESVLUNU-UHFFFAOYSA-N 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 102000002689 Toll-like receptor Human genes 0.000 description 2
- 108020000411 Toll-like receptor Proteins 0.000 description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 2
- 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 2
- YGPZYYDTPXVBRA-RTDBHSBRSA-N [(2r,3s,4r,5r,6s)-2-[[(2r,3r,4r,5s,6r)-3-[[(3r)-3-dodecanoyloxytetradecanoyl]amino]-6-(hydroxymethyl)-5-phosphonooxy-4-[(3r)-3-tetradecanoyloxytetradecanoyl]oxyoxan-2-yl]oxymethyl]-3,6-dihydroxy-5-[[(3r)-3-hydroxytetradecanoyl]amino]oxan-4-yl] (3r)-3-hydr Chemical compound O1[C@H](CO)[C@@H](OP(O)(O)=O)[C@H](OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC)[C@@H](NC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCC)[C@@H]1OC[C@@H]1[C@@H](O)[C@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](O)O1 YGPZYYDTPXVBRA-RTDBHSBRSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- MBMBGCFOFBJSGT-KUBAVDMBSA-N all-cis-docosa-4,7,10,13,16,19-hexaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 description 2
- 229950010482 alpelisib Drugs 0.000 description 2
- 125000000266 alpha-aminoacyl group Chemical group 0.000 description 2
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 229960001561 bleomycin Drugs 0.000 description 2
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 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)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 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 229940022399 cancer vaccine Drugs 0.000 description 2
- 238000009566 cancer vaccine Methods 0.000 description 2
- 229960004117 capecitabine Drugs 0.000 description 2
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 238000009172 cell transfer therapy Methods 0.000 description 2
- 210000003679 cervix uteri Anatomy 0.000 description 2
- HWGQMRYQVZSGDQ-HZPDHXFCSA-N chembl3137320 Chemical compound CN1N=CN=C1[C@H]([C@H](N1)C=2C=CC(F)=CC=2)C2=NNC(=O)C3=C2C1=CC(F)=C3 HWGQMRYQVZSGDQ-HZPDHXFCSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 2
- 229960004316 cisplatin Drugs 0.000 description 2
- 230000008045 co-localization Effects 0.000 description 2
- 238000011278 co-treatment Methods 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 238000004891 communication Methods 0.000 description 2
- FPUGCISOLXNPPC-IOSLPCCCSA-N cordysinin B Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(N)=C2N=C1 FPUGCISOLXNPPC-IOSLPCCCSA-N 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 229960004397 cyclophosphamide Drugs 0.000 description 2
- 229960000684 cytarabine Drugs 0.000 description 2
- 210000000805 cytoplasm Anatomy 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
- 229960000975 daunorubicin Drugs 0.000 description 2
- 238000012350 deep sequencing Methods 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 239000005547 deoxyribonucleotide Substances 0.000 description 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229960003724 dimyristoylphosphatidylcholine Drugs 0.000 description 2
- 229960005160 dimyristoylphosphatidylglycerol Drugs 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- BPHQZTVXXXJVHI-AJQTZOPKSA-N ditetradecanoyl phosphatidylglycerol Chemical compound CCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@@H](O)CO)OC(=O)CCCCCCCCCCCCC BPHQZTVXXXJVHI-AJQTZOPKSA-N 0.000 description 2
- 229960003668 docetaxel Drugs 0.000 description 2
- UKMSUNONTOPOIO-UHFFFAOYSA-N docosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCC(O)=O UKMSUNONTOPOIO-UHFFFAOYSA-N 0.000 description 2
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 2
- 229960004679 doxorubicin Drugs 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 210000003162 effector t lymphocyte Anatomy 0.000 description 2
- 210000004696 endometrium Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229960003265 epirubicin hydrochloride Drugs 0.000 description 2
- 229960000439 eribulin mesylate Drugs 0.000 description 2
- QAMYWGZHLCQOOJ-PWIVHLLHSA-N eribulin mesylate Chemical compound CS(O)(=O)=O.C([C@H]1CC[C@@H]2O[C@@H]3[C@H]4O[C@H]5C[C@](O[C@H]4[C@H]2O1)(O[C@@H]53)CC[C@@H]1O[C@H](C(C1)=C)CC1)C(=O)C[C@@H]2[C@@H](OC)[C@@H](C[C@H](O)CN)O[C@H]2C[C@@H]2C(=C)[C@H](C)C[C@H]1O2 QAMYWGZHLCQOOJ-PWIVHLLHSA-N 0.000 description 2
- MMXKVMNBHPAILY-UHFFFAOYSA-N ethyl laurate Chemical compound CCCCCCCCCCCC(=O)OCC MMXKVMNBHPAILY-UHFFFAOYSA-N 0.000 description 2
- 229960005167 everolimus Drugs 0.000 description 2
- 229960000255 exemestane Drugs 0.000 description 2
- 229960002949 fluorouracil Drugs 0.000 description 2
- 229960002258 fulvestrant Drugs 0.000 description 2
- 229960005277 gemcitabine Drugs 0.000 description 2
- 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 2
- 230000004547 gene signature Effects 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 229960002913 goserelin Drugs 0.000 description 2
- 229940029575 guanosine Drugs 0.000 description 2
- 210000003128 head Anatomy 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 102000051276 human BCHE Human genes 0.000 description 2
- 230000028996 humoral immune response Effects 0.000 description 2
- VKOBVWXKNCXXDE-UHFFFAOYSA-N icosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCC(O)=O VKOBVWXKNCXXDE-UHFFFAOYSA-N 0.000 description 2
- 230000005934 immune activation Effects 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 239000002955 immunomodulating agent Substances 0.000 description 2
- 229940121354 immunomodulator Drugs 0.000 description 2
- 230000028709 inflammatory response Effects 0.000 description 2
- 229960003786 inosine Drugs 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 229960005386 ipilimumab Drugs 0.000 description 2
- 229960002014 ixabepilone Drugs 0.000 description 2
- FABUFPQFXZVHFB-CFWQTKTJSA-N ixabepilone Chemical compound C/C([C@@H]1C[C@@H]2O[C@]2(C)CCC[C@@H]([C@@H]([C@H](C)C(=O)C(C)(C)[C@H](O)CC(=O)N1)O)C)=C\C1=CSC(C)=N1 FABUFPQFXZVHFB-CFWQTKTJSA-N 0.000 description 2
- 229960004891 lapatinib Drugs 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
- 229960003881 letrozole Drugs 0.000 description 2
- HPJKCIUCZWXJDR-UHFFFAOYSA-N letrozole Chemical compound C1=CC(C#N)=CC=C1C(N1N=CN=C1)C1=CC=C(C#N)C=C1 HPJKCIUCZWXJDR-UHFFFAOYSA-N 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 230000021633 leukocyte mediated immunity Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 208000037841 lung tumor Diseases 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229960001786 megestrol Drugs 0.000 description 2
- RQZAXGRLVPAYTJ-GQFGMJRRSA-N megestrol acetate Chemical compound C1=C(C)C2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(C)=O)(OC(=O)C)[C@@]1(C)CC2 RQZAXGRLVPAYTJ-GQFGMJRRSA-N 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 229960005558 mertansine Drugs 0.000 description 2
- 208000037819 metastatic cancer Diseases 0.000 description 2
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 2
- FEFIBEHSXLKJGI-UHFFFAOYSA-N methyl 2-[3-[[3-(6-amino-2-butoxy-8-oxo-7h-purin-9-yl)propyl-(3-morpholin-4-ylpropyl)amino]methyl]phenyl]acetate Chemical compound C12=NC(OCCCC)=NC(N)=C2NC(=O)N1CCCN(CC=1C=C(CC(=O)OC)C=CC=1)CCCN1CCOCC1 FEFIBEHSXLKJGI-UHFFFAOYSA-N 0.000 description 2
- 108091086850 miR-19a-1 stem-loop Proteins 0.000 description 2
- 108091088468 miR-19a-2 stem-loop Proteins 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- GLGLUQVVDHRLQK-WRBBJXAJSA-N n,n-dimethyl-2,3-bis[(z)-octadec-9-enoxy]propan-1-amine Chemical compound CCCCCCCC\C=C/CCCCCCCCOCC(CN(C)C)OCCCCCCCC\C=C/CCCCCCCC GLGLUQVVDHRLQK-WRBBJXAJSA-N 0.000 description 2
- 210000003739 neck Anatomy 0.000 description 2
- 229950008835 neratinib Drugs 0.000 description 2
- JWNPDZNEKVCWMY-VQHVLOKHSA-N neratinib Chemical compound C=12C=C(NC(=O)\C=C\CN(C)C)C(OCC)=CC2=NC=C(C#N)C=1NC(C=C1Cl)=CC=C1OCC1=CC=CC=N1 JWNPDZNEKVCWMY-VQHVLOKHSA-N 0.000 description 2
- 229960000572 olaparib Drugs 0.000 description 2
- FAQDUNYVKQKNLD-UHFFFAOYSA-N olaparib Chemical compound FC1=CC=C(CC2=C3[CH]C=CC=C3C(=O)N=N2)C=C1C(=O)N(CC1)CCN1C(=O)C1CC1 FAQDUNYVKQKNLD-UHFFFAOYSA-N 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 229960004390 palbociclib Drugs 0.000 description 2
- AHJRHEGDXFFMBM-UHFFFAOYSA-N palbociclib Chemical compound N1=C2N(C3CCCC3)C(=O)C(C(=O)C)=C(C)C2=CN=C1NC(N=C1)=CC=C1N1CCNCC1 AHJRHEGDXFFMBM-UHFFFAOYSA-N 0.000 description 2
- SECPZKHBENQXJG-FPLPWBNLSA-N palmitoleic acid Chemical compound CCCCCC\C=C/CCCCCCCC(O)=O SECPZKHBENQXJG-FPLPWBNLSA-N 0.000 description 2
- 229940046231 pamidronate Drugs 0.000 description 2
- WRUUGTRCQOWXEG-UHFFFAOYSA-N pamidronate Chemical compound NCCC(O)(P(O)(O)=O)P(O)(O)=O WRUUGTRCQOWXEG-UHFFFAOYSA-N 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000008823 permeabilization Effects 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 150000003905 phosphatidylinositols Chemical class 0.000 description 2
- 229920000747 poly(lactic acid) Polymers 0.000 description 2
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 150000004804 polysaccharides Chemical class 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 229960002429 proline Drugs 0.000 description 2
- 239000001294 propane Substances 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 238000001959 radiotherapy Methods 0.000 description 2
- BKXVVCILCIUCLG-UHFFFAOYSA-N raloxifene hydrochloride Chemical compound [H+].[Cl-].C1=CC(O)=CC=C1C1=C(C(=O)C=2C=CC(OCCN3CCCCC3)=CC=2)C2=CC=C(O)C=C2S1 BKXVVCILCIUCLG-UHFFFAOYSA-N 0.000 description 2
- 229960002119 raloxifene hydrochloride Drugs 0.000 description 2
- 210000000664 rectum Anatomy 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 229950003687 ribociclib Drugs 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 108700015434 rotavirus NS28 Proteins 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- NLQLSVXGSXCXFE-UHFFFAOYSA-N sitosterol Natural products CC=C(/CCC(C)C1CC2C3=CCC4C(C)C(O)CCC4(C)C3CCC2(C)C1)C(C)C NLQLSVXGSXCXFE-UHFFFAOYSA-N 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 150000003431 steroids Chemical class 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- CCEKAJIANROZEO-UHFFFAOYSA-N sulfluramid Chemical group CCNS(=O)(=O)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F CCEKAJIANROZEO-UHFFFAOYSA-N 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229950004550 talazoparib Drugs 0.000 description 2
- 229960003454 tamoxifen citrate Drugs 0.000 description 2
- FQZYTYWMLGAPFJ-OQKDUQJOSA-N tamoxifen citrate Chemical compound [H+].[H+].[H+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 FQZYTYWMLGAPFJ-OQKDUQJOSA-N 0.000 description 2
- 150000003512 tertiary amines Chemical class 0.000 description 2
- 210000001550 testis Anatomy 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 229960001196 thiotepa Drugs 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 210000001685 thyroid gland Anatomy 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 229960000575 trastuzumab Drugs 0.000 description 2
- 230000001960 triggered effect Effects 0.000 description 2
- 229950003463 tucatinib Drugs 0.000 description 2
- 230000005751 tumor progression Effects 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 229940035893 uracil Drugs 0.000 description 2
- 210000003932 urinary bladder Anatomy 0.000 description 2
- 210000004291 uterus Anatomy 0.000 description 2
- 229960003048 vinblastine Drugs 0.000 description 2
- 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 2
- 230000003612 virological effect Effects 0.000 description 2
- KZJWDPNRJALLNS-VPUBHVLGSA-N (-)-beta-Sitosterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@@H](C(C)C)CC)C)CC4)CC3)CC=2)CC1 KZJWDPNRJALLNS-VPUBHVLGSA-N 0.000 description 1
- JTERLNYVBOZRHI-PPBJBQABSA-N (2-aminoethoxy)[(2r)-2,3-bis[(5z,8z,11z,14z)-icosa-5,8,11,14-tetraenoyloxy]propoxy]phosphinic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC JTERLNYVBOZRHI-PPBJBQABSA-N 0.000 description 1
- IHNKQIMGVNPMTC-UHFFFAOYSA-N (2-hydroxy-3-octadecanoyloxypropyl) 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)COP([O-])(=O)OCC[N+](C)(C)C IHNKQIMGVNPMTC-UHFFFAOYSA-N 0.000 description 1
- XLKQWAMTMYIQMG-SVUPRYTISA-N (2-{[(2r)-2,3-bis[(4z,7z,10z,13z,16z,19z)-docosa-4,7,10,13,16,19-hexaenoyloxy]propyl phosphonato]oxy}ethyl)trimethylazanium Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CC XLKQWAMTMYIQMG-SVUPRYTISA-N 0.000 description 1
- CSVWWLUMXNHWSU-UHFFFAOYSA-N (22E)-(24xi)-24-ethyl-5alpha-cholest-22-en-3beta-ol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(CC)C(C)C)C1(C)CC2 CSVWWLUMXNHWSU-UHFFFAOYSA-N 0.000 description 1
- RQOCXCFLRBRBCS-UHFFFAOYSA-N (22E)-cholesta-5,7,22-trien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CCC(C)C)CCC33)C)C3=CC=C21 RQOCXCFLRBRBCS-UHFFFAOYSA-N 0.000 description 1
- YZSZLBRBVWAXFW-LNYQSQCFSA-N (2R,3R,4S,5R)-2-(2-amino-6-hydroxy-6-methoxy-3H-purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol Chemical compound COC1(O)NC(N)=NC2=C1N=CN2[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O YZSZLBRBVWAXFW-LNYQSQCFSA-N 0.000 description 1
- GRYSXUXXBDSYRT-WOUKDFQISA-N (2r,3r,4r,5r)-2-(hydroxymethyl)-4-methoxy-5-[6-(methylamino)purin-9-yl]oxolan-3-ol Chemical compound C1=NC=2C(NC)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1OC GRYSXUXXBDSYRT-WOUKDFQISA-N 0.000 description 1
- KYJLJOJCMUFWDY-UUOKFMHZSA-N (2r,3r,4s,5r)-2-(6-amino-8-azidopurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol Chemical compound [N-]=[N+]=NC1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O KYJLJOJCMUFWDY-UUOKFMHZSA-N 0.000 description 1
- MQECTKDGEQSNNL-UMCMBGNQSA-N (2r,3r,4s,5r)-2-[6-(14-aminotetradecoxyperoxyperoxyamino)purin-9-yl]-5-(hydroxymethyl)oxolane-3,4-diol Chemical compound C1=NC=2C(NOOOOOCCCCCCCCCCCCCCN)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O MQECTKDGEQSNNL-UMCMBGNQSA-N 0.000 description 1
- PHFMCMDFWSZKGD-IOSLPCCCSA-N (2r,3s,4r,5r)-2-(hydroxymethyl)-5-[6-(methylamino)-2-methylsulfanylpurin-9-yl]oxolane-3,4-diol Chemical compound C1=NC=2C(NC)=NC(SC)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O PHFMCMDFWSZKGD-IOSLPCCCSA-N 0.000 description 1
- KUHSEZKIEJYEHN-BXRBKJIMSA-N (2s)-2-amino-3-hydroxypropanoic acid;(2s)-2-aminopropanoic acid Chemical compound C[C@H](N)C(O)=O.OC[C@H](N)C(O)=O KUHSEZKIEJYEHN-BXRBKJIMSA-N 0.000 description 1
- OEDPHAKKZGDBEV-GFPBKZJXSA-N (2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-2-[[(2r)-3-[2,3-di(hexadecanoyloxy)propylsulfanyl]-2-(hexadecanoylamino)propanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]hexanoyl]amino]hexanoyl]amino]hexanoic acid Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)CCCCCCCCCCCCCCC)CSCC(COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC OEDPHAKKZGDBEV-GFPBKZJXSA-N 0.000 description 1
- MYUOTPIQBPUQQU-CKTDUXNWSA-N (2s,3r)-2-amino-n-[[9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-methylsulfanylpurin-6-yl]carbamoyl]-3-hydroxybutanamide Chemical compound C12=NC(SC)=NC(NC(=O)NC(=O)[C@@H](N)[C@@H](C)O)=C2N=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O MYUOTPIQBPUQQU-CKTDUXNWSA-N 0.000 description 1
- GPTUGCGYEMEAOC-IBZYUGMLSA-N (2s,3r)-2-amino-n-[[9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]purin-6-yl]-methylcarbamoyl]-3-hydroxybutanamide Chemical compound C1=NC=2C(N(C)C(=O)NC(=O)[C@@H](N)[C@H](O)C)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O GPTUGCGYEMEAOC-IBZYUGMLSA-N 0.000 description 1
- RLCKHJSFHOZMDR-UHFFFAOYSA-N (3R, 7R, 11R)-1-Phytanoid acid Natural products CC(C)CCCC(C)CCCC(C)CCCC(C)CC(O)=O RLCKHJSFHOZMDR-UHFFFAOYSA-N 0.000 description 1
- XBBQCOKPWNZHFX-TYASJMOZSA-N (3r,4s,5r)-2-[(2r,3r,4r,5r)-2-(6-aminopurin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy-5-(hydroxymethyl)oxolane-3,4-diol Chemical compound O([C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C=2N=CN=C(C=2N=C1)N)C1O[C@H](CO)[C@@H](O)[C@H]1O XBBQCOKPWNZHFX-TYASJMOZSA-N 0.000 description 1
- YUFFSWGQGVEMMI-JLNKQSITSA-N (7Z,10Z,13Z,16Z,19Z)-docosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCCCC(O)=O YUFFSWGQGVEMMI-JLNKQSITSA-N 0.000 description 1
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-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
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- SSCDRSKJTAQNNB-DWEQTYCFSA-N 1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphoethanolamine Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC SSCDRSKJTAQNNB-DWEQTYCFSA-N 0.000 description 1
- FVXDQWZBHIXIEJ-LNDKUQBDSA-N 1,2-di-[(9Z,12Z)-octadecadienoyl]-sn-glycero-3-phosphocholine Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC FVXDQWZBHIXIEJ-LNDKUQBDSA-N 0.000 description 1
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 1
- DSNRWDQKZIEDDB-SQYFZQSCSA-N 1,2-dioleoyl-sn-glycero-3-phospho-(1'-sn-glycerol) Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@@H](O)CO)OC(=O)CCCCCCC\C=C/CCCCCCCC DSNRWDQKZIEDDB-SQYFZQSCSA-N 0.000 description 1
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 1
- MIXBUOXRHTZHKR-XUTVFYLZSA-N 1-Methylpseudoisocytidine Chemical compound CN1C=C(C(=O)N=C1N)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O MIXBUOXRHTZHKR-XUTVFYLZSA-N 0.000 description 1
- KYEKLQMDNZPEFU-KVTDHHQDSA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,3,5-triazine-2,4-dione Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)N=C1 KYEKLQMDNZPEFU-KVTDHHQDSA-N 0.000 description 1
- MUSPKJVFRAYWAR-XVFCMESISA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)thiolan-2-yl]pyrimidine-2,4-dione Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)S[C@H]1N1C(=O)NC(=O)C=C1 MUSPKJVFRAYWAR-XVFCMESISA-N 0.000 description 1
- BNXGRQLXOMSOMV-UHFFFAOYSA-N 1-[4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-4-(methylamino)pyrimidin-2-one Chemical compound O=C1N=C(NC)C=CN1C1C(OC)C(O)C(CO)O1 BNXGRQLXOMSOMV-UHFFFAOYSA-N 0.000 description 1
- GFYLSDSUCHVORB-IOSLPCCCSA-N 1-methyladenosine Chemical compound C1=NC=2C(=N)N(C)C=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O GFYLSDSUCHVORB-IOSLPCCCSA-N 0.000 description 1
- UTAIYTHAJQNQDW-KQYNXXCUSA-N 1-methylguanosine Chemical compound C1=NC=2C(=O)N(C)C(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O UTAIYTHAJQNQDW-KQYNXXCUSA-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
- FPUGCISOLXNPPC-UHFFFAOYSA-N 2'-O-Methyladenosine Natural products COC1C(O)C(CO)OC1N1C2=NC=NC(N)=C2N=C1 FPUGCISOLXNPPC-UHFFFAOYSA-N 0.000 description 1
- RFCQJGFZUQFYRF-UHFFFAOYSA-N 2'-O-Methylcytidine Natural products COC1C(O)C(CO)OC1N1C(=O)N=C(N)C=C1 RFCQJGFZUQFYRF-UHFFFAOYSA-N 0.000 description 1
- OVYNGSFVYRPRCG-UHFFFAOYSA-N 2'-O-Methylguanosine Natural products COC1C(O)C(CO)OC1N1C(NC(N)=NC2=O)=C2N=C1 OVYNGSFVYRPRCG-UHFFFAOYSA-N 0.000 description 1
- SXUXMRMBWZCMEN-UHFFFAOYSA-N 2'-O-methyl uridine Natural products COC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 SXUXMRMBWZCMEN-UHFFFAOYSA-N 0.000 description 1
- RFCQJGFZUQFYRF-ZOQUXTDFSA-N 2'-O-methylcytidine Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(N)C=C1 RFCQJGFZUQFYRF-ZOQUXTDFSA-N 0.000 description 1
- OVYNGSFVYRPRCG-KQYNXXCUSA-N 2'-O-methylguanosine Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=C(N)NC2=O)=C2N=C1 OVYNGSFVYRPRCG-KQYNXXCUSA-N 0.000 description 1
- HPHXOIULGYVAKW-IOSLPCCCSA-N 2'-O-methylinosine Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC2=O)=C2N=C1 HPHXOIULGYVAKW-IOSLPCCCSA-N 0.000 description 1
- HPHXOIULGYVAKW-UHFFFAOYSA-N 2'-O-methylinosine Natural products COC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 HPHXOIULGYVAKW-UHFFFAOYSA-N 0.000 description 1
- RPROHCOBMVQVIV-UHFFFAOYSA-N 2,3,4,5-tetrahydro-1h-pyrido[4,3-b]indole Chemical compound N1C2=CC=CC=C2C2=C1CCNC2 RPROHCOBMVQVIV-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 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 1
- WALUVDCNGPQPOD-UHFFFAOYSA-M 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCOCC(C[N+](C)(C)CCO)OCCCCCCCCCCCCCC WALUVDCNGPQPOD-UHFFFAOYSA-M 0.000 description 1
- YUCFXTKBZFABID-WOUKDFQISA-N 2-(dimethylamino)-9-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-3h-purin-6-one Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(NC(=NC2=O)N(C)C)=C2N=C1 YUCFXTKBZFABID-WOUKDFQISA-N 0.000 description 1
- DEVCLHVFELRPIU-UHFFFAOYSA-N 2-(ethoxymethyl)-3h-imidazo[4,5-c]quinolin-4-amine Chemical compound NC1=NC2=CC=CC=C2C2=C1NC(COCC)=N2 DEVCLHVFELRPIU-UHFFFAOYSA-N 0.000 description 1
- CWXIOHYALLRNSZ-JWMKEVCDSA-N 2-Thiodihydropseudouridine Chemical compound C1C(C(=O)NC(=S)N1)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O CWXIOHYALLRNSZ-JWMKEVCDSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- XWGKZKKNJOQUSG-SFHVURJKSA-N 2-[[4-[[2-amino-4-[[(3s)-1-hydroxyhexan-3-yl]amino]-6-methylpyrimidin-5-yl]methyl]-3-methoxyphenyl]methyl-(2,2,2-trifluoroethyl)amino]acetic acid Chemical compound CCC[C@@H](CCO)NC1=NC(N)=NC(C)=C1CC1=CC=C(CN(CC(O)=O)CC(F)(F)F)C=C1OC XWGKZKKNJOQUSG-SFHVURJKSA-N 0.000 description 1
- SOEYIPCQNRSIAV-IOSLPCCCSA-N 2-amino-5-(aminomethyl)-7-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1h-pyrrolo[2,3-d]pyrimidin-4-one Chemical compound C1=2NC(N)=NC(=O)C=2C(CN)=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O SOEYIPCQNRSIAV-IOSLPCCCSA-N 0.000 description 1
- MPDKOGQMQLSNOF-GBNDHIKLSA-N 2-amino-5-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1h-pyrimidin-6-one Chemical compound O=C1NC(N)=NC=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 MPDKOGQMQLSNOF-GBNDHIKLSA-N 0.000 description 1
- JRYMOPZHXMVHTA-DAGMQNCNSA-N 2-amino-7-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1h-pyrrolo[2,3-d]pyrimidin-4-one Chemical compound C1=CC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O JRYMOPZHXMVHTA-DAGMQNCNSA-N 0.000 description 1
- BIRQNXWAXWLATA-IOSLPCCCSA-N 2-amino-7-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-4-oxo-1h-pyrrolo[2,3-d]pyrimidine-5-carbonitrile Chemical compound C1=C(C#N)C=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O BIRQNXWAXWLATA-IOSLPCCCSA-N 0.000 description 1
- OTDJAMXESTUWLO-UUOKFMHZSA-N 2-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-oxolanyl]-3H-purine-6-thione Chemical compound C12=NC(N)=NC(S)=C2N=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OTDJAMXESTUWLO-UUOKFMHZSA-N 0.000 description 1
- NTYZLKZZBRSAPT-DBINCYRJSA-N 2-amino-9-[(2r,3r,4r,5r)-3-[(3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxy-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-3h-purin-6-one Chemical compound O([C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C=NC=2C(=O)N=C(NC=21)N)C1O[C@H](CO)[C@@H](O)[C@H]1O NTYZLKZZBRSAPT-DBINCYRJSA-N 0.000 description 1
- HPKQEMIXSLRGJU-UUOKFMHZSA-N 2-amino-9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-7-methyl-3h-purine-6,8-dione Chemical compound O=C1N(C)C(C(NC(N)=N2)=O)=C2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O HPKQEMIXSLRGJU-UUOKFMHZSA-N 0.000 description 1
- QSPOQCXMGPDIHI-UHFFFAOYSA-N 2-amino-n,n-dipropyl-8-[4-(pyrrolidine-1-carbonyl)phenyl]-3h-1-benzazepine-4-carboxamide Chemical compound C1=C2N=C(N)CC(C(=O)N(CCC)CCC)=CC2=CC=C1C(C=C1)=CC=C1C(=O)N1CCCC1 QSPOQCXMGPDIHI-UHFFFAOYSA-N 0.000 description 1
- PBFLIOAJBULBHI-JJNLEZRASA-N 2-amino-n-[[9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]purin-6-yl]carbamoyl]acetamide Chemical compound C1=NC=2C(NC(=O)NC(=O)CN)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O PBFLIOAJBULBHI-JJNLEZRASA-N 0.000 description 1
- MWBWWFOAEOYUST-UHFFFAOYSA-N 2-aminopurine Chemical compound NC1=NC=C2N=CNC2=N1 MWBWWFOAEOYUST-UHFFFAOYSA-N 0.000 description 1
- QCPQCJVQJKOKMS-VLSMUFELSA-N 2-methoxy-5-methyl-cytidine Chemical compound CC(C(N)=N1)=CN([C@@H]([C@@H]2O)O[C@H](CO)[C@H]2O)C1OC QCPQCJVQJKOKMS-VLSMUFELSA-N 0.000 description 1
- TUDKBZAMOFJOSO-UHFFFAOYSA-N 2-methoxy-7h-purin-6-amine Chemical compound COC1=NC(N)=C2NC=NC2=N1 TUDKBZAMOFJOSO-UHFFFAOYSA-N 0.000 description 1
- STISOQJGVFEOFJ-MEVVYUPBSA-N 2-methoxy-cytidine Chemical compound COC(N([C@@H]([C@@H]1O)O[C@H](CO)[C@H]1O)C=C1)N=C1N STISOQJGVFEOFJ-MEVVYUPBSA-N 0.000 description 1
- VWSLLSXLURJCDF-UHFFFAOYSA-N 2-methyl-4,5-dihydro-1h-imidazole Chemical compound CC1=NCCN1 VWSLLSXLURJCDF-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
- FXGXEFXCWDTSQK-UHFFFAOYSA-N 2-methylsulfanyl-7h-purin-6-amine Chemical compound CSC1=NC(N)=C2NC=NC2=N1 FXGXEFXCWDTSQK-UHFFFAOYSA-N 0.000 description 1
- VZQXUWKZDSEQRR-SDBHATRESA-N 2-methylthio-N(6)-(Delta(2)-isopentenyl)adenosine Chemical compound C12=NC(SC)=NC(NCC=C(C)C)=C2N=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O VZQXUWKZDSEQRR-SDBHATRESA-N 0.000 description 1
- QEWSGVMSLPHELX-UHFFFAOYSA-N 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine Chemical compound C12=NC(SC)=NC(NCC=C(C)CO)=C2N=CN1C1OC(CO)C(O)C1O QEWSGVMSLPHELX-UHFFFAOYSA-N 0.000 description 1
- JUMHLCXWYQVTLL-KVTDHHQDSA-N 2-thio-5-aza-uridine Chemical compound [C@@H]1([C@H](O)[C@H](O)[C@@H](CO)O1)N1C(=S)NC(=O)N=C1 JUMHLCXWYQVTLL-KVTDHHQDSA-N 0.000 description 1
- VRVXMIJPUBNPGH-XVFCMESISA-N 2-thio-dihydrouridine Chemical compound OC[C@H]1O[C@H]([C@H](O)[C@@H]1O)N1CCC(=O)NC1=S VRVXMIJPUBNPGH-XVFCMESISA-N 0.000 description 1
- ZVGONGHIVBJXFC-WCTZXXKLSA-N 2-thio-zebularine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=S)N=CC=C1 ZVGONGHIVBJXFC-WCTZXXKLSA-N 0.000 description 1
- RHFUOMFWUGWKKO-XVFCMESISA-N 2-thiocytidine Chemical compound S=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 RHFUOMFWUGWKKO-XVFCMESISA-N 0.000 description 1
- GJTBSTBJLVYKAU-XVFCMESISA-N 2-thiouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=S)NC(=O)C=C1 GJTBSTBJLVYKAU-XVFCMESISA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- SLQKYSPHBZMASJ-QKPORZECSA-N 24-methylene-cholest-8-en-3β-ol Chemical compound C([C@@]12C)C[C@H](O)C[C@@H]1CCC1=C2CC[C@]2(C)[C@@H]([C@H](C)CCC(=C)C(C)C)CC[C@H]21 SLQKYSPHBZMASJ-QKPORZECSA-N 0.000 description 1
- KLEXDBGYSOIREE-UHFFFAOYSA-N 24xi-n-propylcholesterol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CCC)C(C)C)C1(C)CC2 KLEXDBGYSOIREE-UHFFFAOYSA-N 0.000 description 1
- RLCKHJSFHOZMDR-PWCSWUJKSA-N 3,7R,11R,15-tetramethyl-hexadecanoic acid Chemical compound CC(C)CCC[C@@H](C)CCC[C@@H](C)CCCC(C)CC(O)=O RLCKHJSFHOZMDR-PWCSWUJKSA-N 0.000 description 1
- RDPUKVRQKWBSPK-UHFFFAOYSA-N 3-Methylcytidine Natural products O=C1N(C)C(=N)C=CN1C1C(O)C(O)C(CO)O1 RDPUKVRQKWBSPK-UHFFFAOYSA-N 0.000 description 1
- HOEIPINIBKBXTJ-IDTAVKCVSA-N 3-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-4,6,7-trimethylimidazo[1,2-a]purin-9-one Chemical compound C1=NC=2C(=O)N3C(C)=C(C)N=C3N(C)C=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O HOEIPINIBKBXTJ-IDTAVKCVSA-N 0.000 description 1
- BINGDNLMMYSZFR-QYVSTXNMSA-N 3-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6,7-dimethyl-5h-imidazo[1,2-a]purin-9-one Chemical compound C1=NC=2C(=O)N3C(C)=C(C)N=C3NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O BINGDNLMMYSZFR-QYVSTXNMSA-N 0.000 description 1
- SDLWKRZBLTZSEL-UHFFFAOYSA-N 3-[5-amino-2-[2-[4-[2-(3,3-difluoro-3-phosphonopropoxy)ethoxy]-2-methylphenyl]ethyl]benzo[f][1,7]naphthyridin-8-yl]propanoic acid Chemical compound CC1=CC(OCCOCCC(F)(F)P(O)(O)=O)=CC=C1CCC1=CN=C(C(N)=NC=2C3=CC=C(CCC(O)=O)C=2)C3=C1 SDLWKRZBLTZSEL-UHFFFAOYSA-N 0.000 description 1
- RDPUKVRQKWBSPK-ZOQUXTDFSA-N 3-methylcytidine Chemical compound O=C1N(C)C(=N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 RDPUKVRQKWBSPK-ZOQUXTDFSA-N 0.000 description 1
- WFCJCYSSTXNUED-UHFFFAOYSA-N 4-(dimethylamino)-1-[4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]pyrimidin-2-one Chemical compound COC1C(O)C(CO)OC1N1C(=O)N=C(N(C)C)C=C1 WFCJCYSSTXNUED-UHFFFAOYSA-N 0.000 description 1
- ZSIINYPBPQCZKU-BQNZPOLKSA-O 4-Methoxy-1-methylpseudoisocytidine Chemical compound C[N+](CC1[C@H]([C@H]2O)O[C@@H](CO)[C@@H]2O)=C(N)N=C1OC ZSIINYPBPQCZKU-BQNZPOLKSA-O 0.000 description 1
- FGFVODMBKZRMMW-XUTVFYLZSA-N 4-Methoxy-2-thiopseudouridine Chemical compound COC1=C(C=NC(=S)N1)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O FGFVODMBKZRMMW-XUTVFYLZSA-N 0.000 description 1
- HOCJTJWYMOSXMU-XUTVFYLZSA-N 4-Methoxypseudouridine Chemical compound COC1=C(C=NC(=O)N1)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O HOCJTJWYMOSXMU-XUTVFYLZSA-N 0.000 description 1
- DMUQOPXCCOBPID-XUTVFYLZSA-N 4-Thio-1-methylpseudoisocytidine Chemical compound CN1C=C(C(=S)N=C1N)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O DMUQOPXCCOBPID-XUTVFYLZSA-N 0.000 description 1
- YBBDRHCNZBVLGT-FDDDBJFASA-N 4-amino-1-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-2-oxopyrimidine-5-carbaldehyde Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(N)C(C=O)=C1 YBBDRHCNZBVLGT-FDDDBJFASA-N 0.000 description 1
- OCMSXKMNYAHJMU-JXOAFFINSA-N 4-amino-1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-oxopyrimidine-5-carbaldehyde Chemical compound C1=C(C=O)C(N)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 OCMSXKMNYAHJMU-JXOAFFINSA-N 0.000 description 1
- OZHIJZYBTCTDQC-JXOAFFINSA-N 4-amino-1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2-thione Chemical compound S=C1N=C(N)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 OZHIJZYBTCTDQC-JXOAFFINSA-N 0.000 description 1
- PJWBTAIPBFWVHX-FJGDRVTGSA-N 4-amino-1-[(2r,3s,4r,5r)-3-fluoro-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@](F)(O)[C@H](O)[C@@H](CO)O1 PJWBTAIPBFWVHX-FJGDRVTGSA-N 0.000 description 1
- VFOKSTCIRGDTBR-UHFFFAOYSA-N 4-amino-2-butoxy-8-[[3-(pyrrolidin-1-ylmethyl)phenyl]methyl]-5,7-dihydropteridin-6-one Chemical compound C12=NC(OCCCC)=NC(N)=C2NC(=O)CN1CC(C=1)=CC=CC=1CN1CCCC1 VFOKSTCIRGDTBR-UHFFFAOYSA-N 0.000 description 1
- QUZQVVNSDQCAOL-WOUKDFQISA-N 4-demethylwyosine Chemical compound N1C(C)=CN(C(C=2N=C3)=O)C1=NC=2N3[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O QUZQVVNSDQCAOL-WOUKDFQISA-N 0.000 description 1
- LOICBOXHPCURMU-UHFFFAOYSA-N 4-methoxy-pseudoisocytidine Chemical compound COC1NC(N)=NC=C1C(C1O)OC(CO)C1O LOICBOXHPCURMU-UHFFFAOYSA-N 0.000 description 1
- UEALKTCRMBVTFN-UHFFFAOYSA-N 4-nitroanthranilic acid Chemical compound NC1=CC([N+]([O-])=O)=CC=C1C(O)=O UEALKTCRMBVTFN-UHFFFAOYSA-N 0.000 description 1
- SJVVKUMXGIKAAI-UHFFFAOYSA-N 4-thio-pseudoisocytidine Chemical compound NC(N1)=NC=C(C(C2O)OC(CO)C2O)C1=S SJVVKUMXGIKAAI-UHFFFAOYSA-N 0.000 description 1
- CNVRVGAACYEOQI-FDDDBJFASA-N 5,2'-O-dimethylcytidine Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(N)C(C)=C1 CNVRVGAACYEOQI-FDDDBJFASA-N 0.000 description 1
- NMUSYJAQQFHJEW-UHFFFAOYSA-N 5-Azacytidine Natural products O=C1N=C(N)N=CN1C1C(O)C(O)C(CO)O1 NMUSYJAQQFHJEW-UHFFFAOYSA-N 0.000 description 1
- NFEXJLMYXXIWPI-JXOAFFINSA-N 5-Hydroxymethylcytidine Chemical compound C1=C(CO)C(N)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NFEXJLMYXXIWPI-JXOAFFINSA-N 0.000 description 1
- ZAYHVCMSTBRABG-UHFFFAOYSA-N 5-Methylcytidine Natural products O=C1N=C(N)C(C)=CN1C1C(O)C(O)C(CO)O1 ZAYHVCMSTBRABG-UHFFFAOYSA-N 0.000 description 1
- AMMRPAYSYYGRKP-BGZDPUMWSA-N 5-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1-ethylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)N(CC)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 AMMRPAYSYYGRKP-BGZDPUMWSA-N 0.000 description 1
- DDHOXEOVAJVODV-GBNDHIKLSA-N 5-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=S)NC1=O DDHOXEOVAJVODV-GBNDHIKLSA-N 0.000 description 1
- BNAWMJKJLNJZFU-GBNDHIKLSA-N 5-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-4-sulfanylidene-1h-pyrimidin-2-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=O)NC1=S BNAWMJKJLNJZFU-GBNDHIKLSA-N 0.000 description 1
- IPRQAJTUSRLECG-UHFFFAOYSA-N 5-[6-(dimethylamino)purin-9-yl]-2-(hydroxymethyl)-4-methoxyoxolan-3-ol Chemical compound COC1C(O)C(CO)OC1N1C2=NC=NC(N(C)C)=C2N=C1 IPRQAJTUSRLECG-UHFFFAOYSA-N 0.000 description 1
- OZQDLJNDRVBCST-SHUUEZRQSA-N 5-amino-2-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,2,4-triazin-3-one Chemical compound O=C1N=C(N)C=NN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 OZQDLJNDRVBCST-SHUUEZRQSA-N 0.000 description 1
- XUNBIDXYAUXNKD-DBRKOABJSA-N 5-aza-2-thio-zebularine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=S)N=CN=C1 XUNBIDXYAUXNKD-DBRKOABJSA-N 0.000 description 1
- OSLBPVOJTCDNEF-DBRKOABJSA-N 5-aza-zebularine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=CN=C1 OSLBPVOJTCDNEF-DBRKOABJSA-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
- ZXIATBNUWJBBGT-JXOAFFINSA-N 5-methoxyuridine Chemical compound O=C1NC(=O)C(OC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 ZXIATBNUWJBBGT-JXOAFFINSA-N 0.000 description 1
- RPQQZHJQUBDHHG-FNCVBFRFSA-N 5-methyl-zebularine Chemical compound C1=C(C)C=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 RPQQZHJQUBDHHG-FNCVBFRFSA-N 0.000 description 1
- ZAYHVCMSTBRABG-JXOAFFINSA-N 5-methylcytidine Chemical compound O=C1N=C(N)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 ZAYHVCMSTBRABG-JXOAFFINSA-N 0.000 description 1
- USVMJSALORZVDV-UHFFFAOYSA-N 6-(gamma,gamma-dimethylallylamino)purine riboside Natural products C1=NC=2C(NCC=C(C)C)=NC=NC=2N1C1OC(CO)C(O)C1O USVMJSALORZVDV-UHFFFAOYSA-N 0.000 description 1
- OZTOEARQSSIFOG-MWKIOEHESA-N 6-Thio-7-deaza-8-azaguanosine Chemical compound Nc1nc(=S)c2cnn([C@@H]3O[C@H](CO)[C@@H](O)[C@H]3O)c2[nH]1 OZTOEARQSSIFOG-MWKIOEHESA-N 0.000 description 1
- LFMPVTVPXHNXOT-HNNXBMFYSA-N 6-amino-2-[(2s)-pentan-2-yl]oxy-9-(5-piperidin-1-ylpentyl)-7h-purin-8-one Chemical compound C12=NC(O[C@@H](C)CCC)=NC(N)=C2NC(=O)N1CCCCCN1CCCCC1 LFMPVTVPXHNXOT-HNNXBMFYSA-N 0.000 description 1
- AFWWNHLDHNSVSD-UHFFFAOYSA-N 6-methyl-7h-purin-2-amine Chemical compound CC1=NC(N)=NC2=C1NC=N2 AFWWNHLDHNSVSD-UHFFFAOYSA-N 0.000 description 1
- CBNRZZNSRJQZNT-IOSLPCCCSA-O 6-thio-7-deaza-guanosine Chemical compound CC1=C[NH+]([C@@H]([C@@H]2O)O[C@H](CO)[C@H]2O)C(NC(N)=N2)=C1C2=S CBNRZZNSRJQZNT-IOSLPCCCSA-O 0.000 description 1
- RFHIWBUKNJIBSE-KQYNXXCUSA-O 6-thio-7-methyl-guanosine Chemical compound C1=2NC(N)=NC(=S)C=2N(C)C=[N+]1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O RFHIWBUKNJIBSE-KQYNXXCUSA-O 0.000 description 1
- MJJUWOIBPREHRU-MWKIOEHESA-N 7-Deaza-8-azaguanosine Chemical compound NC=1NC(C2=C(N=1)N(N=C2)[C@H]1[C@H](O)[C@H](O)[C@H](O1)CO)=O MJJUWOIBPREHRU-MWKIOEHESA-N 0.000 description 1
- ISSMDAFGDCTNDV-UHFFFAOYSA-N 7-deaza-2,6-diaminopurine Chemical compound NC1=NC(N)=C2NC=CC2=N1 ISSMDAFGDCTNDV-UHFFFAOYSA-N 0.000 description 1
- YVVMIGRXQRPSIY-UHFFFAOYSA-N 7-deaza-2-aminopurine Chemical compound N1C(N)=NC=C2C=CN=C21 YVVMIGRXQRPSIY-UHFFFAOYSA-N 0.000 description 1
- ZTAWTRPFJHKMRU-UHFFFAOYSA-N 7-deaza-8-aza-2,6-diaminopurine Chemical compound NC1=NC(N)=C2NN=CC2=N1 ZTAWTRPFJHKMRU-UHFFFAOYSA-N 0.000 description 1
- SMXRCJBCWRHDJE-UHFFFAOYSA-N 7-deaza-8-aza-2-aminopurine Chemical compound NC1=NC=C2C=NNC2=N1 SMXRCJBCWRHDJE-UHFFFAOYSA-N 0.000 description 1
- LHCPRYRLDOSKHK-UHFFFAOYSA-N 7-deaza-8-aza-adenine Chemical compound NC1=NC=NC2=C1C=NN2 LHCPRYRLDOSKHK-UHFFFAOYSA-N 0.000 description 1
- VJNXUFOTKNTNPG-IOSLPCCCSA-O 7-methylinosine Chemical compound C1=2NC=NC(=O)C=2N(C)C=[N+]1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O VJNXUFOTKNTNPG-IOSLPCCCSA-O 0.000 description 1
- HCAJQHYUCKICQH-VPENINKCSA-N 8-Oxo-7,8-dihydro-2'-deoxyguanosine Chemical compound C1=2NC(N)=NC(=O)C=2NC(=O)N1[C@H]1C[C@H](O)[C@@H](CO)O1 HCAJQHYUCKICQH-VPENINKCSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- IGUVTVZUVROGNX-WOUKDFQISA-O 9-[(2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-7-methyl-2-(methylamino)-1H-purin-9-ium-6-one Chemical compound CNC=1NC(C=2[N+](=CN([C@H]3[C@H](OC)[C@H](O)[C@@H](CO)O3)C=2N=1)C)=O IGUVTVZUVROGNX-WOUKDFQISA-O 0.000 description 1
- OJTAZBNWKTYVFJ-IOSLPCCCSA-N 9-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-2-(methylamino)-3h-purin-6-one Chemical compound C1=2NC(NC)=NC(=O)C=2N=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1OC OJTAZBNWKTYVFJ-IOSLPCCCSA-N 0.000 description 1
- ABXGJJVKZAAEDH-IOSLPCCCSA-N 9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-(dimethylamino)-3h-purine-6-thione Chemical compound C1=NC=2C(=S)NC(N(C)C)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O ABXGJJVKZAAEDH-IOSLPCCCSA-N 0.000 description 1
- ADPMAYFIIFNDMT-KQYNXXCUSA-N 9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-(methylamino)-3h-purine-6-thione Chemical compound C1=NC=2C(=S)NC(NC)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O ADPMAYFIIFNDMT-KQYNXXCUSA-N 0.000 description 1
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- PEMQXWCOMFJRLS-UHFFFAOYSA-N Archaeosine Natural products C1=2NC(N)=NC(=O)C=2C(C(=N)N)=CN1C1OC(CO)C(O)C1O PEMQXWCOMFJRLS-UHFFFAOYSA-N 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 102000008096 B7-H1 Antigen Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 235000021357 Behenic acid Nutrition 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- OILXMJHPFNGGTO-NRHJOKMGSA-N Brassicasterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@](C)([C@H]([C@@H](/C=C/[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 OILXMJHPFNGGTO-NRHJOKMGSA-N 0.000 description 1
- DPUOLQHDNGRHBS-UHFFFAOYSA-N Brassidinsaeure Natural products CCCCCCCCC=CCCCCCCCCCCCC(O)=O DPUOLQHDNGRHBS-UHFFFAOYSA-N 0.000 description 1
- 102100024217 CAMPATH-1 antigen Human genes 0.000 description 1
- 108010029697 CD40 Ligand Proteins 0.000 description 1
- 102100032937 CD40 ligand Human genes 0.000 description 1
- 108010065524 CD52 Antigen Proteins 0.000 description 1
- 102100025221 CD70 antigen Human genes 0.000 description 1
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical class [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- SGNBVLSWZMBQTH-FGAXOLDCSA-N Campesterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 SGNBVLSWZMBQTH-FGAXOLDCSA-N 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 108700000434 Cannabis sativa edestin Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- LPZCCMIISIBREI-MTFRKTCUSA-N Citrostadienol Natural products CC=C(CC[C@@H](C)[C@H]1CC[C@H]2C3=CC[C@H]4[C@H](C)[C@@H](O)CC[C@]4(C)[C@H]3CC[C@]12C)C(C)C LPZCCMIISIBREI-MTFRKTCUSA-N 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 229940046168 CpG oligodeoxynucleotide Drugs 0.000 description 1
- 229920001651 Cyanoacrylate Polymers 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FDKWRPBBCBCIGA-UWTATZPHSA-N D-Selenocysteine Natural products [Se]C[C@@H](N)C(O)=O FDKWRPBBCBCIGA-UWTATZPHSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- COLNVLDHVKWLRT-MRVPVSSYSA-N D-phenylalanine Chemical compound OC(=O)[C@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-MRVPVSSYSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- ARVGMISWLZPBCH-UHFFFAOYSA-N Dehydro-beta-sitosterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)CCC(CC)C(C)C)CCC33)C)C3=CC=C21 ARVGMISWLZPBCH-UHFFFAOYSA-N 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 108091027757 Deoxyribozyme Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- YKWUPFSEFXSGRT-JWMKEVCDSA-N Dihydropseudouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1C(=O)NC(=O)NC1 YKWUPFSEFXSGRT-JWMKEVCDSA-N 0.000 description 1
- 235000021294 Docosapentaenoic acid Nutrition 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 description 1
- URXZXNYJPAJJOQ-UHFFFAOYSA-N Erucic acid Natural products CCCCCCC=CCCCCCCCCCCCC(O)=O URXZXNYJPAJJOQ-UHFFFAOYSA-N 0.000 description 1
- 102100038595 Estrogen receptor Human genes 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108010040721 Flagellin Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 101150063370 Gzmb gene Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- BTEISVKTSQLKST-UHFFFAOYSA-N Haliclonasterol Natural products CC(C=CC(C)C(C)(C)C)C1CCC2C3=CC=C4CC(O)CCC4(C)C3CCC12C BTEISVKTSQLKST-UHFFFAOYSA-N 0.000 description 1
- 206010019695 Hepatic neoplasm Diseases 0.000 description 1
- 108091027305 Heteroduplex Proteins 0.000 description 1
- 241001272567 Hominoidea Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000934356 Homo sapiens CD70 antigen Proteins 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 description 1
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 1
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 1
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 1
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- 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 1
- 102100022297 Integrin alpha-X Human genes 0.000 description 1
- 108010014726 Interferon Type I Proteins 0.000 description 1
- 102000002227 Interferon Type I Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 102000013462 Interleukin-12 Human genes 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 102100021592 Interleukin-7 Human genes 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- 125000000998 L-alanino group Chemical group [H]N([*])[C@](C([H])([H])[H])([H])C(=O)O[H] 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 1
- ZKZBPNGNEQAJSX-REOHCLBHSA-N L-selenocysteine Chemical compound [SeH]C[C@H](N)C(O)=O ZKZBPNGNEQAJSX-REOHCLBHSA-N 0.000 description 1
- 125000000510 L-tryptophano group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C(C([H])([H])[C@@]([H])(C(O[H])=O)N([H])[*])C2=C1[H] 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 239000005551 L01XE03 - Erlotinib Substances 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 239000005639 Lauric acid Substances 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 239000007987 MES buffer Substances 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 101100264040 Mus musculus Wnt2b gene Proteins 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- ZBYRSRLCXTUFLJ-IOSLPCCCSA-O N(2),N(7)-dimethylguanosine Chemical compound CNC=1NC(C=2[N+](=CN([C@H]3[C@H](O)[C@H](O)[C@@H](CO)O3)C=2N=1)C)=O ZBYRSRLCXTUFLJ-IOSLPCCCSA-O 0.000 description 1
- SLEHROROQDYRAW-KQYNXXCUSA-N N(2)-methylguanosine Chemical compound C1=NC=2C(=O)NC(NC)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O SLEHROROQDYRAW-KQYNXXCUSA-N 0.000 description 1
- NIDVTARKFBZMOT-PEBGCTIMSA-N N(4)-acetylcytidine Chemical compound O=C1N=C(NC(=O)C)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NIDVTARKFBZMOT-PEBGCTIMSA-N 0.000 description 1
- WVGPGNPCZPYCLK-WOUKDFQISA-N N(6),N(6)-dimethyladenosine Chemical compound C1=NC=2C(N(C)C)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O WVGPGNPCZPYCLK-WOUKDFQISA-N 0.000 description 1
- USVMJSALORZVDV-SDBHATRESA-N N(6)-(Delta(2)-isopentenyl)adenosine Chemical compound C1=NC=2C(NCC=C(C)C)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O USVMJSALORZVDV-SDBHATRESA-N 0.000 description 1
- VQAYFKKCNSOZKM-IOSLPCCCSA-N N(6)-methyladenosine Chemical compound C1=NC=2C(NC)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O VQAYFKKCNSOZKM-IOSLPCCCSA-N 0.000 description 1
- WVGPGNPCZPYCLK-UHFFFAOYSA-N N-Dimethyladenosine Natural products C1=NC=2C(N(C)C)=NC=NC=2N1C1OC(CO)C(O)C1O WVGPGNPCZPYCLK-UHFFFAOYSA-N 0.000 description 1
- UNUYMBPXEFMLNW-DWVDDHQFSA-N N-[(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl]threonine Chemical compound C1=NC=2C(NC(=O)N[C@@H]([C@H](O)C)C(O)=O)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O UNUYMBPXEFMLNW-DWVDDHQFSA-N 0.000 description 1
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 1
- SLLVJTURCPWLTP-UHFFFAOYSA-N N-[9-[3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]purin-6-yl]acetamide Chemical compound C1=NC=2C(NC(=O)C)=NC=NC=2N1C1OC(CO)C(O)C1O SLLVJTURCPWLTP-UHFFFAOYSA-N 0.000 description 1
- PYUSHNKNPOHWEZ-YFKPBYRVSA-N N-formyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC=O PYUSHNKNPOHWEZ-YFKPBYRVSA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- LZCNWAXLJWBRJE-ZOQUXTDFSA-N N4-Methylcytidine Chemical compound O=C1N=C(NC)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 LZCNWAXLJWBRJE-ZOQUXTDFSA-N 0.000 description 1
- GOSWTRUMMSCNCW-UHFFFAOYSA-N N6-(cis-hydroxyisopentenyl)adenosine Chemical compound C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1OC(CO)C(O)C1O GOSWTRUMMSCNCW-UHFFFAOYSA-N 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- VQAYFKKCNSOZKM-UHFFFAOYSA-N NSC 29409 Natural products C1=NC=2C(NC)=NC=NC=2N1C1OC(CO)C(O)C1O VQAYFKKCNSOZKM-UHFFFAOYSA-N 0.000 description 1
- VZQXUWKZDSEQRR-UHFFFAOYSA-N Nucleosid Natural products C12=NC(SC)=NC(NCC=C(C)C)=C2N=CN1C1OC(CO)C(O)C1O VZQXUWKZDSEQRR-UHFFFAOYSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- WIHSZOXPODIZSW-KJIWEYRQSA-N PE(18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z)) Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/C\C=C/C\C=C/CC WIHSZOXPODIZSW-KJIWEYRQSA-N 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 235000021319 Palmitoleic acid Nutrition 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010081690 Pertussis Toxin Proteins 0.000 description 1
- 229920002845 Poly(methacrylic acid) Polymers 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 229930185560 Pseudouridine Natural products 0.000 description 1
- PTJWIQPHWPFNBW-UHFFFAOYSA-N Pseudouridine C Natural products OC1C(O)C(CO)OC1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-UHFFFAOYSA-N 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 1
- 101150099493 STAT3 gene Proteins 0.000 description 1
- 235000019485 Safflower oil Nutrition 0.000 description 1
- 101710084578 Short neurotoxin 1 Proteins 0.000 description 1
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 231100000632 Spindle poison Toxicity 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 101150037787 Sting gene Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- BPEGJWRSRHCHSN-UHFFFAOYSA-N Temozolomide Chemical compound O=C1N(C)N=NC2=C(C(N)=O)N=CN21 BPEGJWRSRHCHSN-UHFFFAOYSA-N 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 102100039360 Toll-like receptor 4 Human genes 0.000 description 1
- 101710182532 Toxin a Proteins 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 102100035100 Transcription factor p65 Human genes 0.000 description 1
- 241000219793 Trifolium Species 0.000 description 1
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 1
- OILXMJHPFNGGTO-ZRUUVFCLSA-N UNPD197407 Natural products C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)C=C[C@H](C)C(C)C)[C@@]1(C)CC2 OILXMJHPFNGGTO-ZRUUVFCLSA-N 0.000 description 1
- HZYXFRGVBOPPNZ-UHFFFAOYSA-N UNPD88870 Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)=CCC(CC)C(C)C)C1(C)CC2 HZYXFRGVBOPPNZ-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- JCZSFCLRSONYLH-UHFFFAOYSA-N Wyosine Natural products N=1C(C)=CN(C(C=2N=C3)=O)C=1N(C)C=2N3C1OC(CO)C(O)C1O JCZSFCLRSONYLH-UHFFFAOYSA-N 0.000 description 1
- 241001441550 Zeiformes Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical class [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- SUTHKQVOHCMCCF-QZNUWAOFSA-N [(2r)-3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-docosa-2,4,6,8,10,12-hexaenoyloxypropyl] docosa-2,4,6,8,10,12-hexaenoate Chemical compound CCCCCCCCCC=CC=CC=CC=CC=CC=CC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)C=CC=CC=CC=CC=CC=CCCCCCCCCC SUTHKQVOHCMCCF-QZNUWAOFSA-N 0.000 description 1
- HOOMGTNENMZAFP-NYNCVSEMSA-N [(2r,3r,5s)-2-(5-amino-2-oxo-[1,3]thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)oxolan-3-yl] acetate Chemical compound CC(=O)O[C@@H]1C[C@@H](CO)O[C@H]1N1C(=O)SC2=CN=C(N)N=C21 HOOMGTNENMZAFP-NYNCVSEMSA-N 0.000 description 1
- UZQJVUCHXGYFLQ-AYDHOLPZSA-N [(2s,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-4-[(2r,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-6-(hydroxymethyl)-4-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-6-(hy Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O)O[C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2[C@@]1(C=O)C)C)(C)CC(O)[C@]1(CCC(CC14)(C)C)C(=O)O[C@H]1[C@@H]([C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O[C@H]4[C@@H]([C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)[C@H](O)[C@@H](CO)O4)O)[C@H](O)[C@@H](CO)O3)O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UZQJVUCHXGYFLQ-AYDHOLPZSA-N 0.000 description 1
- 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 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 108091006088 activator proteins Proteins 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000009824 affinity maturation Effects 0.000 description 1
- 229940042992 afinitor Drugs 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 150000001294 alanine derivatives Chemical class 0.000 description 1
- 229960000548 alemtuzumab Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 description 1
- 230000003281 allosteric effect Effects 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical class [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 229940024545 aluminum hydroxide Drugs 0.000 description 1
- 229940024546 aluminum hydroxide gel Drugs 0.000 description 1
- SMYKVLBUSSNXMV-UHFFFAOYSA-K aluminum;trihydroxide;hydrate Chemical compound O.[OH-].[OH-].[OH-].[Al+3] SMYKVLBUSSNXMV-UHFFFAOYSA-K 0.000 description 1
- 125000000909 amidinium group Chemical group 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003474 anti-emetic effect Effects 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000000611 antibody drug conjugate Substances 0.000 description 1
- 229940049595 antibody-drug conjugate Drugs 0.000 description 1
- 230000005904 anticancer immunity Effects 0.000 description 1
- 239000002111 antiemetic agent Substances 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 235000021342 arachidonic acid Nutrition 0.000 description 1
- 229940114079 arachidonic acid Drugs 0.000 description 1
- PEMQXWCOMFJRLS-RPKMEZRRSA-N archaeosine Chemical compound C1=2NC(N)=NC(=O)C=2C(C(=N)N)=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O PEMQXWCOMFJRLS-RPKMEZRRSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229960003852 atezolizumab Drugs 0.000 description 1
- 230000003190 augmentative effect Effects 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 229950002916 avelumab Drugs 0.000 description 1
- 229960002756 azacitidine Drugs 0.000 description 1
- 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 1
- 230000001580 bacterial effect Effects 0.000 description 1
- SLQKYSPHBZMASJ-UHFFFAOYSA-N bastadin-1 Natural products CC12CCC(O)CC1CCC1=C2CCC2(C)C(C(C)CCC(=C)C(C)C)CCC21 SLQKYSPHBZMASJ-UHFFFAOYSA-N 0.000 description 1
- HDRGJRSISASRAJ-WKPMUQCKSA-N bazlitoran Chemical compound CO[C@@H]1[C@H](O)[C@@H](COP(=O)(S)O[C@@H]2[C@@H](COP(=O)(S)O[C@H]3C[C@@H](O[C@@H]3COP(=O)(S)O[C@H]4C[C@@H](O[C@@H]4COP(=O)(S)O[C@H]5C[C@@H](O[C@@H]5COP(=O)(S)O[C@H]6C[C@@H](O[C@@H]6COP(=O)(S)O[C@H]7C[C@@H](O[C@@H]7COP(=O)(S)O[C@H]8C[C@@H](O[C@@H]8COP(=O)(S)O[C@H]9C[C@@H](O[C@@H]9COP(=O)(S)O[C@H]%10C[C@@H](O[C@@H]%10COP(=O)(S)O[C@@H]%11[C@@H](COP(=O)(S)O[C@@H]%12[C@@H](COP(=O)(S)O[C@H]%13C[C@@H](O[C@@H]%13COP(=O)(S)O[C@H]%14C[C@@H](O[C@@H]%14COP(=O)(S)O[C@H]%15C[C@@H](O[C@@H]%15COP(=O)(S)O[C@H]%16C[C@@H](O[C@@H]%16COP(=O)(S)O[C@H]%17C[C@@H](O[C@@H]%17COP(=O)(S)O[C@H]%18C[C@@H](O[C@@H]%18CO)N%19C=CC(=NC%19=O)N)N%20C=C(C)C(=O)NC%20=O)n%21cnc%22c(N)ncnc%21%22)N%23C=C(C)C(=O)NC%23=O)N%24C=CC(=NC%24=O)N)N%25C=C(C)C(=O)NC%25=O)O[C@H]([C@@H]%12OC)n%26cnc%27C(=O)NC(=Nc%26%27)N)O[C@H]([C@@H]%11OC)N%28C=CC(=O)NC%28=O)N%29C=C(C)C(=NC%29=O)N)n%30ccc%31C(=O)NC(=Nc%30%31)N)N%32C=C(C)C(=O)NC%32=O)N%33C=C(C)C(=O)NC%33=O)N%34C=CC(=NC%34=O)N)N%35C=C(C)C(=O)NC%35=O)N%36C=CC(=NC%36=O)N)N%37C=C(C)C(=O)NC%37=O)O[C@H]([C@@H]2OC)n%38cnc%39C(=O)NC(=Nc%38%39)N)O[C@H]1N%40C=CC(=O)NC%40=O HDRGJRSISASRAJ-WKPMUQCKSA-N 0.000 description 1
- 229940116226 behenic acid Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- WGDUUQDYDIIBKT-UHFFFAOYSA-N beta-Pseudouridine Natural products OC1OC(CN2C=CC(=O)NC2=O)C(O)C1O WGDUUQDYDIIBKT-UHFFFAOYSA-N 0.000 description 1
- MJVXAPPOFPTTCA-UHFFFAOYSA-N beta-Sistosterol Natural products CCC(CCC(C)C1CCC2C3CC=C4C(C)C(O)CCC4(C)C3CCC12C)C(C)C MJVXAPPOFPTTCA-UHFFFAOYSA-N 0.000 description 1
- MVCRZALXJBDOKF-JPZHCBQBSA-N beta-hydroxywybutosine 5'-monophosphate Chemical compound C1=NC=2C(=O)N3C(CC(O)[C@H](NC(=O)OC)C(=O)OC)=C(C)N=C3N(C)C=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O MVCRZALXJBDOKF-JPZHCBQBSA-N 0.000 description 1
- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 description 1
- 229960000397 bevacizumab Drugs 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 229930189065 blasticidin Natural products 0.000 description 1
- 229960003008 blinatumomab Drugs 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- OILXMJHPFNGGTO-ZAUYPBDWSA-N brassicasterol 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)/C=C/[C@H](C)C(C)C)[C@@]1(C)CC2 OILXMJHPFNGGTO-ZAUYPBDWSA-N 0.000 description 1
- 235000004420 brassicasterol Nutrition 0.000 description 1
- 229960000455 brentuximab vedotin Drugs 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- SGNBVLSWZMBQTH-PODYLUTMSA-N campesterol 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)CC[C@@H](C)C(C)C)[C@@]1(C)CC2 SGNBVLSWZMBQTH-PODYLUTMSA-N 0.000 description 1
- 235000000431 campesterol Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229960004562 carboplatin Drugs 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000008568 cell cell communication Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 229940121420 cemiplimab Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940106189 ceramide Drugs 0.000 description 1
- 150000001783 ceramides Chemical class 0.000 description 1
- 229960005395 cetuximab Drugs 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- BHYOQNUELFTYRT-DPAQBDIFSA-N cholesterol sulfate Chemical compound C1C=C2C[C@@H](OS(O)(=O)=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 BHYOQNUELFTYRT-DPAQBDIFSA-N 0.000 description 1
- 150000001841 cholesterols Chemical class 0.000 description 1
- WLNARFZDISHUGS-MIXBDBMTSA-N cholesteryl hemisuccinate Chemical compound C1C=C2C[C@@H](OC(=O)CCC(O)=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 WLNARFZDISHUGS-MIXBDBMTSA-N 0.000 description 1
- 229940080277 cholesteryl sulfate Drugs 0.000 description 1
- SECPZKHBENQXJG-UHFFFAOYSA-N cis-palmitoleic acid Natural products CCCCCCC=CCCCCCCCC(O)=O SECPZKHBENQXJG-UHFFFAOYSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000011254 conventional chemotherapy Methods 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 239000002385 cottonseed oil Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- NLCKLZIHJQEMCU-UHFFFAOYSA-N cyano prop-2-enoate Chemical class C=CC(=O)OC#N NLCKLZIHJQEMCU-UHFFFAOYSA-N 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 229960003901 dacarbazine Drugs 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000001687 destabilization Effects 0.000 description 1
- 150000001982 diacylglycerols Chemical class 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 150000001985 dialkylglycerols Chemical class 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- XRWMGCFJVKDVMD-UHFFFAOYSA-M didodecyl(dimethyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCC XRWMGCFJVKDVMD-UHFFFAOYSA-M 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 229960003983 diphtheria toxoid Drugs 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 239000003534 dna topoisomerase inhibitor Substances 0.000 description 1
- 235000020669 docosahexaenoic acid Nutrition 0.000 description 1
- 229940090949 docosahexaenoic acid Drugs 0.000 description 1
- 231100000294 dose-dependent toxicity Toxicity 0.000 description 1
- 229950009791 durvalumab Drugs 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 1
- 229960005135 eicosapentaenoic acid Drugs 0.000 description 1
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000010201 enrichment analysis Methods 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
- 230000001973 epigenetic effect Effects 0.000 description 1
- RRCFLRBBBFZLSB-XIFYLAFSSA-N epoxyqueuosine Chemical compound C1=C(CN[C@@H]2[C@H]([C@@H](O)[C@@H]3O[C@@H]32)O)C=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O RRCFLRBBBFZLSB-XIFYLAFSSA-N 0.000 description 1
- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 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
- DPUOLQHDNGRHBS-KTKRTIGZSA-N erucic acid Chemical compound CCCCCCCC\C=C/CCCCCCCCCCCC(O)=O DPUOLQHDNGRHBS-KTKRTIGZSA-N 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Chemical compound CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000002191 fatty alcohols Chemical class 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 238000009459 flexible packaging Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 229960000578 gemtuzumab Drugs 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000012178 germinal center formation Effects 0.000 description 1
- 229910001679 gibbsite Inorganic materials 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol Substances OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 229960004956 glycerylphosphorylcholine Drugs 0.000 description 1
- 150000002332 glycine derivatives Chemical class 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- ZKVLEFBKBNUQHK-UHFFFAOYSA-N helium;molecular nitrogen;molecular oxygen Chemical compound [He].N#N.O=O ZKVLEFBKBNUQHK-UHFFFAOYSA-N 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 102000049782 human ESS2 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- DOUYETYNHWVLEO-UHFFFAOYSA-N imiquimod Chemical compound C1=CC=CC2=C3N(CC(C)C)C=NC3=C(N)N=C21 DOUYETYNHWVLEO-UHFFFAOYSA-N 0.000 description 1
- 230000005931 immune cell recruitment Effects 0.000 description 1
- 230000008088 immune pathway Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000000899 immune system response Effects 0.000 description 1
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 230000002480 immunoprotective effect Effects 0.000 description 1
- 230000003116 impacting effect Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 238000010212 intracellular staining Methods 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 1
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 1
- 229940043355 kinase inhibitor Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 229960004488 linolenic acid Drugs 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 229920001427 mPEG Polymers 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 238000011880 melting curve analysis Methods 0.000 description 1
- 210000001806 memory b lymphocyte Anatomy 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 230000000696 methanogenic effect Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- GWKIZNPISGBQGY-GNLDREGESA-N methyl (2S)-4-[4,6-dimethyl-9-oxo-3-[(2R,3R,4S,5R)-2,3,4-trihydroxy-5-(hydroxymethyl)oxolan-2-yl]imidazo[1,2-a]purin-7-yl]-2-(methoxycarbonylamino)butanoate Chemical class O[C@@]1([C@H](O)[C@H](O)[C@@H](CO)O1)N1C=NC=2C(=O)N3C(CC[C@@H](C(=O)OC)NC(=O)OC)=C(C)N=C3N(C)C21 GWKIZNPISGBQGY-GNLDREGESA-N 0.000 description 1
- WCNMEQDMUYVWMJ-UHFFFAOYSA-N methyl 4-[3-[3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-4,6-dimethyl-9-oxoimidazo[1,2-a]purin-7-yl]-3-hydroperoxy-2-(methoxycarbonylamino)butanoate Chemical compound C1=NC=2C(=O)N3C(CC(C(NC(=O)OC)C(=O)OC)OO)=C(C)N=C3N(C)C=2N1C1OC(CO)C(O)C1O WCNMEQDMUYVWMJ-UHFFFAOYSA-N 0.000 description 1
- 125000000250 methylamino group Chemical group [H]N(*)C([H])([H])[H] 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000003122 modulative effect Effects 0.000 description 1
- 238000009126 molecular therapy Methods 0.000 description 1
- 238000013365 molecular weight analysis method Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000002487 multivesicular body Anatomy 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- CYDFBLGNJUNSCC-QCNRFFRDSA-N n-[1-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-2-oxopyrimidin-4-yl]acetamide Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(NC(C)=O)C=C1 CYDFBLGNJUNSCC-QCNRFFRDSA-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
- YZOQZEXYFLXNKA-UHFFFAOYSA-N n-[4-(4-amino-2-ethylimidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide Chemical compound C1=CC=CC2=C(N(C(CC)=N3)CCCCNS(C)(=O)=O)C3=C(N)N=C21 YZOQZEXYFLXNKA-UHFFFAOYSA-N 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229960003301 nivolumab Drugs 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000030147 nuclear export Effects 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 229960002450 ofatumumab Drugs 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 235000021313 oleic acid Nutrition 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 230000014207 opsonization Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 229940124641 pain reliever Drugs 0.000 description 1
- 229960001972 panitumumab Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 229960002621 pembrolizumab Drugs 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 229960002087 pertuzumab Drugs 0.000 description 1
- 150000002994 phenylalanines Chemical class 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000008103 phosphatidic acids Chemical class 0.000 description 1
- 229940067605 phosphatidylethanolamines Drugs 0.000 description 1
- 239000003757 phosphotransferase inhibitor Substances 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 229920002627 poly(phosphazenes) Polymers 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 238000012809 post-inoculation Methods 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000003998 progesterone receptors Human genes 0.000 description 1
- 108090000468 progesterone receptors Proteins 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 229940048914 protamine Drugs 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- PTJWIQPHWPFNBW-GBNDHIKLSA-N pseudouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-GBNDHIKLSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-O pyridinium Chemical compound C1=CC=[NH+]C=C1 JUJWROOIHBZHMG-UHFFFAOYSA-O 0.000 description 1
- 150000004728 pyruvic acid derivatives Chemical class 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- QQXQGKSPIMGUIZ-AEZJAUAXSA-N queuosine Chemical compound C1=2C(=O)NC(N)=NC=2N([C@H]2[C@@H]([C@H](O)[C@@H](CO)O2)O)C=C1CN[C@H]1C=C[C@H](O)[C@@H]1O QQXQGKSPIMGUIZ-AEZJAUAXSA-N 0.000 description 1
- 238000003156 radioimmunoprecipitation Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 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
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- DWRXFEITVBNRMK-JXOAFFINSA-N ribothymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 DWRXFEITVBNRMK-JXOAFFINSA-N 0.000 description 1
- 229960004641 rituximab Drugs 0.000 description 1
- RHFUOMFWUGWKKO-UHFFFAOYSA-N s2C Natural products S=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 RHFUOMFWUGWKKO-UHFFFAOYSA-N 0.000 description 1
- 235000005713 safflower oil Nutrition 0.000 description 1
- 239000003813 safflower oil Substances 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 229940055619 selenocysteine Drugs 0.000 description 1
- ZKZBPNGNEQAJSX-UHFFFAOYSA-N selenocysteine Natural products [SeH]CC(N)C(O)=O ZKZBPNGNEQAJSX-UHFFFAOYSA-N 0.000 description 1
- 235000016491 selenocysteine Nutrition 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- 229960002930 sirolimus Drugs 0.000 description 1
- 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 1
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol 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)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
- 229950005143 sitosterol Drugs 0.000 description 1
- 235000015500 sitosterol Nutrition 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 229940032091 stigmasterol Drugs 0.000 description 1
- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol 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)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 description 1
- 235000016831 stigmasterol Nutrition 0.000 description 1
- BFDNMXAIBMJLBB-UHFFFAOYSA-N stigmasterol Natural products CCC(C=CC(C)C1CCCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(C)C BFDNMXAIBMJLBB-UHFFFAOYSA-N 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 238000012385 systemic delivery Methods 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- ZZIZZTHXZRDOFM-XFULWGLBSA-N tamsulosin hydrochloride Chemical compound [H+].[Cl-].CCOC1=CC=CC=C1OCCN[C@H](C)CC1=CC=C(OC)C(S(N)(=O)=O)=C1 ZZIZZTHXZRDOFM-XFULWGLBSA-N 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 229960004964 temozolomide Drugs 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 210000002303 tibia Anatomy 0.000 description 1
- 230000003614 tolerogenic effect Effects 0.000 description 1
- 229940044693 topoisomerase inhibitor Drugs 0.000 description 1
- 229960005267 tositumomab Drugs 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 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
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000013024 troubleshooting Methods 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 230000010472 type I IFN response Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 150000003667 tyrosine derivatives Chemical class 0.000 description 1
- 150000003668 tyrosines Chemical class 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 229960003636 vidarabine Drugs 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- QAOHCFGKCWTBGC-QHOAOGIMSA-N wybutosine Chemical compound C1=NC=2C(=O)N3C(CC[C@H](NC(=O)OC)C(=O)OC)=C(C)N=C3N(C)C=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O QAOHCFGKCWTBGC-QHOAOGIMSA-N 0.000 description 1
- QAOHCFGKCWTBGC-UHFFFAOYSA-N wybutosine Natural products C1=NC=2C(=O)N3C(CCC(NC(=O)OC)C(=O)OC)=C(C)N=C3N(C)C=2N1C1OC(CO)C(O)C1O QAOHCFGKCWTBGC-UHFFFAOYSA-N 0.000 description 1
- JCZSFCLRSONYLH-QYVSTXNMSA-N wyosin Chemical compound N=1C(C)=CN(C(C=2N=C3)=O)C=1N(C)C=2N3[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O JCZSFCLRSONYLH-QYVSTXNMSA-N 0.000 description 1
- RPQZTTQVRYEKCR-WCTZXXKLSA-N zebularine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=CC=C1 RPQZTTQVRYEKCR-WCTZXXKLSA-N 0.000 description 1
- 239000011701 zinc Chemical class 0.000 description 1
- 229910052725 zinc Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
-
- 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
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/31—Combination therapy
Definitions
- polypeptides comprising a tetramerization motif and a C-terminal cytoplasmic domain of a stimulator of interferon genes (STING) protein.
- the polypeptide further comprises a linker between the tetramerization motif and the C-terminal cytoplasmic domain of a STING protein.
- the linker is a flexible linker.
- polynucleotide encoding the described polypeptide.
- the polynucleotide is RNA.
- the polynucleotide is mRNA.
- the polynucleotide comprises at least one chemically modified nucleotide.
- the at least one chemically modified nucleotide comprises a modified uracil, a 5 -methylcytosine or a combination thereof.
- compositions comprising the described polypeptide or polynucleotide.
- the delivery vehicle comprises a lipid nanoparticle encapsulating the polynucleotide or polypeptide.
- the lipid nanoparticle comprises a cationic lipid, a neutral and/or non-cationic lipid, a sterol, or any combination thereof.
- the non-cationic lipid comprises a phospholipid.
- the sterol comprises cholesterol or a modification or ester thereof.
- the lipid nanoparticle comprises a polyethylene glycol (PEG)-lipid conjugate.
- kits for treating a disease or disorder comprise administering the described polypeptide, polynucleotide, composition or vaccine or medicament to a subject in need thereof.
- the disease or disorder comprises cancer.
- the subject has cancer, has had cancer, is predisposed to cancer, is suspected of having cancer, or has a family history of cancer.
- the cancer comprises breast cancer, lung cancer, skin cancer, liver cancer, or a combination thereof.
- the administering comprises an initial immunization and at least one subsequent immunization. In some embodiments, the administering comprises intratumoral administration and/or systemic administration.
- a disease or disorder or inducing an immune response in a subject comprising administering an effective amount of a Wnt2b blocking agent to a subject in need thereof.
- the administering comprises an initial administration and at least one subsequent administration of the Wnt2b blocking agent.
- the administering comprises intratumoral administration and/or systemic administration.
- the Wnt2b blocking agent is an antibody or an interfering RNA to Wnt2b.
- the disease or disorder comprises cancer.
- the subject has cancer, has had cancer, is predisposed to cancer, is suspected of having cancer, or has a family history of cancer.
- the cancer comprises breast cancer, lung cancer, skin cancer, liver cancer, or a combination thereof.
- FIG. 1 is a schematic of the uniSTING-mRNA/LNP induced constitutive STING activation and the EV-mediated intercommunication between tumor cells and DCs.
- uniSTING-mRNA/LNPs treatment results in the expression of a universal STING mimic in tumor cells and DC cells, which self-assembles into a tetrameric subunit followed by the formation of a higher-order STING architecture for efficient downstream phosphorylation of IRF3 and subsequent release of type I IFNs and ISG cytokines.
- EVs released by uniSTING-treated tumor cells further sensitize DCs’ function in the TME by the delivery of miRNAs including miR-13O-3p, miR-15b-5p, and miR-16-3p that target Wnt2b and reduce immunosuppressive signaling molecules.
- P phosphorylation.
- MVBs multivesicular bodies.
- FIGS. 2A-2L show the characterization of tetramer-based uniSTING as a universal STING activator independent of cGAMP or endogenous STING.
- FIG. 2A is a schematic for uniSTING protein construction by genetically fusing a 52-residue tetramerization motif with the C-terminal cytoplasmic domain of STING. A Flag tag was engineered at the N-terminus to facilitate purification and detection.
- FIG. 2B is a confocal microscopy image showing TBK1 and IRF3 colocalized with uniSTING in the cytosol. TBK1-GFP and IRF3-HA were co-expressed with Flag-uniSTING in HEK293T cells.
- FIG. 2C shows coimmunoprecipitation of uniSTING.
- Cell lysates from DC2.4 cells were incubated with uniSTING bound to anti-Flag M2 resin.
- the interaction of indicated proteins was determined by using anti- phospho-TBKl, anti-pho spho-IRF3, and anti-Flag tag antibody, respectively.
- FIG. 2D is a graph of the top seven enriched Gene Ontology (GO) pathways in DC2.4 cells with uniSTING treatment.
- FIG. 2E is a Venn diagram and volcano plot of altered DEGs in uniSTING-treated group versus Mock in 4T1 tumor cells, showing the percentages of DEGs that are associated with either pIRF3 binding or p-p65 binding.
- FIG. 2F is a graph of selected IRF3 -dependent and NF-KB -dependent pathways in 4T1 tumor cells. Enriched pathways are categorized based on enrichment p values (-log(p value) > 1.3).
- FIG. 2H is immunoblot analysis of STING signaling activation in DC2.4 cells, 4T1 tumor cells, and ES2 tumor cells treated with PBS, mock mRNA (1 pg/ml), mSTING mRNA (1 pg/ml), uniSTING mRNA (1 pg/ml), or 2’3’-cGAMP (5 or 10 pg/ml).
- FIGS. 3A-3O show that the cytosolic delivery of uniSTING-mRNA based on LNPs promotes tumor inhibition in vivo through intratumoral administration.
- FIG. 3A is a schematic of mRNA loaded SS-OP LNPs and a representative Cryo-EM image of uniSTING-mRNA/LNPs. Scale bar, 100 nm.
- FIGS. 3B and 3C show that mRNA is mainly expressed in tumor cells (4T1-GFP) and DCs (CD45 + CDl lc + ) post intratumoral injection of mCherry-mRNA/LNPs (mCherry mRNA: 0.5 mg/kg) as quantified by flow cytometry (FIG. 3B).
- FIG. 3A is a schematic of mRNA loaded SS-OP LNPs and a representative Cryo-EM image of uniSTING-mRNA/LNPs. Scale bar, 100 nm.
- FIGS. 3B and 3C show
- FIG. 3D shows a comparison of in vivo transfection efficiency between SS-OP LNPs and MC3 LNPs.
- the bioluminescence signal was measured by IVIS imaging 24 h and 72 h post intratumoral administration of luciferase-mRNA/LNPs (luciferase mRNA: 0.5 mg/kg) in murine 4T1 tumor models.
- FIG. 4E is a treatment scheme for orthotopic Hepal-6 HCC tumor-bearing mice with the indicated formulations.
- 5xl0 7 of bioluminescence intensity was used as the endpoint criteria in 4T1 liver metastatic tumor model and a 30% weight loss was used as the endpoint criteria in HCC tumor model.
- Each line represents one survival curve for each group; Log-rank (Mantcl-Cox) test.
- Data arc represented as the mean ⁇ s.d. Significant differences were assessed using a one-way ANOVA and Tukey’s multiple comparisons test. Results are presented as mean ⁇ s.d.
- FIGS. 5A-5H show that uniSTING-mRNA/LNPs promotes DC maturation and robust CD8 + T cell responses.
- FIG. 5B shows the impact of intratumoral delivery of uniSTING-mRNA/LNPs on IFN-y and granzyme B (GzmB) expression in CD8 + T cells.
- FIG. 5G is a representative histogram of the flow cytometry analysis of CD62L and CD44 markers in PBMCs with indicated treatment.
- FIG. 6C shows the relative mRNA expression of IFN-P and CXCL10 (FIG. 6D) in DC2.4 cells after co-culturing with supernatant (SUPmock or SUPunisriNG) or EVs (
- FIG. 6F is a scatter plot for miRNA arrays of EVs derived from CM of mock- or uniSTING-treated 4T1 tumor cells with cutoff at threefold. The black line indicates the highly enriched exosomal miRNAs from uniSTING-treated tumor cells.
- Significant differences were assessed using a one-way ANOVA and Tukey’s multiple comparisons test (FIGS. 6B-6D and 6H) and two-way ANOVA with multiple comparisons (FIGS. 6E and 61). Results are presented as mean ⁇ s.d.
- FIG. 7E is graph of integrated scores that predict the association between miRNAs with Wnt2b gene. Higher scores indicate a stronger association.
- FIGS. 7G and 71 show Wnt2b expression in DC2.4 cells after treatment with EVs collected via ultracentrifugation of the CM from 4T1 tumor cells treated with mock + miRiCtr, mock + miRi Pool, uniSTING + miRiCtr, and uniSTING + miRi Pool, analyzed by western blotting (FIG. 7H) and qRT-PCR (FIG. 71).
- FIGS. 8A-8H show that the a-Wnt2b antibody enhances in vivo antitumor activity of STING activation.
- FIG. 8 A is a treatment scheme for 4T1-Luc2 tumor-bearing mice with the indicated formulations.
- FIG. 8 A is a treatment scheme for 4T1-Luc2 tumor-bearing mice with the indicated formulations.
- FIG. 8D is a treatment scheme for LLC tumor-bearing mice with the indicated formulations.
- FIG. 8E is Kaplan-Meier survival curves of LLC tumor-bearing mice treated with indicated formulations.
- FIGS. 8F-8H show the impact of indicated treatments on CD8 + frequency in CD45 + cells (FIG. 8F) and the percentage of TCF1 + CD27 + memory CD8 + T cells (FIG. 8G) in lung tumor bed. The percentage of GzmB expression in CD8 + T cells is shown in FIG. 8H.
- n 3 biologically independent samples. Gating strategies for flow cytometry were displayed in FIG. 25. Significant differences were assessed using a one-way ANOVA and Tukey’s multiple comparisons test (FIGS. 8C and 8F). Results are presented as mean ⁇ s.d.
- FIGS. 10A- 10C show the activity of uniSTING in DC2.4 cells and E0771 tumor cells.
- FIG. 10A is the GSEA enrichment of IFN-a and IFN-y response signatures in DC2.4 cells treated with either mock or uniSTING mRNA. EGFP mRNA was used as mock mRNA control.
- FIG. 10B is immunoblot analysis of STING signaling activation in E0771 tumor cells treated with PBS, mock mRNA (1 pg/ml), mSTING mRNA (1 pg/ml), uniSTING mRNA (1 pg/ml), or 2’3’-cGAMP (5 pg/ml or 10 pg/ml).
- FIG. 10A is the GSEA enrichment of IFN-a and IFN-y response signatures in DC2.4 cells treated with either mock or uniSTING mRNA. EGFP mRNA was used as mock mRNA control.
- FIG. 10B is immunoblot analysis of STING signaling activ
- FIGS. 14A-14D show characterization of SS-OP LNP delivery system in vitro and in vivo.
- FIG. 14A is a graph of size distribution of mRNA-loaded LNPs measured by DLS.
- FIG. 14B shows the quantification of luciferase activity in the DC2.4 cells, E0771, and 4T1 tumor cells 12 h after the transfection of luciferase mRNA loaded SS-OP LNPs (mRNA: Ipg/ml), measured by luciferase assay kit.
- n 6 biologically independent samples.
- One-way ANOVA and Tukey’ s multiple comparisons test.
- FIG. 14A is a graph of size distribution of mRNA-loaded LNPs measured by DLS.
- FIG. 14B shows the quantification of luciferase activity in the DC2.4 cells, E0771, and 4T1 tumor cells 12 h after the transfection of luciferase mRNA loaded SS-OP LNPs (mRNA:
- FIG. 14C shows the gating strategies used for flow cytometry analysis of mCherry + cells in the 4T1 tumor tissue.
- FIGS. 15A-15D show the comparison of monomeric STING with uniSTING in vivo.
- FIG. 15A is a treatment scheme for 4T1-Luc2 tumor-bearing mice with the indicated formulations.
- FIG. 15A is a treatment scheme for 4T1-Luc2 tumor-bearing mice with the indicated formulations.
- FIG. 15B is spider plots of individual tumor growth curves.
- FIG. 15C is a graph of tumor growth burden of mice bearing 4T1-Luc2 tumors receiving different treatments. Two-way ANOVA with multiple comparisons.
- FIG. 19B is a graph of the impact of intratumoral delivery of uSTING LNPs (administered as described in FIG. 3F) on E0771 tumor infiltrating NK1.1 + cells.
- FIG. 19C is a graph of the impact of intratumoral delivery of uSTING LNPs (administered as described in FIG.
- FIGS. 20A and 20B show high level of miR-130a, miR-19a, and miR-16 in human breast cancer correlates with prolonged survival time of cancer patients.
- One-way ANOVA and Tukey’ s multiple comparisons test. Data are represented as the mean ⁇ s.d.
- FIGS. 21A-21F show exosomes derived from uniSTING-treated tumor cells primed antitumor immunity by blocking Wnt2b signaling in DCs.
- FIGS. 21B and 21C show Wnt2b mRNA expression in DC2.4 (FIG. 21B) or BMDC (FIG. 21C) cells pretreated with mock or uniSTING mRNA, upon EXOmock or EXOUSTING addition.
- dual therapy of uniSTING-mRNA/LNP with a-Wnt2b could further replenished CD8 + T cell and its memory subpopulation in tumor local bed, as well as cytotoxic GzmB + CD8 + T cells, compared to that of the combination of ADU-S 100 with a-Wnt2b.
- the robust antitumor immunity makes the uniSTING-mRNA/LNPs an attractive candidate for translational applications, especially for the treatment of STING deficient tumor types.
- Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors.
- Chemotherapeutic agents include compounds used in “targeted therapy” and conventional chemotherapy.
- chemotherapeutic agents include, but are not limited to: cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazinc, cisplatin, carboplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib, everolimus, alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, erlotinib, toremifene, fulvestrant, letrozole, gemcitabine, goserelin, ixabepilone, emtansine, lapatinib, olaparib, megestrol, neratinib, palbociclib, ribociclib
- nucleic acid or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)).
- the present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like.
- the polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced.
- the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single- stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.
- a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat.
- LNA locked nucleic acid
- cyclohcxcnyl nucleic acids see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000), and/or a ribozyme.
- nucleic acid or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand.
- nucleic acid refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
- a “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds.
- Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies.
- the terms “polypeptide” and “protein” are used interchangeably herein.
- sequence identity refers to the extent that sequences are identical on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
- a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (e.g., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
- the identical nucleic acid base e.g., A, T, C, G, I
- the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys,
- sequence similarity or sequence identity between sequences can be performed as follows.
- the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
- the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
- amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions arc then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
- the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
- the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
- the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using an NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
- Another exemplary set of parameters includes a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
- the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
- Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).
- the default parameters of the respective programs e.g., XBLAST and NBLAST can be used.
- amino acid or “any amino acid” as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D- amino acids occur in bacterial envelopes and some antibiotics.
- non-standard natural amino acids include, for example, pyrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts).
- “Unnatural” or “nonnatural” amino acids are non-proteinogenic amino acids (e.g., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible.
- a peptide inhibitor comprises an intramolecular bond between two amino acid residues present in the peptide inhibitor. It is understood that the amino acid residues that form the bond will be altered somewhat when bonded to each other as compared to when not bonded to each other. Reference to a particular amino acid is meant to encompass that amino acid in both its unbonded and bonded state.
- the amino acid residue homoSerine (hSer) or homoSerine(Cl) in its unbonded form may take the form of 2-aminobutyric acid (Abu) when participating in an intramolecular bond according to the present invention.
- amino acids arc referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.).
- L-amino acid refers to the “L” isomeric form of a peptide
- D-amino acid refers to the “D” isomeric form of a peptide (e.g., Dphe, (D)Phe, D-Phe, or U F for the D isomeric form of Phenylalanine).
- Amino acid residues in the D isomeric form can be substituted for any L- amino acid residue, as long as the desired function is retained by the peptide.
- the terms “providing,” “administering,” and “introducing” are used interchangeably herein and refer to the placement of the nanocomplexes and/or compositions of the present disclosure into a subject by a method or route which results in at least partial localization to a desired site.
- mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
- non-mammals include, but are not limited to, birds, fish, and the like.
- the mammal is a human.
- “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a peptide or composition described herein to an appropriate subject. The term also includes a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the disease.
- “treating” means an application or administration of the peptides or compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.
- the present disclosure provides polypeptides comprising STING protein mimics.
- the polypeptides comprise a tetramerization motif and a C-terminal cytoplasmic domain of a STING protein.
- the tetramerization motif and a C-terminal cytoplasmic domain of a STING protein may be linked in any configuration.
- tetramerization domain as used herein is defined as a domain that mediates the formation of a tetramer out of four monomeric proteins or parts thereof. Suitable tetramerization domains include, but are not limited to, tetramerization domains derived from the tetrabrachion protein in Staphylothermus marinus and related organisms, tetramerization domains derived from human potassium voltage-gated channel subfamily A members or orthologs thereof, tetramerization domains derived from human potassium voltage-gated channel subfamily KCNQ1, tetramerization domains derived from human potassium voltage-gated channel subfamily KQT member, tetramerization domains derived from human P53, tetramerization domains derived from human vasodilator- stimulated phosphoprotein, tetramerization domains derived from human acetylcholinesterase, tetramerization domains derived from human butyrylcho
- the tetramerization domain may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-37.
- the tetramerization domain sequence comprises one, two, three, four, five, six, seven, eight, nine, or ten substitutions, truncations, or additions as compared to the sequences disclosed herein.
- the C-terminal cytoplasmic domain of STING comprises an amino acid sequence of at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 98%, at least 99%) similarity to SEQ ID NOs: 38 or 39.
- the C-terminal cytoplasmic domain of STING comprises an amino acid sequence of SEQ ID NO: 38 or 39.
- the amino acid sequence of the C-terminal cytoplasmic domain of STING comprises one or more substitutions, truncations, or additions as compared to SEQ ID NO: 38 or 39.
- amino acid “substitution” or “replacement” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence.
- Amino acids arc broadly grouped as “aromatic” or “aliphatic.”
- An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp).
- Non- aromatic amino acids are broadly grouped as “aliphatic.”
- “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or He), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).
- conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained.
- “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same subgroup.
- substitution of aspartic acid for asparagine, or asparagine for lysine involves amino acids within the same group, but different sub-groups.
- “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.
- the polynucleotide is DNA.
- the polynucleotide is RNA.
- the polynucleotide is a mRNA.
- the disclosure further provides a mRNA comprising an open reading frame encoding the disclosed polypeptide, and compositions thereof.
- the disclosure also provides polynucleotide segments encoding the polynucleotide or mRNA, vectors containing these segments, and cells containing the vectors. The vectors may be used to propagate the segment in an appropriate cell and/or to allow expression from the segment (e.g., an expression vector).
- an expression vector The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.
- the polynucleotide is optimized for enhanced expression, productive co-translational protein folding, increased stability, or a combination thereof.
- the polynucleotide or mRNA is codon-optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; rcmovc/add post translation modification sites in encoded protein (c.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide.
- the polynucleotide or mRNA may further comprise other structures and sequences necessary for proper functionality or stability, including but not limited to a 5’ untranslated region (UTR), a 5’ cap, a 3’ UTR, a 3’ tailing sequence, or any combination thereof.
- UTR untranslated region
- the disclosure provides a mRNA comprising: a 5’ UTR, an open reading frame encoding the disclosed polypeptide, and a 3’ UTR.
- the polynucleotide or mRNA may comprise a 5’ and 3’ untranslated region (UTR). Sequence elements within the UTRs affect translational efficiency and RNA stability but do not encode a polypeptide.
- the 3’ UTR relates to the region located at the 3’ end of the mRNA, downstream of the termination codon of a protein-encoding region, which is transcribed but not translated into an amino acid sequence.
- the 5 ’UTR refers to the region directly upstream from the initiation codon.
- Eukaryotic 5’ UTRs contain the Kozak consensus sequence (ACCAUGG), which contains the initiation codon.
- the polynucleotide or mRNA may comprise a 3’ tailing sequence.
- the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.
- the polynucleotide or mRNA further comprises a triple helix forming sequence.
- a tiple helix is formed after the binding of a third strand to the major groove of a duplex nucleic acid through Hoogsteen base pairing (e.g., hydrogen bonds) while maintaining the duplex structure of two strands making the major groove.
- Pyrimidine-rich and purine-rich sequences e.g., two pyrimidine tracts and one purine tract or vice versa
- can form stable triplex structures as a consequence of the formation of triplets e.g., A-U-A and C-G-C).
- the triple helix sequence is derived from the 3’ terminal triple helix sequences of triple helix terminators from a long non-coding RNAs (IncRNAs), e.g., metastasis-associated lung adenocarcinoma transcript 1 (MALAT1).
- IncRNAs long non-coding RNAs
- MALAT1 metastasis-associated lung adenocarcinoma transcript 1
- the polynucleotide or mRNA further comprises a tRNA-like sequence.
- the tRNA-like sequences are those sequences which form similar overall secondary and tertiary structure to tRNA.
- the tRNA-like sequence is derived from a long noncoding RNAs (IncRNAs), e.g., MALAT1.
- the tRNA-like sequence derived from IncRNAs may be truncated or modified as long as they retain the clover secondary structure.
- the MALAT1 sequences for the triple helix or the tRNA-like sequence can be from any species.
- the MALAT1 sequences are from a human.
- the MALAT1 sequences are from a mouse.
- the MALAT1 sequences are from a non-human primate.
- the 3’ poly(A) sequence of mRNA is important for nuclear export, RNA stability and translational efficiency of eukaryotic messenger RNA (mRNA).
- the poly(A) tail is a segment of RNA at the 3’ end of the molecule that has only adenine bases.
- a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates.
- the poly(A) tail may contain two segments of only adenine bases separated by a linker.
- the polynucleotide or mRNA comprises at least one chemical modification or chemically modified base, nucleoside, or nucleotide.
- the chemical modifications may comprise any modification which is not naturally present in said RNA or any naturally-occurring modification of adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribonucleosides.
- A adenosine
- G guanosine
- U uridine
- C cytidine
- a single polynucleotide or mRNA may include both naturally-occurring and non-naturally- occurring modifications.
- the at least one chemical modification comprises a modified uridine residue.
- modified uridine residues include, but are not limited to, pseudouridine, 1- methylpseudouridine, 1 -ethylpseudouridine, 2-thiouridine, 4’- thiouridine, 5-methyluridine, 2-thio-l - methyl- 1-deaza-pseudouridine, 2- thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2’-0-methyl uridine
- the at least one chemical modification comprises a modified cytosine residue.
- exemplary nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5- methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza-pseudoisocytidine, 1-
- the at least one chemical modification comprises a modified adenine residue.
- exemplary nucleosides having a modified adenine include 2-amino-purine, 2,6- diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido-adenosine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7- deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl-adenosine, 2-methylthio- N6-isopentenyl-aden
- the at least one chemical modification comprises a modified guanine residue.
- exemplary nucleosides having a modified guanine include inosine, 1-methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, undermodified hydroxywybutosine, 7-deaza-guanosine, queuosine, epoxy queuo sine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deaza-guanosine, 7- aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
- compositions comprising the described polypeptides or polynucleotides.
- the compositions may further comprise excipients or pharmaceutically acceptable carriers.
- excipients or pharmaceutically acceptable carriers will depend on factors including, but not limited to, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
- Excipients and carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents.
- materials which can serve as excipients and/or carriers are sugars including, but not limited to, lactose, glucose and sucrose; starches including, but not limited to, com starch and potato starch; cellulose and its derivatives including, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients including, but not limited to, cocoa butter and suppository waxes; oils including, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; including propylene glycol; esters including, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents including, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’
- polypeptides, polynucleotides (e.g., mRNA), or compositions thereof described herein may be used to prepare vaccines or another medicament.
- adjuvants such as cytokines (e.g., GM-CSF or interleukin-2, -7, or -12), interferons, or tumor necrosis factor, may also be used as adjuvants.
- cytokines e.g., GM-CSF or interleukin-2, -7, or -12
- interferons e.g., interferon-associated antigen, tumor necrosis factor
- tumor necrosis factor e.g., tumor necrosis factor
- Protein and polypeptide adjuvants may be obtained from natural or recombinant sources according to methods well known to those skilled in the art. When obtained from recombinant sources, the adjuvant may comprise a protein fragment comprising at least the immunostimulatory portion of the molecule.
- the vaccines or medicaments may further comprise a delivery vehicle.
- exemplary delivery vehicles include, but are not limited to, microparticle compositions comprising poly(lactic acid) (PLA) and/or poly(lactic-co-glycolic acid) (PLGA), albumin nanoparticles, and liposomal compositions.
- the vaccines comprise a lipid nanoparticle encapsulating the disclosed polypeptides, polynucleotides (e.g., mRNA).
- Lipid nanoparticle compositions of the disclosure may include one or more cationic and/or ionizable lipids, phospholipids, neutral or non-cationic lipids, polyethylene glycol (PEG)-lipid conjugates, and/or sterols.
- the lipid nanoparticle comprises a cationic lipid and/or ionizable lipid, a neutral or non-cationic lipid, and cholesterol.
- DOTMA 1.2-di-O-octadecenyl-3 -trimethylammonium propane
- DOTAP l,2-dioleoyl-3- trimethylammonium propane
- DMTAP 1 ,2-dimyristoyl-3 -trimethylammonium propane
- Ionizable lipids include, but are not limited to, 1 ,2-dioleyloxy-3 -dimethylamino-propane (DODMA).
- a neutral or non-cationic lipid may include one or more phospholipids.
- Phospholipids include a phospholipid moiety and one or more fatty acid moieties.
- a phospholipid moiety may include, but is not limited to, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin.
- Phospholipids suitable for use in the compositions may include, but are not limited to, phosphatidylglycerol (PG) including dimyristoyl phosphatidylglycerol (DMPG) and 1,2-dioleoyl-sn- glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecano
- the positively charged lipid structures described herein may also include other components typically used in the formation of vesicles (e.g., for stabilization).
- other components includes, without being limited thereto, fatty alcohols, fatty acids, and/or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the lipid into the lipid assembly.
- the vaccine or medicament of the present disclosure may also contain other compounds, which may be biologically active or inactive.
- one or more immunogenic and antigenic portions of polypeptides or polynucleotides encoding immunogenic polypeptides, or nucleic acid(s) encoding thereof may be present within the vaccines or medicaments.
- the vaccine or medicament may generally be used for prophylactic and therapeutic purposes.
- compositions, vaccines, or medicaments may be prepared, packaged, or sold in a form suitable for bolus administration or sold in unit dosage forms, such as in ampules or multi-dose containers.
- the compositions and vaccines contain a preservative.
- a humoral immune response refers generally to antibody production, and to all of the processes that accompany antibody production, including, but not limited to, B lymphocyte (B cell) activation, affinity maturation, differentiation into plasma cells, and memory B cell generation, germinal center formation and isotype switching, and T helper cell activation, signaling, and cytokine production, as well as effector functions of antibodies, which include neutralization, classical complement activation, and opsonization.
- B lymphocyte B cell activation
- affinity maturation differentiation into plasma cells
- memory B cell generation germinal center formation and isotype switching
- T helper cell activation signaling
- cytokine production as well as effector functions of antibodies, which include neutralization, classical complement activation, and opsonization.
- the administration may comprise an initial immunization or dose and at least one subsequent immunization or booster dose, following known standard immunization protocols.
- the boosting doses will be adequately spaced at such times where the levels of circulating antibody fall below a desired level.
- Boosting doses may consist of the compositions or vaccines disclosed herein and may comprise alternative carriers and/or adjuvants.
- the booster dosage levels may be the same or different that those of the initial dosage.
- the second therapy (e.g., an immunotherapy) may be administered at the same time as the initial therapy.
- the second therapy may be administered in the same composition or in a separate composition administered at substantially the same time as the initial therapy.
- the second therapy may precede or follow administration of the disclosed polynucleotides, polypeptides, compositions, vaccines, medicaments, and methods by time intervals ranging from hours to months.
- the methods further comprise administration of a Wnt2b antibody or fragment thereof or an interfering RNA to Wnt2b.
- the Wnt2b inhibitor comprises an antisense oligonucleotide, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a guide RNA (gRNA), and combinations thereof.
- the disease or disorder comprises cancer.
- the subject is a human.
- the subject has cancer, has had cancer, is predisposed to cancer, is suspected of having cancer, or has a family history of cancer.
- the cancer comprises a solid tumor.
- cancers that comprise solid tumors include, but are not limited to, pancreatic, bladder, non-small cell lung cancer (NSCLC), breast and ovarian cancers.
- the cancer is metastatic cancer.
- the methods result in decreased tumor growth.
- the methods result in tumor regression.
- the methods result in decreased numbers of tumor.
- the methods result in decreased tumor growth.
- the methods prevent tumor recurrence.
- the methods result in increases in overall subject survival.
- the methods result in long-term anti-tumor efficacy.
- the methods comprise an initial administration and at least one subsequent administration of the Wnt2b blocking agent.
- administration may be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and/or intraperitoneal administration to a subject in need thereof, some embodiments, administration includes parenteral administration (including, but not limited to, subcutaneous, intramuscular, intravenous, intraperitoneal, intracardiac and intraarticular injections).
- the administration is intratumoral.
- the administration is systemic.
- a wide range of second therapies may be used in conjunction with the Wnt2b blocking agent.
- the second therapy may be administration of an additional active agent or may be a second therapy not connected to administration of another agent.
- Such second therapies include, but are not limited to, surgery, immunotherapy, radiotherapy.
- Exemplary immune modulators include: STING agonists, signal transducer and activator of transcription 3 (Stat3) inhibitors and analogs thereof, toll-like receptor (TLR) agonists and analogs thereof, SM-360320, TMX-101, TMX-202, TMX-302, TMX-306, GSK2245035, CL097, 852A, AZD- 8848, DSP-3025, GS-9620, R07020531, RO6871765, ANA773, DSP-0509, NJH395, BNT411, TQ- A3334, JNJ-4964, LHC165, CV8102, VTX-1463, VTX-2337, IMO-8400, IMO-3100, IRS-954, and analogs thereof; and statins or other lipid-lowering medications and analogs thereof.
- Stat3 signal transducer and activator of transcription 3
- TLR toll-like receptor
- the additional active agent comprises at least one chemotherapeutic agent.
- chemotherapeutic or “anti-cancer drug” includes any small molecule or other drug used in cancer treatment or prevention.
- Chemotherapeutic s include, but are not limited to, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazine, cisplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib,
- Armolimus alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, toremifene, fulvestrant, letrozole, gemcitabine, gose
- the immunotherapy may comprise an antibody targeting, for example, CD20, CD33, CD52, CD30, HER (also referred to as erbB or EGFR), VEGF, CTLA-4 (also referred to as CD 152), epithelial cell adhesion molecule (EpCAM, also referred to as CD326), and PD- 1/PD-L1.
- an antibody targeting for example, CD20, CD33, CD52, CD30, HER (also referred to as erbB or EGFR), VEGF, CTLA-4 (also referred to as CD 152), epithelial cell adhesion molecule (EpCAM, also referred to as CD326), and PD- 1/PD-L1.
- the additional therapeutic agent may comprise anti-PD-l/PD-Ll antibodies, including, but not limited to, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab.
- the antibodies may also be linked to a chemotherapeutic agent.
- the antibody is an antibody-drug conjugate.
- compositions, polypeptides, polynucleotides, vaccines, or medicaments disclosed herein are also disclosed herein.
- kits can also comprise other agents and/or products co-packaged, co-formulated, and/or co-delivered with other components.
- a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed peptide and/or product and another agent (e.g., a chemotherapeutic, a monoclonal antibody, a pain reliever, a steroid, an antiemetic) for delivery to a patient or a cell.
- another agent e.g., a chemotherapeutic, a monoclonal antibody, a pain reliever, a steroid, an antiemetic
- kits can also comprise instructions for using the components of the kit.
- the instructions are relevant materials or methodologies pertaining to the kit.
- the materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents.
- Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
- kits can be employed in connection with the disclosed methods.
- the kit may further contain containers or devices for use with the methods or compositions disclosed herein.
- the kits optionally may provide additional components such as buffers and disposable single-use equipment (e.g., pipettes, cell culture plates, flasks).
- the kits comprise a delivery device or container.
- the delivery device or container comprises a syringe or syringe vial.
- the delivery device or container is pre-filled with the composition.
- kits provided herein are in suitable packaging.
- suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Individual member components of the kits may be physically packaged together or separately.
- Cell culture 4T1 wild-type, 4T1-Luc2, and HEK293T cell lines were obtained from ATCC.
- ExpiCHO cell line was obtained from ThemoFisher Scientific.
- DC2.4 cell line was obtained from Millipore Sigma.
- E0771 cell line was from Dr. Jenny P.-Y. Ting’s lab at UNC Chapel Hill.
- 4T1 wildtype cells were stably transfected with a vector carrying the GFP and the puromycin resistance gene.
- 4T1-Luc2 cells were cultured in RPMI1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 8 pg/ml blasticidin at 37 °C and 5% CO2 in a humidified atmosphere.
- FBS fetal bovine serum
- 4TLGFP cells were cultured in RPMI1640 supplemented with 10% FBS and 1 pg/ml puromycin at 37 °C and 5% CO2 in a humidified atmosphere.
- 4T1 wild-type cells were cultured in RPMI1640 supplemented with 10% FBS at 37 °C and 5% CO2 in a humidified atmosphere.
- DC2.4 cells were cultured with RPMI1640 supplemented with 10% FBS, lx non-essential amino acids (Gibco), lx HEPES buffer (Gibco), and 0.0054x p -mercaptoethanol (Gibco).
- bone marrow cells were resuspended in RPMI1640 medium containing 10% heat-inactivated FBS, murine recombinant GM- CSF (20 ng/ml, BioLegend) and 1% antibiotic-antimycotic (Gibco) and distributed into 100 mm Petri- dishes at a density of 3xl0 6 cells per dish for 6 days. Every 2 days, cells were washed and resupplemented with fresh complete medium. On day 6, highly adherent cells were discarded and CD1 lc + F4/80“ cells were sorted.
- mice that could be cured were inoculated with IxlO 5 B16F10 cells for tumor rechallenging. Tumor size was directly measured by using a digital caliper.
- 5xl0 6 Hepal-6 cells in 30 pl of PBS were inoculated at the subcapsular region of the left lobes of the liver in C57BL/6J.
- mice were randomly assigned to treatment groups. The investigators were blinded to the group allocation during the animal experiments.
- the constitutively active STING mimic protein was designed by genetically fusing a thermostable tetramerization motif (GIINETADDIVYRLTVIIDDRYESLKNLITLRADRLEMIINDNVSTILASIG - SEQ ID NO: 1) with the C-terminal cytoplasmic domain of STING (residues 138-378 of murine STING).
- a Flag tag was engineered at the N-terminus to facilitate protein purification and detection.
- the expression vectors encoding the fusion protein were generated by inserting synthetic DNAs (codon optimized for expression in mammalian cells) into pCDNA3.4 vector.
- the purified proteins were analyzed on 4-12% SDS-PAGE gel (Invitrogen) with Coomassie G-250 (Bio-Rad) stain. Molecular weight analysis was performed by FPLC (GE AKTATM) by using a Superdex 200 10/300 GL gel filtration column (MilliporeSigma). 200 pg of each standard (FIG. 9B) and 400 pg of uniSTING protein was dissolved in 200 pl of TBS (50 mM Tris HC1, 150 mM NaCl, pH 7.4) buffer with 0.02% Tween 80 and filtered through 0.45 pm filter, respectively.
- TBS 50 mM Tris HC1, 150 mM NaCl, pH 7.4
- the protein samples were then injected to the column and separated at a flow rate of 0.3 ml/min and detected at 280 nm— .
- Molecular weight of uniSTING was finally determined from the standard curve.
- the melting temperature (T m ) of uniSTING was measured by using Protein Thermal ShiftTM (PTS) kit (ThermoFisher Scientific) according to the manufacturer’s protocol. Briefly, uniSTING was mixed with different buffers (FIG. 9C) and PTS dye, and a mclt-curvc experiment was run on a real-time PCR instrument (Analytik Jena, qTOWER 3 G). The T m was calculated from the melt curve based on Protein Thermal ShiftTM Software (ThermoFisher Scientific).
- ELISA For ELISA, flat-bottomed 96-well plates (ThermoFisher Scientific) were precoated with anti-Flag-tag monoclonal antibody (ThermoFisher Scientific) at a concentration of 0.5 pg/ml per well in 100 mM carbonate buffer (pH 9.6) at 4 °C overnight. 5% BSA in PBST was then used to block the non-specific binding for 1 h at room temperature. The samples were diluted 50 times in PBST buffer and added to the wells.
- IFN-0 expression in DCs and tumor cells after treatment with either PBS, EGFP-mRNA (mock), uniSTING-mRNA, mSTING- mRNA, or free cGAMP was also measured by using mouse IFN-P ELISA Kit (R&D systems) according to the manufacturer’s instructions.
- Flow cytometry assay Tumor tissues were harvested and digested in RPMI1640 medium containing 2% heat-inactivated FBS, collagenase type I (200 U/ml, Invitrogen), collagenase type IV (200 U/ml, Invitrogen), and DNAase I (100 pg/ml, Invitrogen) in 2% FBS at 37 °C for 1 h to generate single-cell suspensions.
- the tumor-infiltrating lymphocytes were isolated by ficoll-paque density gradient centrifugation and diluted to IxlO 6 cells/ml for staining with LIVE/DEADTM Fixable Near-IR dye, followed by surface staining with fluorescently conjugated antibodies.
- BMDCs For sorting BMDCs, cells were stained with BV510 anti-mouse CD45, APC anti-mouse CD11c, and PE anti-mouse F4/80 and sorted according to the gating strategy displayed in FIGS. 20 and 18. Dead cells were excluded via LIVE/DEAD fixable dye staining.
- STING high mobility allows its trafficking through subcellular organelle membranes which initiates a chain reaction of cellular immune pathways. It is reported proinflammatory NF-KB signal occurs when STING is in the ER or ERGIC which is not necessary for IRF3 transcriptional activity.
- uniSTING was designed to form a tetrameric structure independent of membrane association. NF-KB phosphorylation was observed in the group treated with 2’3’-cGAMP, whereas there was no substantial activation of NF-KB signaling after treatment with uniSTING (FIG. 2F).
- RNA-seq was performed on 4T1 tumor cells via an unbiased analysis.
- IRF3 -dependent signalings were extensively upregulated by uniSTING treatment (p ⁇ 0.01) while NF-KB target signaling was unaffected by uniSTING.
- both uniSTING and cGAMP treatments elevated IFN- and CXCE10 transcripts (FIG. 2G).
- cGAMP treatment increased the production of IE6 and TNF-a, whereas uniSTING treatment showed no discernible change in these downstream molecules of NF-KB signaling (FIG. 2G).
- mRNA encoding uniSTING was encapsulated into LNPs (uniSTING mRNA/LNPs) based on ionizable lipid- COATSOME SS-OP for efficient intracellular delivery.
- SS-OP contains two sensing motifs: a tertiary amine that is responsive for endosomal membrane destabilization and a disulfide bond that can be cleaved by GSH in a reductive environment (cytoplasm) for spontaneous particle collapse.
- the resulting mRNA/LNPs represent surface-neutral particles with a median diameter of -100 nm (FIGS. 3A and 14A).
- uniSTING was enriched in tumors compared to that in nontumor tissues or plasma.
- Intratumoral injection of 10 pg of LNP-formulated mRNA resulted in peak production of uniSTING protein after 4 hrs with decreased yet detectable measurements that lasted up to day 7 (FIG. 3E).
- uniSTING protein was detected in the liver from 2 hr to 72 hr post injection (FIG. 9D). This solved the poor stability problem of the current STING agonist by demonstrating continuous translation of the mRNA inside the transfected cells prior to its degradation.
- An orthotopic HCC liver cancer model was also constructed by inoculating three million Hcpal-6 tumor cells into the subcapsular region of the left lobes of the liver.
- Intravenous administration with uniSTING-mRNA/LNPs significantly reduced HCC tumor burden and prolonged survival in comparison with PBS, mock, or cGAMP groups (FIGS. 4E-4G).
- uniSTING-mRNA/LNPs effectively restrained advanced orthotopic/metastatic tumors by systemic administration.
- mice were vaccinated with or without uniSTING-mRNA/LNPs combined with ovalbumin (OVA) antigens.
- OVA ovalbumin
- FIG. 5D ADU-S100, a STING agonist that has been used in clinical trials, was used as a control. Consistent with tumor studies (FIG. 5A), uniSTING-mRNA/LNPs increased CD8 + T cells in PBMC compared to the ADU-S100 treated group (FIG. 5E). Additionally, uniSTING treatment decreased the level of CD44 CD62L + naive CD8 T cell without obvious change for CD44 + CD62L + central memory subpopulation (FIG. 5F).
- RNA-seq of DC2.4 cells was conducted post exposure to EXOuniSTiNG or EXOmock. More than 2000 significantly altered genes were identified. Indeed, EXOuniSTiNG regulated the expression of a major set of genes involved in IFN signaling responses, including Ifit2, Ifit3, Isgl5, and Uspl8 (FIG. 7A). GSEA results showed IFN- a response genes were markedly enriched in recipient DC2.4 cells exposed to EXOunisi iNG (FIG. 7A).
- DC2.4 cells drastically downregulated Wnt signaling-associated genes, particularly Wnt2b and Snail, after exposure to EXOuniSTiNG (FIG. 7B). It has been demonstrated that Wnt signaling activation in TME can drive DCs into tolerogenic regulatory state, increase infiltration of regulatory T cells, and impair differentiation of CD8 + effector T cells. Wnt2b transcripts were examined in DC2.4 cells after indicated treatments to validate the RNA-seq result.
- Wnt2b serves as a regulatory signal for type I IFN response, in line with the earlier finding that Wnt2b negatively regulated the IFN-P signaling.
- Wnt2b levels were correlated with poor prognosis in human breast cancer (FIG. 21F).
- Wnt2b blockade enhances in vivo antitumor activity of uniSTING
- Wnt signaling is one of the well-known oncogenic drivers in many cancer types, notably suppressing the maturation and differentiation of DCs and T cells. Whether Wnt2b blockade further enhanced antitumor activity of uniSTING was investigated. The therapeutic efficacy of uniSTING- mRNA/LNP or/and a-Wnt2b antibody was evaluated by treating mice bearing 4T1-Luc2 tumors (FIGS. 8A-8C).
- Wnt2b blockade together with uniSTING-mRNA/LNPs further drastically increased CD8 + T cells frequency in CD45 + cells as well as the TCF1 + CD27 + memory subpopulation in lung tumor bed (FIGS. 8F-8G).
- Tetramerization domains derived from human potassium voltage-gated channel subfamily A members (the orthologs of these sequences in other organisms, including in mouse and rat can also be used)
- Tetramerization domain derived from measles virus phosphoprotein YDDELFSDVQDIKTALAKIHEDNQKIISKLESLLLLKGEVESIKKQINRQNISI - SEQ ID NO: 17
- the underlined sequence is the tetramerization domain/motif that can be replaced with other tetramerization domain/motif disclosed herein.
- the italic sequence is the flexible linker.
Abstract
The present disclosure provides STING protein mimics, polynucleotides encoding thereof, and compositions, vaccines and kits comprising thereof for stimulating an immune response and treating a disease or disorder, particularly cancer.
Description
CONSTITUTIVELY ACTIVE POLYMERIC STING MIMICS FOR ANTITUMOR IMMUNITY
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/350,714, filed June 9, 2022, the content of which is herein incorporated by reference in its entirety.
SEQUENCE LISTING STATEMENT
[0002] The contents of the electronic sequence listing titled 40903_601_ST26.xml (Size: 42.073 bytes; and Date of Creation: June 2, 2023) is herein incorporated by reference in its entirety.
FIELD
[0003] The present disclosure relates to STING protein mimics, polynucleotides encoding thereof, and compositions, vaccines and kits comprising thereof for stimulating an immune response and treating a disease or disorder, particularly cancer.
BACKGROUND
[0004] Stimulator of interferon genes (STING) is a major regulator that induces the release of type I interferon (IFN) and other proinflammatory cytokines in response to cytosolic DNAs derived from pathogens and cancer cells. Recent studies have demonstrated that the activation of STING and the secretion of type I IFN play a critical role in anticancer immunity. STING can be potently activated by its natural ligand cyclic guanosine monophosphate- adenosine monophosphate (cGAMP) or by cyclic dinucleotide (CDN) analogs. The therapeutic potential of CDN-based STING agonists is being examined in several clinical trials. However, the poor stability and dose-dependent toxicity of these compounds make their clinical applications a major undertaking. Recently, orally available nonnucleotide STING agonists with improved antitumor efficacy and broadened indications have been reported. Despite therapeutic promise, all the CDN- and non-nucleotide-based STING agonists rely on the presence of sufficient endogenous STING in the tumor microenvironment (TME) to take effect. Several studies have shown that STING signaling pathways in tumor cells are suppressed due to the epigenetic silencing of either STING or its upstream regulator, the cyclic GMP-AMP synthase (cGAS), which catalyzes the synthesis of cGAMP. The low expression and deficiency of STING in TME suppress downstream signaling responses despite treatment with small molecule STING agonists. Thus, there is an urgent unmet need to develop novel STING stimulators that can activate the STING-mediated immune response independent of the availability of endogenous STING, aiming at a universal STING activation strategy that can be applied to a wide variety of cancer types.
SUMMARY
[0005] Disclosed herein are polypeptides comprising a tetramerization motif and a C-terminal cytoplasmic domain of a stimulator of interferon genes (STING) protein. In some embodiments, the polypeptide further comprises a linker between the tetramerization motif and the C-terminal cytoplasmic domain of a STING protein. In some embodiments, the linker is a flexible linker.
[0006] In some embodiments, the tetramerization motif is N-terminal to the C-terminal cytoplasmic domain of a STING protein. In some embodiments, the tetramerization motif comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-37. In some embodiments, tetramerization motif comprises an amino acid sequence of SEQ ID NO: 1.
[0007] In some embodiments, the C-terminal cytoplasmic domain of a STING protein comprises an amino acid sequence of at least 70% similarity to SEQ ID NOs: 38 or 39. In some embodiments, the C- tcrminal cytoplasmic domain of a STING protein comprises an amino acid sequence of SEQ ID NO: 38 or 39.
[0008] Also disclosed herein is a polynucleotide encoding the described polypeptide. In some embodiments, the polynucleotide is RNA. In some embodiments, the polynucleotide is mRNA.
[0009] In some embodiments, the polynucleotide further comprises or encodes: a 5’ untranslated region (UTR), a 5’ cap, a 3’ UTR, a 3’ tailing sequence, or any combination thereof. In some embodiments, the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.
[0010] In some embodiments, the polynucleotide comprises at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide comprises a modified uracil, a 5 -methylcytosine or a combination thereof.
[0011] Further provided herein are compositions comprising the described polypeptide or polynucleotide.
[0012] Additionally provided are vaccines or medicaments comprising the described polypeptide, polynucleotide, or composition. In some embodiments, the vaccines or medicaments further comprise at least one adjuvant. In some embodiments, the vaccines or medicaments further comprise a delivery vehicle.
[0013] In some embodiments, the delivery vehicle comprises a lipid nanoparticle encapsulating the polynucleotide or polypeptide. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a neutral and/or non-cationic lipid, a sterol, or any combination thereof. In some embodiments, the non-cationic lipid comprises a phospholipid. In some embodiments, the sterol comprises cholesterol or
a modification or ester thereof. In some embodiments, the lipid nanoparticle comprises a polyethylene glycol (PEG)-lipid conjugate.
[0014] In another aspect, provided herein are methods of treating a disease or disorder and methods of inducing an immune response. The methods comprise administering the described polypeptide, polynucleotide, composition or vaccine or medicament to a subject in need thereof.
[0015] In some embodiments, the disease or disorder comprises cancer. In some embodiments, the subject has cancer, has had cancer, is predisposed to cancer, is suspected of having cancer, or has a family history of cancer. In some embodiments, the cancer comprises breast cancer, lung cancer, skin cancer, liver cancer, or a combination thereof.
[0016] In some embodiments, the methods further comprise administration of a Wnt2b blocking agent based on an antibody or non-immunoglobulin scaffold, or an interfering RNA to Wnt2b.
[0017] In some embodiments, the administering comprises an initial immunization and at least one subsequent immunization. In some embodiments, the administering comprises intratumoral administration and/or systemic administration.
[0018] Additionally disclosed are methods for treating a disease or disorder or inducing an immune response in a subject comprising administering an effective amount of a Wnt2b blocking agent to a subject in need thereof. In some embodiments, the administering comprises an initial administration and at least one subsequent administration of the Wnt2b blocking agent. In some embodiments, the administering comprises intratumoral administration and/or systemic administration. In some embodiments, the Wnt2b blocking agent is an antibody or an interfering RNA to Wnt2b.
[0019] In some embodiments, the disease or disorder comprises cancer. In some embodiments, the subject has cancer, has had cancer, is predisposed to cancer, is suspected of having cancer, or has a family history of cancer. In some embodiments, the cancer comprises breast cancer, lung cancer, skin cancer, liver cancer, or a combination thereof.
[0020] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a schematic of the uniSTING-mRNA/LNP induced constitutive STING activation and the EV-mediated intercommunication between tumor cells and DCs. uniSTING-mRNA/LNPs treatment results in the expression of a universal STING mimic in tumor cells and DC cells, which self-assembles into a tetrameric subunit followed by the formation of a higher-order STING architecture for efficient downstream phosphorylation of IRF3 and subsequent release of type I IFNs
and ISG cytokines. EVs released by uniSTING-treated tumor cells further sensitize DCs’ function in the TME by the delivery of miRNAs including miR-13O-3p, miR-15b-5p, and miR-16-3p that target Wnt2b and reduce immunosuppressive signaling molecules. P, phosphorylation. MVBs, multivesicular bodies.
[0022] FIGS. 2A-2L show the characterization of tetramer-based uniSTING as a universal STING activator independent of cGAMP or endogenous STING. FIG. 2A is a schematic for uniSTING protein construction by genetically fusing a 52-residue tetramerization motif with the C-terminal cytoplasmic domain of STING. A Flag tag was engineered at the N-terminus to facilitate purification and detection. FIG. 2B is a confocal microscopy image showing TBK1 and IRF3 colocalized with uniSTING in the cytosol. TBK1-GFP and IRF3-HA were co-expressed with Flag-uniSTING in HEK293T cells. Immunofluorescence staining was performed by using anti-HA antibody (purple), anti-Flag tag antibody (red), and DAPI for the nucleus (blue). Scale bar, 10 pm (left images). Magnified images of the areas indicated by the white boxes. Scale bar, 2 pm (Right images). FIG. 2C shows coimmunoprecipitation of uniSTING. Cell lysates from DC2.4 cells were incubated with uniSTING bound to anti-Flag M2 resin. The interaction of indicated proteins was determined by using anti- phospho-TBKl, anti-pho spho-IRF3, and anti-Flag tag antibody, respectively. FIG. 2D is a graph of the top seven enriched Gene Ontology (GO) pathways in DC2.4 cells with uniSTING treatment. FIG. 2E is a Venn diagram and volcano plot of altered DEGs in uniSTING-treated group versus Mock in 4T1 tumor cells, showing the percentages of DEGs that are associated with either pIRF3 binding or p-p65 binding. FIG. 2F is a graph of selected IRF3 -dependent and NF-KB -dependent pathways in 4T1 tumor cells. Enriched pathways are categorized based on enrichment p values (-log(p value) > 1.3). FIG. 2G is a graphs of the expression of transcripts of IFN- , CXCL10, IL6, and TNF-a in 4T1 tumor cells 24 h after indicated treatments, n = 6 biologically independent samples. FIG. 2H is immunoblot analysis of STING signaling activation in DC2.4 cells, 4T1 tumor cells, and ES2 tumor cells treated with PBS, mock mRNA (1 pg/ml), mSTING mRNA (1 pg/ml), uniSTING mRNA (1 pg/ml), or 2’3’-cGAMP (5 or 10 pg/ml). FIG. 21 is graphs of IFN-P levels measured by ELISA in murine DCs, 4T1 tumor cells, and human ES2 tumor cells 24 h after indicated treatments (mSTING: monomeric (no tetramerization motif) STING mRNA; mock: EGFP mRNA; uniSTING: polymeric STING mRNA; 1 pg mRNA or 5 pg/10 pg 2’3’-cGAMP per well in a 12-well plate), n = 5 biologically independent samples. FIG. 2J shows the gating strategy of FACS sorting of BMDCs (CD1 lc+F4/80+ cells gated from CD45+CD1 lb’) and mRNA expression of IFN-P and CXCL10 in BMDC WT, BMDC Tmeml?^-, BMDC /r/3 A, and BMDC Ifnarl 1- cells 24 h after PBS, mock mRNA (1 pg/ml)), uniSTING mRNA (1 pg/ml), or
cGAMP (10 p.g/ml) treatment, n = 5 biologically independent samples. Significant differences were assessed using a one-way ANOVA and Tukey’s multiple comparisons test (FIGS. 2G and 21) and two- way ANOVA with multiple comparisons (FIG. 21). Results are presented as mean + s.d.
[0023] FIGS. 3A-3O show that the cytosolic delivery of uniSTING-mRNA based on LNPs promotes tumor inhibition in vivo through intratumoral administration. FIG. 3A is a schematic of mRNA loaded SS-OP LNPs and a representative Cryo-EM image of uniSTING-mRNA/LNPs. Scale bar, 100 nm. FIGS. 3B and 3C show that mRNA is mainly expressed in tumor cells (4T1-GFP) and DCs (CD45+CDl lc+) post intratumoral injection of mCherry-mRNA/LNPs (mCherry mRNA: 0.5 mg/kg) as quantified by flow cytometry (FIG. 3B). FIG. 3C is representative confocal microscopy images of mCherry+ cells expressed in 4T1 tumor tissue 6 h post injection, n = 6 biologically independent mice per group. Confocal microscopy images were taken under 40x magnification (Scale bar, 10 pm) and its regional magnification in the tumor site was shown. Scale bar, 2 pm.
Immunofluorescence staining using anti-CDllc antibody (white) and DAPI (blue) was performed for confocal images. FIG. 3D shows a comparison of in vivo transfection efficiency between SS-OP LNPs and MC3 LNPs. The bioluminescence signal was measured by IVIS imaging 24 h and 72 h post intratumoral administration of luciferase-mRNA/LNPs (luciferase mRNA: 0.5 mg/kg) in murine 4T1 tumor models. FIG. 3E is graphs of in vivo uniSTING expression after intratumoral administration of uniSTING-mRNA/LNPs (mRNA: 1 mg/kg). n = 8 biologically independent samples. uniSTING expression in tumor tissue, normal organs, and serum 6 h post dosing. The uniSTING mRNA was mainly expressed in tumor tissue. Time course of uniSTING expression in tumor tissues. The maximal protein concentration in tumor tissue was observed 4 h after injection, followed by a decrease yet detectable measurements up to day 7. FIG. 3F is a treatment scheme for 4T1-Luc2 tumor-bearing mice with the indicated formulations. Kaplan-Meier survival curves of mice treated with indicated formulations (for PBS, mock-mRNA/LNPs, cGAMP, and uniSTING-mRNA/LNPs, n = 10, 10, 12, and 8 biologically independent samples, respectively). FIG. 3G is a graph of average tumor weight in 4T1-Luc2 tumor models after indicated treatments, n = 8 biologically independent sample. FIG. 3H is spider plots of individual tumor growth curves, measured by bioluminescence signals, n = 10 biologically independent samples in each group. FIG. 31 is a treatment scheme for LLC tumor-bearing mice with the indicated formulations and Kaplan-Meier survival curves of mice treated with indicated formulations (n = 7 biologically independent samples). FIG. 31 is a graph of the average tumor weight in LLC tumor models after indicated treatments, n = 8 biologically independent sample. FIG. 3K is spider plots of individual tumor growth curves, n = 7 biologically independent samples in each group.
FIG. 3L is a treatment scheme for B16F10 tumor-bearing mice with the indicated formulations and Kaplan-Meier survival curves of mice treated with indicated formulations (n = 8 biologically independent samples). FIG. 3M is spider plots of individual tumor growth curves, n = 6 biologically independent samples in each group. In all the survival studies, a 1,000 mm3 tumor volume was used as the endpoint criteria. Each line represents one survival curve for each group; Log-rank (Mantel-Cox) test. The cured mice were inoculated with IxlO5 B16F10 tumor cells. Shown in FIG. 3N is the mean rechallenging tumor growth and Kaplan-Meier survival curves, data are the combination of three independent experiments. FIG. 30 is a treatment scheme for E0771 tumor-bearing mice (WT, Tmeml73^ . and Ijha 1' mice) with the indicated formulations and Kaplan-Meier survival curves of mice treated with indicated formulations (for PBS and uniSTING-mRNA/LNPs in WT, Tmeml73~'~ and Ifnaf1' mice, n = 7, 8, 5, 7, 5 and 6 biologically independent samples, respectively). FIG. 3P is a graph of the average tumor weight in E0771 tumor models after indicated formulation treatments (for PBS and uniSTING-mRNA/LNPs in WT, Tmem S 1' , and Ifnar1' mice, n = 6, 8, 7, 8, 5 and 6 biologically independent samples, respectively). Data are represented as the mean ± s.d. Significant differences were assessed using a one-way ANOVA and Tukey’s multiple comparisons test (FIGS. 3G and 3 J) and two-way ANOVA with multiple comparisons (FIG. 3P). Results are presented as mean ± s.d.
[0024] FIGS. 4A-4G show that systemic uniSTING treatment exerts potent antitumor effects on orthotopic/metastatic tumors. FIG. 4A is a treatment scheme for 4T1-Luc2 liver metastatic tumor model with the indicated formulations. FIG .4B is spider plots of individual tumor growth curves measured by bioluminescence intensity, n = 5 biologically independent samples in each group. FIG. 4C is in vivo bioluminescence imaging of mice bearing liver metastatic tumors on Day 1 and Day 10 post treatment (n - 5 biologically independent samples, respectively). FIG. 4D is Kaplan-Meier survival curves of mice treated with indicated formulations (n = 8 biologically independent samples). FIG. 4E is a treatment scheme for orthotopic Hepal-6 HCC tumor-bearing mice with the indicated formulations. FIG. 4F is a graph of average tumor weight in HCC tumor models after indicated treatments, n = 7 biologically independent sample. Fig. 4G is Kaplan-Meier survival curves of mice treated with indicated formulations (n = 8 biologically independent samples). For survival studies, 5xl07 of bioluminescence intensity was used as the endpoint criteria in 4T1 liver metastatic tumor model and a 30% weight loss was used as the endpoint criteria in HCC tumor model. Each line represents one survival curve for each group; Log-rank (Mantcl-Cox) test. Data arc represented as the
mean ± s.d. Significant differences were assessed using a one-way ANOVA and Tukey’s multiple comparisons test. Results are presented as mean ± s.d.
[0025] FIGS. 5A-5H show that uniSTING-mRNA/LNPs promotes DC maturation and robust CD8+ T cell responses. FIG. 5A is graphs showing the impact of intratumoral delivery of uniSTING- mRNA/LNPs (administered as described in FIG. 3F) on tumor infiltrating CD45, DC, CD8+ T, CD4+ T, and NK1.1+ cells, assessed by flow cytometry, n = 4 biologically independent samples. FIG. 5B shows the impact of intratumoral delivery of uniSTING-mRNA/LNPs on IFN-y and granzyme B (GzmB) expression in CD8+ T cells. Representative flow dots and the percentage of IFN-y and granzyme B expression in CD8+ T cells were shown, n = 4 biologically independent samples. Gating strategies for flow cytometry were displayed in FIGS. 23 and 24. FIG. 5C is graphs of the relative mRNA expression of IFN-P, IFN-y, and IL12A in 4T1 TME 4 days following treatments indicated in FIG. 3P. FIGS. 5D-5F show C57BL/6 mice immunized with OVA alone or OVA mixed with ADU- S100 or uniSTING/mock- mRNA/LNPs on days 0 and 21. On day 30, PBMCs were collected and CD8+T cells (FIG. 5E), CD62L+, CD44+, and CD44+ CD62L+ (FIG. 5F) were analyzed by flow cytometry, n = 3 biologically independent samples. FIG. 5G is a representative histogram of the flow cytometry analysis of CD62L and CD44 markers in PBMCs with indicated treatment. FIG. 5H is a graph of the percentage of CD44+TCFl+CD27+in CD8+ T cells, n = 3 biologically independent samples. Gating strategies for flow cytometry were displayed in FIG. 25. Significant differences were analyzed by one-way ANOVA and Tukey’s multiple comparisons test. Data are represented as the mean ± s.d.
[0026] FIGS. 6A-6I show that uniSTING-treated tumor cells impact immune activation of TME via exosomal microRNAs. FIG. 6A shows differential ultracentrifugation for isolating EVs and supernatant from CM of 4T1 tumor cells after uniSTING or Mock treatment and incubation with DC2.4 cells. The size distribution of purified EVs was measured by TEM image and Nanoparticle Tracking Analysis (NTA). Cells (Ctr) and EVs were blotted for the exosomal markers CD9 and CD81. FIG. 6B is a graph of the relative mRNA expression of IFN-P and CXCL10 in DC2.4 cells after treatment with CM from either mock- or uniSTING-mRNA treated 4T1 tumor cells, n = 6 biologically independent samples per group. FIG. 6C and 6D show the relative mRNA expression of IFN-P (FIG. 6C) and CXCL10 (FIG. 6D) in DC2.4 cells after co-culturing with supernatant (SUPmock or SUPunisriNG) or EVs (EXOmock or EXOuniSTiNG) collected via the ultracentrifugation of CM from either mock- or uniSTING-treated 4T1 cells, n = 6 biologically independent samples per group. FIG. 6E is graphs of tumor growth burden (Left) of mice bearing 4T1 tumors after intratumoral administration of
EXOmock or EXOuniSTiNG (5 mg/kg, every 3 days for 4 times; n = 5 biologically independent samples per group) and relative mRNA expression (Right) of IFN-P, IFN-y, IL12A, TNF-a, IL6, TGF- , and MRC1 in 4T1 tumor tissues 2 days after intratumoral injection of EXOmock or EXOuniSTiNG (5 mg/kg; n - 6 biologically independent samples). FIG. 6F is a scatter plot for miRNA arrays of EVs derived from CM of mock- or uniSTING-treated 4T1 tumor cells with cutoff at threefold. The black line indicates the highly enriched exosomal miRNAs from uniSTING-treated tumor cells. FIG. 6G is a heatmap output for miRNA arrays of EVs derived from CM of mock- or uniSTING-treated 4T1 tumor cells. Values represent Iogio2-ACL, where the change in threshold cycle ACt = CtmiRNA - Ctcontroi_miRNA. Ct, threshold cycle. FIG. 6H is a graph of levels of miRNAs assessed by qRT-PCR for EXOunisiiNG treated with RNase, n = 6 biologically independent samples. FIG. 61 is a graph of the relative mRNA expression of IFN- , CXCL10, TNF-a, TGF-P, IL10, and MRC1 in DC2.4 cells at 24 h after treatments with PBS, synthetic miRNA pool (mixture of miR-130b-3p, miR-130a-3p, and miR-19a-3p mimics, 25 nM miRNAs per well in 6-well plates) or miRNA mimic negative control, n = 8 biologically independent samples. Significant differences were assessed using a one-way ANOVA and Tukey’s multiple comparisons test (FIGS. 6B-6D and 6H) and two-way ANOVA with multiple comparisons (FIGS. 6E and 61). Results are presented as mean ± s.d.
[0027] FIGS. 7A-7J show that exosomal miRNAs derived from uniSTING-treated tumor cells prime antitumor immunity by blocking Wnt2b signaling in DCs. FIG. 7A is RNAseq analysis of IFN-a pathway-associated genes in DC2.4 cells treated with EXOmock or EXOuniSTiNG. Heatmap of two DC biological replicates with EXOmock or EXOuniSTiNG treatments was presented. The panel shows upregulated (red) or downregulated (blue) genes with the highest significant fold changes. Enrichment of IFN-a response signatures was analyzed by GSEA of RNA-seq datasets. FIG. 7B is a volcano plot showing fold changes for genes differentially expressed between EXOmock-treated and EXOunisriNG- treated DC2.4 cells (log2 fold change >1.5; adjusted P < 0.05). Pronounced downregulation of Wnt signaling-associated genes especially Wnt2b and Snail was found in DC2.4 cells after exposure to EXOuniSTiNG- FIG. 7C is a graph of Wnt2b mRNA expression in DC2.4 cells pretreated with PBS, mock, or uniSTING-mRNA, upon EXOmock or EXOuniSTiNG addition, n = 6 biologically independent samples per group. FIG. 7D is graphs of mRNA expression of Wnt2b, IFN-0, CXCL9, CXCL10, TGF- P, and IL10 in DC2.4 cells 48 h after treatment with siRNA against Wnt2b, a-Wnt2b antibody, PBS, or siRNA negative control, respectively, n = 6 biologically independent samples per group. FIG. 7E is graph of integrated scores that predict the association between miRNAs with Wnt2b gene. Higher scores indicate a stronger association. Among the fourteen top-ranked Wnt2b associated miRNAs,
miR-130b-3p, miR-130a-3p, miR-16-5p, and miR-15b-5p as detected in EXOuniSTiNG were highly correlated with Wnt2b. FIG. 7F is a graph of Wnt2b expression in DC2.4 cells after treatments with synthetic miRNA pool (mixture of miR-130b-3p, miR-130a-3p, and miR-19a-3p mimics, 25 nM miRNAs per well in 6-well plates) or miR mimic negative control, upon EXOm0ckor EXOuniSTiNG addition, n = 5 biologically independent samples per group. FIG. 7G is graphs of relative miR-130a-3p and miR-130b-3p expression in mork/uniSTING treated tumor cells transfected with miRiCtr or miRi Pool (miRi 130a-3p; miRi 130b-3p). n = 5 biologically independent samples per group. FIGS. 7H and 71 show Wnt2b expression in DC2.4 cells after treatment with EVs collected via ultracentrifugation of the CM from 4T1 tumor cells treated with mock + miRiCtr, mock + miRi Pool, uniSTING + miRiCtr, and uniSTING + miRi Pool, analyzed by western blotting (FIG. 7H) and qRT-PCR (FIG. 71). Nontreated DC2.4 cells as a control, n = 6 biologically independent samples per group. FIG. 7J are graphs of the relative mRNA expression of IFN-|L CXCE10, CD80, and CD86 in DC2.4 cells after treatment with EVs collected via ultracentrifugation of the CM from 4T1 tumor cells treated with mock + miRiCtr, mock + miRi Pool, uniSTING + miRiCtr, and uniSTING + miRi Pool. Non-treated DC2.4 cells were used as control, n = 5 biologically independent samples per group. Significant differences were assessed using a one-way ANOVA and Tukey’s multiple comparisons test and two-way ANOVA with multiple comparisons. Results arc presented as mean ± s.d.
[0028] FIGS. 8A-8H show that the a-Wnt2b antibody enhances in vivo antitumor activity of STING activation. FIG. 8 A is a treatment scheme for 4T1-Luc2 tumor-bearing mice with the indicated formulations. FIG. 8B is a graph of average tumor weight in 4T1-Luc2 tumor models after indicated treatments, n = 10 biologically independent sample. FIG. 8C is Kaplan-Meier survival curves of 4T1- Luc2 tumor-bearing mice treated with indicated formulations (for PBS, mock-mRNA/LNP, a-Wnt2b, uniSTING-mRNA/LNP, uniSTING-mRNA/LNP + a-Wnt2b, cGAMP, and cGAMP + a-Wnt2b, n = 10, 10, 10, 10, 9, 7, and 11 biologically independent samples, respectively). FIG. 8D is a treatment scheme for LLC tumor-bearing mice with the indicated formulations. FIG. 8E is Kaplan-Meier survival curves of LLC tumor-bearing mice treated with indicated formulations. In all the survival analysis, an 800 mm3 tumor volume was used as the endpoint criteria. Each line represents one survival curve for each group; Log-rank (Mantel-Cox) test. FIGS. 8F-8H show the impact of indicated treatments on CD8+ frequency in CD45+ cells (FIG. 8F) and the percentage of TCF1+CD27+ memory CD8+ T cells (FIG. 8G) in lung tumor bed. The percentage of GzmB expression in CD8+ T cells is shown in FIG. 8H. n = 3 biologically independent samples. Gating strategies for flow cytometry were
displayed in FIG. 25. Significant differences were assessed using a one-way ANOVA and Tukey’s multiple comparisons test (FIGS. 8C and 8F). Results are presented as mean ± s.d.
[0029] FIGS. 9A-9C show the characterization of uniSTING protein. FIG. 9A is a Coomassie stained SDS-PAGE of the expressed uniSTING protein in the presence of reducing agent DTT. FIG. 9B is molecular weight profiles by size-exclusion FPLC of uniSTING protein. The absorbance at 280 nm was represented. FIG. 9C is melting temperature (Tm) curves of uniSTING protein in different buffers.
[0030] FIGS. 10A- 10C show the activity of uniSTING in DC2.4 cells and E0771 tumor cells. FIG. 10A is the GSEA enrichment of IFN-a and IFN-y response signatures in DC2.4 cells treated with either mock or uniSTING mRNA. EGFP mRNA was used as mock mRNA control. FIG. 10B is immunoblot analysis of STING signaling activation in E0771 tumor cells treated with PBS, mock mRNA (1 pg/ml), mSTING mRNA (1 pg/ml), uniSTING mRNA (1 pg/ml), or 2’3’-cGAMP (5 pg/ml or 10 pg/ml). FIG. 10C is IFN-0 levels measured by ELISA in human monocyte THP-1 24 h after indicated treatments (mock: EGFP mRNA; uSTING: huSTING mRNA; 1 pg mRNA or 5 pg/10 pg 2’3’-cGAMP per well in a 12-well plate), n = 5 biologically independent samples. Data are represented as the mean ± s.d.
[0031] FIG. 11 shows the activity of uniSTING in DCs and tumor cells after inhibition of cGAS. mRNA expression of IFN-|3 and CXCL10 in DC2.4 cells, 4T1 tumor cells, and E0771 tumor cells 24 h after indicated treatments. uniSTING mRNA (1 pg/ml) or cGAMP (10 pg/ml) was added 2 h after beginning of the incubation with cGAS inhibitor (RU.521, 2.5 pg/ml). n = 4 biologically independent samples. Data are represented as the mean ± s.d.
[0032] FIG. 12 shows immunoblot analysis of STING signaling activation in wild-type, Tmeml73, Irf3, and IFNaR knockout BMDCs treated with PBS, uniSTING mRNA (1 pg/ml), or 2’3’-cGAMP (10 pg/ml).
[0033] FIGS. 13A and 13B show that the low expression level of STING protein in human breast cancer correlates with poor prognosis. FIG. 13A is a graph of the Relapse-Free Survival (RFS) of breast cancer patients expressing high or low levels of TEME173 (STING), from Kaplan-meier plotter database with auto-selected best cutoff (kmplot.com/analysis/index.php ?p=background). mRNA data collected with RNA-seq; Log-rank (Mantel-Cox) test. FIG. 13B shows STING gene expression profile across breast cancer tissues (T) and paired normal tissues (N) from breast cancer patients (GEPIA2 database, gcpia2.cancer-pku.en/#index).
[0034] FIGS. 14A-14D show characterization of SS-OP LNP delivery system in vitro and in vivo. FIG. 14A is a graph of size distribution of mRNA-loaded LNPs measured by DLS. FIG. 14B shows the quantification of luciferase activity in the DC2.4 cells, E0771, and 4T1 tumor cells 12 h after the transfection of luciferase mRNA loaded SS-OP LNPs (mRNA: Ipg/ml), measured by luciferase assay kit. n = 6 biologically independent samples. One-way ANOVA and Tukey’s multiple comparisons test. FIG. 14C shows the gating strategies used for flow cytometry analysis of mCherry+ cells in the 4T1 tumor tissue. FIG. 14D is a time course of uniSTING expression in liver tissues, n = 8 biologically independent samples. Data are represented as the mean ± s.d.
[0035] FIGS. 15A-15D show the comparison of monomeric STING with uniSTING in vivo. FIG. 15A is a treatment scheme for 4T1-Luc2 tumor-bearing mice with the indicated formulations. FIG.
15B is spider plots of individual tumor growth curves. FIG. 15C is a graph of tumor growth burden of mice bearing 4T1-Luc2 tumors receiving different treatments. Two-way ANOVA with multiple comparisons. FIG. 15D is Kaplan-Meier survival curves of mice treated with indicated formulations using a 1,000 mm3 tumor volume as the endpoint criteria. Each line represents one survival curve for each group; Log-rank (Mantel-Cox) test. For PBS, mSTING, and uniSTING, n = 5, 5, 6 biologically independent samples.
[0036] FIG. 16 is in vivo bioluminescence imaging of mice bearing 4T1 tumors on Day 15 and Day 23 post inoculation (n = 10 biologically independent samples, respectively). Treatment scheme with the indicated formulations was shown in FIG. 3F.
[0037] FIGS. 17A-17D show the antitumor efficacy of uniSTING-mRNA/LNPs in E0771 tumor model. FIG. 17A is a treatment scheme for E0771 tumor-bearing mice with the indicated formulations. FIG. 17B shows the average tumor weight in E0771 tumor models after indicated treatments, n = 7 biologically independent sample. FIG. 17C is Kaplan-Meier survival curves of mice treated with indicated formulations (for PBS, mock-mRNA/LNPs, cGAMP, and uniSTING-mRNA/LNPs, n = 7, 7, 7, and 8 biologically independent samples, respectively). FIG. 17D is spider plots of individual tumor growth curves, n = 7 biologically independent samples in each group. Data are represented as the mean ± s.d.
[0038] FIG. 18 shows antitumor efficacy of uniSTING-mRNA/LNPs in WT, TmemlVS 1', and Ifnar /_ mice bearing E0771 tumors, respectively. Spider plots of individual tumor growth curves (for PBS and uniSTING-mRNA/LNPs in WT, Tmeml73~f and Ifnar mice, n = 7, 8, 5, 7, 5 and 6 biologically independent samples, respectively). Treatment scheme was shown in FIG. 3M.
[0039] FIGS. 19A-19D are graphs showing the impact of uSTING LNP administration on antitumor immunity in TME. FIG. 19A is graphs of the impact of intratumoral delivery of uSTING LNPs (administered as described in FIG. 3F) on E0771 tumor infiltrating CD8+ T cell and its intracellular cytokine expression, determined by flow cytometry, n = 5 biologically independent samples. FIG. 19B is a graph of the impact of intratumoral delivery of uSTING LNPs (administered as described in FIG. 3F) on E0771 tumor infiltrating NK1.1+ cells. FIG. 19C is a graph of the impact of intratumoral delivery of uSTING LNPs (administered as described in FIG. 3F) on E0771 tumor infiltrating CD4+ T cell, determined by flow cytometry, n = 5 biologically independent samples. FIG. 19D is graphs of the impact of intratumoral delivery of uSTING LNPs (administered as described in FIG. 3F) on E0771 tumor infiltrating DC cell and its surface CD86 and CD80 expression, determined by flow cytometry. n = 5 biologically independent samples. Significant differences were assessed using a one-way ANOVA and Tukcy’s multiple comparisons test. Results arc presented as mean ± s.d.
[0040] FIGS. 20A and 20B show high level of miR-130a, miR-19a, and miR-16 in human breast cancer correlates with prolonged survival time of cancer patients. FIG. 20A are graphs of the relative miR-130a-3p, miR-130b-3p, and miR-19a-3p expression in DC2.4 cells after transfection with miRNA pool (mixture of miR-130b-3p, miR-130a-3p, and miR-19a-3p mimics, 25 nM miRNAs per well in 6- well plates) or scrambled-miR mimic as negative control, n = 8 biologically independent samples per group. One-way ANOVA and Tukey’s multiple comparisons test. Data are represented as the mean ± s.d. FIG. 20B shows the overall survival (OS) of breast cancer patients expressing high or low levels of indicated miRNAs, from Kaplan-Meier plotter database with auto-selected best cutoff (kmplot.com/analysis/index.php ?p=background). Log-rank (Mantel-Cox) test.
[0041] FIGS. 21A-21F show exosomes derived from uniSTING-treated tumor cells primed antitumor immunity by blocking Wnt2b signaling in DCs. FIG. 21 A shows graphs of Wnt2b mRNA expression in BMDC cells pretreated with mock or uniSTING mRNA, upon EXOmock or EXOUSTING addition. Exosomes derived from 4T1 tumor cells; n = 6 biologically independent samples per group. FIGS. 21B and 21C show Wnt2b mRNA expression in DC2.4 (FIG. 21B) or BMDC (FIG. 21C) cells pretreated with mock or uniSTING mRNA, upon EXOmock or EXOUSTING addition. Exosomes derived from E0771 tumor cells; n = 6 biologically independent samples per group. FIGS. 21D-21E show mRNA expression of Wnt2b, IFN- , CXCL9, CXCL10, TGF-p, and IL10 in DC2.4 cells 24 h after treatments with siRNA against Wnt2b, a-Wnl2h antibody, PBS, or siRNA negative control, n = 6 biologically independent samples per group. FIG. 2 IF shows RFS of breast cancer patients expressing high or low Wnt2b levels, from Kaplan-Meier plotter database with auto-selected best cutoff
(kmplot.com/analysis/index.php ?p=background). mRNA data collected with RNA-seq; Log-rank (Mantel-Cox) test. Significant differences were assessed using a one-way ANOVA and Tukey’s multiple comparisons test (FIGS. 21A-21D) and two-way ANOVA with multiple comparisons (FIG. 2 IE). Data are represented as the mean ± s.d.
[0042] FIGS. 22A-22H show that the a-Wnt2b antibody enhanced in vivo antitumor activity of STING activation. FIG. 22A is a treatment scheme for E0771 tumor-bearing mice with the indicated formulations. FIG. 22B is a graph of average tumor weight in E0771 tumor models after indicated treatments, n = 8 biologically independent sample. FIG. 22C is spider plots of individual E0771 tumor growth curves (for PBS, mock-mRNA/LNP, a-Wnt2b, uSTING-mRNA/LNP, uSTING-mRNA/LNP + a-Wnt2b, cGAMP, and cGAMP + a-Wnt2b, n = 8, 8, 10, 9, 8, 8 and 8 biologically independent samples, respectively). FIG. 22D is Kaplan-Meier survival curves of E0771 tumor-bearing mice treated with indicated formulations. In all the survival analysis, a 1,000 mm3 tumor volume was used as the endpoint criteria. Each line represents one survival curve for each group; Log-rank (Mantel- Cox) test. Significant differences were assessed using a one-way ANOVA and Tukey’s multiple comparisons test. Results are presented as mean ± s.d.
[0043] FIG. 23 shows gating strategies used for flow cytometry analysis of DCs (CD45+CDllc+MHCII+) in the E0771 tumor tissue.
[0044] FIG. 24 shows gating strategies used for flow cytometry analysis of CD8 T (CD45+CD3+CD8+), CD4 T (CD45+CD3+CD8+), NK1.1 (CD45+CD3’NK1.1+) in the E0771 tumor tissue.
[0045] FIG. 25 shows gating strategies used for flow cytometry analysis of memory CD8 T cells.
DETAILED DESCRIPTION
[0046] Disclosed herein are compounds, compositions, and methods for activation of STING signaling pathways. The clinical potential of current small molecule STING agonists is limited by their poor stability, deliverability, undesired side toxicity post systemic delivery, as well as the low level, downregulation, and/or loss of endogenous STING in TME. Herein is described a universal, constitutively active STING mimic with a robust tetrameric subunit for the activation of STING signaling pathways, favoring IRF3/IFN-I activity over the pro-inflammatory NF-KB signaling in a manner independent of the endogenous STING, referred to herein as uniSTING or uSTING, and referred to previously in U.S. Provisional Application No. 63/380,714 as pSTING. Through LNP- mediated delivery of the mRNA encoding this constitutively active STING mimic protein, the resulting uniSTING potently promoted antitumor immunity and activated NK1.1+ cells, CD8 T+ cells, and DCs
within tumors. uniSTING administration reduced tumor burden in multiple poorly immunogenic murine tumors, including TNBC, LLC, B16F10 melanoma, or advanced orthotopic/metastatic liver tumor models that showed negligible response when treated with soluble cGAMP. Mechanistic analysis showed that uniSTING activated STING signaling remained in either Tmeml 73~'~ BMDCs in vitro or tumor-bearing Tmeml73~ mice mirroring the downregulation or loss of STING in TME. Surprisingly, uniSTING selectively induced IRF3 -dependent signaling activation fostering the specific CD8+ T cell antitumor response, but not the undesired pro-inflammatory NF-KB signaling. Current STING agonists activate both IRF3 signaling and the double-edged, pro-tumor NF-kB pathways, such that the disclosed uniSTING provides beneficial results without potential activation of pro-tumor responses. uniSTING-mRNA/LNP potently boosted DCs activation and cytotoxic CD8+ T cells antitumor response. Significantly, increased frequency of CD8+ T cell and promoted differentiation of its memory subpopulation were stimulated by the uniSTING-mRNA/LNPs. Significantly, uniSTING triggered the crosstalk between tumor cells and DCs mediated by EVs originated from uniSTING treated tumor cells in TME. Additionally, Wnt2b signaling-mediated negative feedback was unveiled in DCs in response to STING activation, which can be relieved by exosomal miRNAs derived from uniSTING-treated tumor cells. A combination of uniSTING-mRNA/LNP with a-Wnt2b antibody synergistically inhibited tumor growth and prolonged survival time of tumor-bearing mice.Mechanically, dual therapy of uniSTING-mRNA/LNP with a-Wnt2b could further replenished CD8+ T cell and its memory subpopulation in tumor local bed, as well as cytotoxic GzmB+ CD8+ T cells, compared to that of the combination of ADU-S 100 with a-Wnt2b. The robust antitumor immunity makes the uniSTING-mRNA/LNPs an attractive candidate for translational applications, especially for the treatment of STING deficient tumor types.
[0047] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
1. Definitions
[0048] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
[0049] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
[0050] A “chemotherapeutic” or “chemotherapeutic agent” as used herein, refers to a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in “targeted therapy” and conventional chemotherapy. Examples of chemotherapeutic agents include, but are not limited to: cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazinc, cisplatin, carboplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib, everolimus, alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, erlotinib, toremifene, fulvestrant, letrozole, gemcitabine, goserelin, ixabepilone, emtansine, lapatinib, olaparib, megestrol, neratinib, palbociclib, ribociclib, talazoparib, thiotepa, toremifene, methotrexate, trastuzumab, temozolomide, rapamycin, and tucatinib.
[0051] As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single- stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlcstcdt ct al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohcxcnyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and/or a ribozyme. Hence, the term “nucleic
acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
[0052] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide” and “protein” are used interchangeably herein.
[0053] The recitations “sequence identity,” “percent identity,” “percent homology,” “percent similarity,” or, for example, comprising a “sequence 50% identical to” or “sequence with at least 50% similarity to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (e.g., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
[0054] Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) can be performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In certain embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions arc then compared. When a position in the first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
[0055] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
[0056] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using an NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Another exemplary set of parameters includes a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
[0057] The peptide sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
[0058] The term “amino acid” or “any amino acid” as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino
acids, and non-natural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D- amino acids occur in bacterial envelopes and some antibiotics. The “non-standard,” natural amino acids include, for example, pyrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). “Unnatural” or “nonnatural” amino acids are non-proteinogenic amino acids (e.g., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible. Examples of “unnatural” amino acids include P-amino acids (P3 and p2), homo-amino acids, proline and pyruvic acid derivatives, 3- substitutcd alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, alpha-methyl amino acids and N- methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. “Modified” amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally present on the amino acid. According to certain embodiments, a peptide inhibitor comprises an intramolecular bond between two amino acid residues present in the peptide inhibitor. It is understood that the amino acid residues that form the bond will be altered somewhat when bonded to each other as compared to when not bonded to each other. Reference to a particular amino acid is meant to encompass that amino acid in both its unbonded and bonded state. For example, the amino acid residue homoSerine (hSer) or homoSerine(Cl) in its unbonded form may take the form of 2-aminobutyric acid (Abu) when participating in an intramolecular bond according to the present invention.
F0059] For the most part, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out in “Nomenclature of a- Amino Acids (Recommendations, 1974)” Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues employed in this specification and appended claims differ from those suggestions, they will be made clear to the reader.
[0060] Throughout the present specification, unless naturally occurring amino acids arc referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or
single-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). The term “L-amino acid,” as used herein, refers to the “L” isomeric form of a peptide, and conversely the term “D-amino acid” refers to the “D” isomeric form of a peptide (e.g., Dphe, (D)Phe, D-Phe, or UF for the D isomeric form of Phenylalanine). Amino acid residues in the D isomeric form can be substituted for any L- amino acid residue, as long as the desired function is retained by the peptide.
[0061] As used herein, the terms “providing,” “administering,” and “introducing” are used interchangeably herein and refer to the placement of the nanocomplexes and/or compositions of the present disclosure into a subject by a method or route which results in at least partial localization to a desired site.
[0062] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include cither adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.
[0063] As used herein, “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a peptide or composition described herein to an appropriate subject. The term also includes a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the disease. As such, “treating” means an application or administration of the peptides or compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.
[0064] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
[0065] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
2. Polypeptides
[0066] The present disclosure provides polypeptides comprising STING protein mimics. The polypeptides comprise a tetramerization motif and a C-terminal cytoplasmic domain of a STING protein. The tetramerization motif and a C-terminal cytoplasmic domain of a STING protein may be linked in any configuration. For example, the N-terminus of the C-terminal cytoplasmic domain of a STING protein may be linked to the C- terminus of the tetramerization motif, or the N-terminus of the tetramerization motif may be linked to the C-terminus of the C-terminal cytoplasmic domain of a STING protein, or the C-terminus of the C-terminal cytoplasmic domain of a STING protein may be linked to the C-terminus of the tetramerization motif.
[0067] In some embodiments, the polypeptide further comprises a linker between the tetramerization motif and the C-terminal cytoplasmic domain of a STING protein. The linker is preferably a polypeptide linker of 1-20 amino acids. The linker may comprise any amino acid sequence. The linker may be flexible such that it does not constrain either the tetramerization motif or the C-terminal cytoplasmic domain of a STING protein. The linkers may essentially act as a spacer. In some embodiments, the linker is glycine-rich. The linker may comprise glycine- serine polymers, glycine-alanine polymers, alanine-serine polymers and/or other flexible linkers known in the art. In some embodiments, the linker includes but not limited to (GGS)n, (GGGS)n (SEQ ID NO: 43) or (PQPQPK)n (SEQ ID NO: 44), where n is an integer of at least one.
[0068] The term “tetramerization domain” as used herein is defined as a domain that mediates the formation of a tetramer out of four monomeric proteins or parts thereof. Suitable tetramerization domains include, but are not limited to, tetramerization domains derived from the tetrabrachion protein in Staphylothermus marinus and related organisms, tetramerization domains derived from human potassium voltage-gated channel subfamily A members or orthologs thereof, tetramerization domains derived from human potassium voltage-gated channel subfamily KCNQ1, tetramerization domains derived from human potassium voltage-gated channel subfamily KQT member, tetramerization domains derived from human P53, tetramerization domains derived from human vasodilator- stimulated phosphoprotein, tetramerization domains derived from human acetylcholinesterase,
tetramerization domains derived from human butyrylcholinesterase, tetramerization domains derived from measles virus phosphoprotein, tetramerization domains derived from Nipah virus phosphoprotein, tetramerization domains derived from human metapneumo virus phosphoprotein, tetramerization domains derived from rotavirus NSP4, tetramerization domains derived from Sendai virus nucleocapsid phosphoprotein, tetramerization domains of Bacillus cereus TubY, tetramerization domains derived from Cauliflower mosaic virus VAP, tetramerization domains derived from Newcastle disease virus hemagglutinin-neuraminidase, tetramerization domains derived from Enterococcus phage N- acetylmuramoyl-L-alanine amidase, tetramerization domains derived from Pseudomonas phage 201phi2-lp060, tetramerization domains derived from Drosophila anastral spindle 2, tetramerization domains derived from Gibberella zeae TRP channel analog, tetramerization domains derived from yeast GCN4, or engineered tetramerization domains.
[0069] The tetramerization domain may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-37. In some embodiments, the tetramerization domain sequence comprises one, two, three, four, five, six, seven, eight, nine, or ten substitutions, truncations, or additions as compared to the sequences disclosed herein.
[0070] STING is comprised of 4 transmembrane regions (TMs) and a C-terminal domain cytoplasmic domain. The C-terminal domain includes a dimerization domain (DD) and the carboxyterminal tail. The C-terminal cytoplasmic domain (e.g., non-membrane-bound domain) of STING may be derived from any STING protein. For example, the C-terminal cytoplasmic domain of STING may comprise residues 138-378 of murine STING, residues 138-379 of human STING, or an equivalent region from an ortholog thereof. In some embodiments, the C-terminal cytoplasmic domain of STING is derived from human STING protein.
[0071] In some embodiments, the C-terminal cytoplasmic domain of STING comprises an amino acid sequence of at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 98%, at least 99%) similarity to SEQ ID NOs: 38 or 39. In some embodiments, the C-terminal cytoplasmic domain of STING comprises an amino acid sequence of SEQ ID NO: 38 or 39. In some embodiments, the amino acid sequence of the C-terminal cytoplasmic domain of STING comprises one or more substitutions, truncations, or additions as compared to SEQ ID NO: 38 or 39. [0072] An amino acid “substitution” or “replacement” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Amino acids arc broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or
His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non- aromatic amino acids are broadly grouped as “aliphatic.” Examples of “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or He), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).
[0073] The amino acid replacement or substitution can be conservative, semi-conservative, or nonconservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer- Vcrlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra).
[0074] Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained. “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same subgroup. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.
3. Polynucleotides
[0075] Disclosed herein are polynucleotides encoding the disclosed polypeptide. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In select embodiments, the polynucleotide is a mRNA. Thus, the disclosure further provides a mRNA comprising an open reading frame encoding the disclosed polypeptide, and compositions thereof. [0076] The disclosure also provides polynucleotide segments encoding the polynucleotide or mRNA, vectors containing these segments, and cells containing the vectors. The vectors may be used
to propagate the segment in an appropriate cell and/or to allow expression from the segment (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.
[0077] In some embodiments, the polynucleotide is optimized for enhanced expression, productive co-translational protein folding, increased stability, or a combination thereof.
[0078] In some embodiments, the polynucleotide or mRNA is codon-optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; rcmovc/add post translation modification sites in encoded protein (c.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide.
[0079] The polynucleotide or mRNA may further comprise other structures and sequences necessary for proper functionality or stability, including but not limited to a 5’ untranslated region (UTR), a 5’ cap, a 3’ UTR, a 3’ tailing sequence, or any combination thereof. Thus, in some embodiments, the disclosure provides a mRNA comprising: a 5’ UTR, an open reading frame encoding the disclosed polypeptide, and a 3’ UTR.
[0080] The polynucleotide or mRNA may comprise a 5’ and 3’ untranslated region (UTR). Sequence elements within the UTRs affect translational efficiency and RNA stability but do not encode a polypeptide. The 3’ UTR relates to the region located at the 3’ end of the mRNA, downstream of the termination codon of a protein-encoding region, which is transcribed but not translated into an amino acid sequence. The 5 ’UTR refers to the region directly upstream from the initiation codon. Eukaryotic 5’ UTRs contain the Kozak consensus sequence (ACCAUGG), which contains the initiation codon. The eukaryotic 5’ UTR may also contain d.s- acting regulatory elements called upstream open reading frames (uORFs) and upstream AUGs (uAUGs) and termination codons, which can impact translation regulation. In some embodiments, the 5’ UTR, 3’ UTR, or both is heterologous to the sequence or open read frame encoding the disclosed polypeptide.
[0081] 5’ -cap refers to the structure found on the 5 ’-end of an mRNA which ordinarily consists of a guanosine nucleotide connected to the mRNA via a 5’ to 5’ triphosphate linkage. The guanosine may
be methylated at the 7-position creating a 7-methylguanosine cap (m7G). In some embodiments, the 5’ cap may be a naturally occurring 5’ cap. In some embodiments, the 5’ cap is a 5’ cap analog or a modified 5’ cap structure which is non-naturally occurring (e.g., phosphorothioate-cap-analogs).
[0082] The polynucleotide or mRNA may comprise a 3’ tailing sequence. The 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.
[0083] In some embodiments, the polynucleotide or mRNA further comprises a triple helix forming sequence. A tiple helix is formed after the binding of a third strand to the major groove of a duplex nucleic acid through Hoogsteen base pairing (e.g., hydrogen bonds) while maintaining the duplex structure of two strands making the major groove. Pyrimidine-rich and purine-rich sequences (e.g., two pyrimidine tracts and one purine tract or vice versa) can form stable triplex structures as a consequence of the formation of triplets (e.g., A-U-A and C-G-C). In some embodiments, the triple helix sequence is derived from the 3’ terminal triple helix sequences of triple helix terminators from a long non-coding RNAs (IncRNAs), e.g., metastasis-associated lung adenocarcinoma transcript 1 (MALAT1).
[0084] In some embodiments, the polynucleotide or mRNA further comprises a tRNA-like sequence. The tRNA-like sequences are those sequences which form similar overall secondary and tertiary structure to tRNA. In some embodiments, the tRNA-like sequence is derived from a long noncoding RNAs (IncRNAs), e.g., MALAT1. The tRNA-like sequence derived from IncRNAs may be truncated or modified as long as they retain the clover secondary structure.
[0085] As the MALAT1 sequences are highly conserved evolutionarily, the MALAT1 sequences for the triple helix or the tRNA-like sequence can be from any species. In one embodiment, the MALAT1 sequences are from a human. In another embodiment, the MALAT1 sequences are from a mouse. In another embodiment, the MALAT1 sequences are from a non-human primate.
[0086] The 3’ poly(A) sequence of mRNA is important for nuclear export, RNA stability and translational efficiency of eukaryotic messenger RNA (mRNA). The poly(A) tail is a segment of RNA at the 3’ end of the molecule that has only adenine bases. For example, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. The poly(A) tail may contain two segments of only adenine bases separated by a linker.
[0087] In some embodiments, the polynucleotide or mRNA comprises at least one chemical modification or chemically modified base, nucleoside, or nucleotide. The chemical modifications may comprise any modification which is not naturally present in said RNA or any naturally-occurring
modification of adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribonucleosides. For example, a single polynucleotide or mRNA may include both naturally-occurring and non-naturally- occurring modifications.
[0088] Chemical modifications may be located in any portion of the polynucleotide or mRNA molecule and the polynucleotide or mRNA molecule may contain any percentage of modified nucleosides (1-100%). In some embodiments, every particular base or nucleoside may be modified (e.g., everjr uridine is a modified uridine). In some embodiments, at least 80% of any single nucleotide (e.g., uracil) in the of the polynucleotide or mRNA is chemically modified. In some embodiments, a particular modification is used for every particular type of nucleoside or base (e.g., every uridine is modified to a 1-methyl-pseudouridine). Exemplary RNA modifications can be found in the RNA modification database (See mods.rna.albany.edu/home).
[0089] In some embodiments, the at least one chemical modification comprises a modified uridine residue. Exemplary modified uridine residues include, but are not limited to, pseudouridine, 1- methylpseudouridine, 1 -ethylpseudouridine, 2-thiouridine, 4’- thiouridine, 5-methyluridine, 2-thio-l - methyl- 1-deaza-pseudouridine, 2- thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2’-0-methyl uridine.
[0090] In some embodiments, the at least one chemical modification comprises a modified cytosine residue. Exemplary nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5- methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza-pseudoisocytidine, 1- methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio- zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, 4-methoxy- 1-methyl-pseudoisocytidine, lysidine, a-thio-cytidine, 2’-O-methyl- cytidine, 5,2’-O-dimethyl-cytidine, N4-acetyl-2’-O-methyl-cytidine, N4,2’-O-dimethyl-cytidine, 5- formyl-2’-O-methyl-cytidine, N4,N4,2’-O-trimethyl-cytidine, 1 -thio-cytidine, 2’-F-aracytidine, 2’-F- cytidine, and 2’-OH-aracytidine.
[0091] In some embodiments, the at least one chemical modification comprises a modified adenine residue. Exemplary nucleosides having a modified adenine include 2-amino-purine, 2,6-
diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido-adenosine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7- deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl-adenosine, 2-methylthio- N6-isopentenyl-adenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6- dimethyl-adenosine, N6-hydroxynoryalylcarbamoyl-adenosine, 2-methylthio-N6- hydroxynoryalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2’-O-methyl-adenosine, N6,2’-O-dimethyl-adenosine, N6,N6,2’-O-trimethyl-adenosine, l,2’-O-dimethyl-adenosine, 2’-O-ribosyladenosine (phosphate), 2- amino-N6-mcthyl-purinc, 1-thio-adcnosinc, 8-azido-adcnosinc, 2’-F-ara-adcnosinc, 2’-F-adcnosinc, 2’-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.
[0092] In some embodiments, the at least one chemical modification comprises a modified guanine residue. Exemplary nucleosides having a modified guanine include inosine, 1-methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, undermodified hydroxywybutosine, 7-deaza-guanosine, queuosine, epoxy queuo sine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deaza-guanosine, 7- aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7- methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2-methyl-guanosine, N2, ^-dimethylguanosine, N2,7-dimethyl-guanosine, N2,N2,7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio- guanosine, a-thio-guanosine, 2’-O-methyl-guanosine, N2-methyl-2’-O-methyl-guanosine, N2,N2- dimethyl-2’-O-methyl-guanosine, l-methyl-2’-O-methyl-guanosine, N2,7-dimethyl-2’-O-methyl- guanosine, 2’-O-methyl-inosine, l,2’-O-dimethyl-inosine, and 2’-O-ribosylguanosine (phosphate).
4. Compositions
[0093] Also disclosed herein are compositions comprising the described polypeptides or polynucleotides. The compositions may further comprise excipients or pharmaceutically acceptable carriers. The choice of excipients or pharmaceutically acceptable carriers will depend on factors including, but not limited to, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
[0094] Excipients and carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Some examples of materials which can serve as excipients and/or carriers are sugars including, but not limited to, lactose, glucose and sucrose; starches including, but not limited to, com starch and potato starch; cellulose and its derivatives including, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients including, but not limited to, cocoa butter and suppository waxes; oils including, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; including propylene glycol; esters including, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents including, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants including, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants. The compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).
[0095] The compositions may be formulated for any particular mode of administration including for example, systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral).
5. Vaccines or Medicaments
[0096] The polypeptides, polynucleotides (e.g., mRNA), or compositions thereof described herein may be used to prepare vaccines or another medicament.
[0097] The vaccine or medicament may comprise an adjuvant or immuno stimulant, or a polynucleotide encoding an adjuvant or immunostimulant (e.g., an adjuvantive polypeptide). Adjuvants and immunostimulants are compounds or compositions that either directly or indirectly stimulate the immune system’s response to a co-administered antigen.
[0098] Suitable adjuvants are commercially available as, for example, Glucopyranosyl Lipid Adjuvant (GLA); Pam3CSK4; Freund’s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.I.); AS-2 (SmithKlinc Beecham); mineral salts (for example, aluminum, silica, kaolin, and carbon); aluminum salts such as aluminum hydroxide gel (alum), A1K(SO4)2, AlNa(SO4)2, A1NH4(SO4), and Al(0H)3; salts of calcium (e.g., Ca3(PO4)i), iron or zinc; an insoluble suspension of acylated tyrosine; acylated
sugars; cationically or anionically derivatized polysaccharides; polynucleotides (for example, poly IC, poly AU acids, and CpG oligodeoxynucleotides (e.g., Class A or B)); polyphosphazenes; cyanoacrylates; polymerase-(DL-lactide-co- glycoside); bovine serum albumin; diphtheria toxoid; tetanus toxoid; edestin; keyhole-limpet hemocyanin; Pseudomonal Toxin A; choleragenoid; cholera toxin; pertussis toxin; viral proteins; Quil A; aminoalkyl glucosamine phosphate compounds. In addition, adjuvants such as cytokines (e.g., GM-CSF or interleukin-2, -7, or -12), interferons, or tumor necrosis factor, may also be used as adjuvants. Protein and polypeptide adjuvants may be obtained from natural or recombinant sources according to methods well known to those skilled in the art. When obtained from recombinant sources, the adjuvant may comprise a protein fragment comprising at least the immunostimulatory portion of the molecule.
[0099] Other known immuno stimulatory macromolecules which can be used include, but are not limited to, polysaccharides, tRNA, non-mctabolizablc synthetic polymers such as polyvinylaminc, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4’,4-diaminodiphenylmethane-3,3’-dicarboxylic acid and 4-nitro-2- aminobenzoic acid (See, Sela, M., Science 166: 1365-1374 (1969)) or glycolipids, lipids, or carbohydrates.
[00100] In some embodiments, the adjuvantive polypeptide comprises immune activator proteins, such as CD70, CD40 ligand, and constitutively active TLR4, or polycationic peptides (e.g., protamine). In some embodiments, the adjuvantive polypeptide is a flagellin polypeptide. Commercially available mRNA encoding adjuvantive polypeptides are available, for example, as TriMix (See Bonehill, A. et al. Mol. Ther. 16, 1170-1180 (2008), incorporated herein by reference).
[00101] The vaccines or medicaments may further comprise a delivery vehicle. Exemplary delivery vehicles include, but are not limited to, microparticle compositions comprising poly(lactic acid) (PLA) and/or poly(lactic-co-glycolic acid) (PLGA), albumin nanoparticles, and liposomal compositions. In some embodiments, the vaccines comprise a lipid nanoparticle encapsulating the disclosed polypeptides, polynucleotides (e.g., mRNA).
[00102] Lipid nanoparticle compositions of the disclosure may include one or more cationic and/or ionizable lipids, phospholipids, neutral or non-cationic lipids, polyethylene glycol (PEG)-lipid conjugates, and/or sterols. In some embodiments, the lipid nanoparticle comprises a cationic lipid and/or ionizable lipid, a neutral or non-cationic lipid, and cholesterol.
[00103] Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be readily protonated and may have a positive or partial positive charge at physiological pH due to a pKa value between pH 5 and 8. The polar headgroup of the cationic lipids preferably comprises amine
derivatives such as primary, secondary, and/or tertiary amines, quaternary ammonium, various combinations of amines, amidinium salts, or guanidine and/or imidazole groups as well as pyridinium, piperazine and amino acid headgroups such as lysine, arginine, ornithine and/or tryptophan. Cationic lipids include, but are not limited to, l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC),
1.2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA) and/or l,2-dioleoyl-3- trimethylammonium propane (DOTAP), 1 ,2-dimyristoyl-3 -trimethylammonium propane (DMTAP),
2.3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide (DMRIE), didodecyl(dimethyl) ammonium bromide (DDAB), l,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 3P-[N — (N\N’-dimethylamino-ethane)carbamoyl]cholesterol (DC- Chol) or dioleyl ether phosphatidylcholine (DOEPC). Ionizable lipids include, but are not limited to, 1 ,2-dioleyloxy-3 -dimethylamino-propane (DODMA).
[00104] In some embodiments, the lipid nanoparticlc comprises a polyethylene glycol (PEG)-lipid conjugate. A PEG-lipid conjugate may include, but is not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG- modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-DMG (l,2-dimyristoyl-rac-glycero-3- methoxypoly ethylene glycol), PEG-c-DOMG (R-3-[(co-methoxy poly (ethylene glycol)2000)carbamoyl)]-l,2-dimyristyloxlpropyl-3-amine), PEG-DMA (PEG-dimethacrylate), PEG- DLPE (l,2-didodecanoyl-sn-glycero-3-phosphoethanolamine-PEG), PEG-DMPE (PEG- 1,2- dimyristoyl-sn-glycero-3 -phosphoethanolamine), PEG-DPPC (PEG-dipalmitoyl phosphatidylcholine), PEG-N,N-di(tetradecyl)acetamide, or a PEG-DSPE (1, 2-distearoyl-sn-glycero-3- phosphoethanolamine-poly(ethylene glycol)) lipid. In some embodiments, the lipid nanoparticle comprises PEG-DMG and/or PEG-N,N-di(tetradecyl)acetamide.
[00105] The sterol may comprise cholesterol, fecosterol, ergosterol, campesterol, sitosterol, stigmasterol, brassicasterol or a sterol ester, such as cholesteryl hemisuccinate, cholesteryl sulfate, or any other derivatives of cholesterol.
[00106] A neutral or non-cationic lipid may include one or more phospholipids. Phospholipids include a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety may include, but is not limited to, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin. A fatty acid moiety may include, but is not limited to, lauric acid, myristic acid, myristolcic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid,
arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
[00107] Phospholipids suitable for use in the compositions may include, but are not limited to, phosphatidylglycerol (PG) including dimyristoyl phosphatidylglycerol (DMPG) and 1,2-dioleoyl-sn- glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3- phosphocholinc (C16 Lyso PC), l,2-dilinolcnoyl-sn-glyccro-3-phosphocholinc, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3 -phosphocholine; phosphatidylethanolamine (PE) including l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dilinolenoyl-sn- glycero-3-phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine; phosphatidic acid (PA); phosphatidylinositol (PI); phosphatidylserine (PS); and sphingomyelin (SM).
[00108] The positively charged lipid structures described herein may also include other components typically used in the formation of vesicles (e.g., for stabilization). Examples of such other components includes, without being limited thereto, fatty alcohols, fatty acids, and/or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the lipid into the lipid assembly.
[00109] The vaccine or medicament of the present disclosure may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic and antigenic portions of polypeptides or polynucleotides encoding immunogenic polypeptides, or nucleic acid(s) encoding thereof, may be present within the vaccines or medicaments. The vaccine or medicament may generally be used for prophylactic and therapeutic purposes.
[00110] The vaccines or medicaments may be formulated for any appropriate manner of administration, and thus administered, including for example, oral, nasal, intravenous, intravaginal,
epicutaneous, sublingual, intracranial, intradermal, intraperitoneal, intratumoral, subcutaneous, intramuscular administration, or via inhalation.
[00111] The vaccines or medicaments may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, vaccines or medicaments may be formulated as a lyophilizate.
[00112] The compositions, vaccines, or medicaments may be prepared, packaged, or sold in a form suitable for bolus administration or sold in unit dosage forms, such as in ampules or multi-dose containers. In some embodiments, the compositions and vaccines contain a preservative.
6. Methods
[00113] The present disclosure provides methods for treating, reducing, or preventing a disease or disorder in a subject in need thereof.
[00114] In some embodiments, the disease or disorder comprises cancer. In some embodiments, the subject is a human. In some embodiments, the subject is female. In some embodiments, the subject has cancer, has had cancer, is predisposed to cancer, is suspected of having cancer, or has a family history of cancer.
[00115] The methods disclosed herein are suitable for any cancer type or subtype. In some embodiments, the cancer may be a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. The cancer may be a cancer of the bladder, blood, bone, brain, breast, cervix, colon/rectum, endometrium, head and neck, kidney, liver, lung, muscle tissue, ovary, pancreas, prostate, skin, spleen, stomach, testicle, thyroid, or uterus. In certain embodiments, the cancer is of the colon, breast, bladder, prostate, or kidney. In some embodiments, the cancer comprises breast cancer, lung cancer, skin cancer, and/or liver cancer.
[00116] In some embodiments, the cancer comprises a solid tumor. Examples of cancers that comprise solid tumors include, but are not limited to, pancreatic, bladder, non-small cell lung cancer (NSCLC), breast and ovarian cancers. In some embodiments, the cancer is metastatic cancer. In some embodiments, the methods result in decreased tumor growth. In some embodiments, the methods result in tumor regression. In some embodiments, the methods result in decreased numbers of tumor. In some embodiments, the methods result in decreased tumor growth. In some embodiments, the methods
prevent tumor recurrence. In some embodiments, the methods result in increases in overall subject survival. In some embodiments, the methods result in long-term anti-tumor efficacy.
[00117] The present disclosure also provides methods of inducing and sustaining an immune response in a subject. Generally, the immune response can include a humoral immune response, a cell- mediated immune response, or both. In some embodiments, the induction of the immune response is due to activation of the STING pathway.
[00118] A humoral immune response refers generally to antibody production, and to all of the processes that accompany antibody production, including, but not limited to, B lymphocyte (B cell) activation, affinity maturation, differentiation into plasma cells, and memory B cell generation, germinal center formation and isotype switching, and T helper cell activation, signaling, and cytokine production, as well as effector functions of antibodies, which include neutralization, classical complement activation, and opsonization.
[00119] A cell-mediated immune response refers generally to the response to an antigen of immune cells including T lymphocytes (including cytotoxic T lymphocytes (CTL)), dendritic cells, macrophages, and natural killer cells, and to all of the processes that accompany such responses, including, but not limited to, activation and proliferation of these cells, CTL effector functions, cytokine production that influences the function of other cells involved in adaptive immune responses and innate immune responses, and memory T cell generation. In some embodiments, the methods result in induction of strong immune memory and/or increased memory T cell differentiation.
[00120] The methods include administering to the subject an effective amount of the compositions, polypeptides, polynucleotides (e.g., mRNA), vaccines, or medicaments disclosed herein. An “effective amount” is an amount that is delivered to a subject, either in a single dose or as part of a series, which is effective for inducing a response in the subject. This amount varies depending upon the health and physical condition of the subject to be treated, the capacity of the subject’s immune system to synthesize antibodies, the formulation of the compositions, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined by one of skill in the art through routine trials.
[00121] The compositions, polypeptides, polynucleotides (e.g., mRNA), vaccines, or medicaments disclosed herein can be administered in a wide variety of therapeutic dosage forms. The route and regimen of administration will vary depending upon the subject and the indication and is to be determined by the skilled practitioner. For example, the compositions, vaccines, or medicaments
disclosed herein may be administered parentally, e.g., intravenous (either by bolus or infusion methods), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form. [00122] The compositions, polypeptides, and polynucleotides may be delivered by any suitable means. Methods of delivering polypeptides and polynucleotides to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference); or viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the polynucleotides can be delivered by any method appropriate for introducing nucleic acids into a cell. In some embodiments, the polynucleotide is a DNA molecule. In some embodiments, the polynucleotide is in a DNA vector and may be electroporated to cells. In some embodiments, the polynucleotide is an RNA molecule, which may be electroporated to cells.
[00123] Additionally, delivery vehicles such as nanoparticle- and lipid-based polynucleotide or protein delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int I Pharm. 2014 Jan 1 ;459(1 -2):70-83), incorporated herein by reference.
[00124] The administration may comprise an initial immunization or dose and at least one subsequent immunization or booster dose, following known standard immunization protocols. The boosting doses will be adequately spaced at such times where the levels of circulating antibody fall below a desired level. Boosting doses may consist of the compositions or vaccines disclosed herein and may comprise alternative carriers and/or adjuvants. The booster dosage levels may be the same or different that those of the initial dosage.
[00125] In any of the uses or methods described herein, administration may be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and/or intraperitoneal administration to a subject in need thereof, in some embodiments, administration includes parenteral administration (including, but not limited to, subcutaneous, intramuscular, intravenous, intraperitoneal, intracardiac and intraarticular
injections). In some embodiments, the administration is intratumoral. In some embodiments, the administration is systemic.
[00126] The specific dose level may depend upon a variety of factors including the activity of the polynucleotides (e.g., mRNA), polypeptides, composition, vaccine, or medicament, the age, body weight, general health, and diet of the subject, time of administration, and route of administration. For prophylaxis purposes, the amount of the polynucleotide(s) (e.g., mRNA) in each dose is an amount which induces an immunoprotective response without significant adverse side effects.
[00127] A wide range of second therapies may be used in conjunction with the polynucleotides, compositions, vaccines, medicaments, and methods of the present disclosure. The second therapy may be administration of a therapeutic agent or may be a second therapy not connected to administration of another agent. Such second therapies include, but are not limited to, surgery, immunotherapy, radiotherapy, or a chemotherapeutic or anti-cancer agent.
[00128] The second therapy (e.g., an immunotherapy) may be administered at the same time as the initial therapy. The second therapy may be administered in the same composition or in a separate composition administered at substantially the same time as the initial therapy. In some embodiments, the second therapy may precede or follow administration of the disclosed polynucleotides, polypeptides, compositions, vaccines, medicaments, and methods by time intervals ranging from hours to months.
[00129] In some embodiments, the second therapy includes immunotherapy. Immunotherapies include chimeric antigen receptor (CAR) T-cell or T-cell transfer therapies, cytokine therapy, immunomodulators, cancer vaccines, or administration of antibodies (e.g., monoclonal antibodies). [00130] In some embodiments, the second therapy includes administration of an agent which downregulates or inhibits Wnt2b (e.g., a Wnt2b inhibitor or Wnt2b blocking agent). Wnt2b inhibitors may block access of a substrate or ligand to Wnt2b, disrupt the expression of Wnt2b, block activity of the Wnt2b by binding an allosteric site, or degrade or destabilize Wnt2b. Suitable Wnt2b inhibitors include, but are not limited to, gene silencing oligonucleotides (e.g., an siRNA, an antisense oligonucleotide, dominant-negative, a short-hairpin RNA, a miRNA, a dicer-substrate RNA, a DNAzyme, an guide RNA, or an aptamer targeting the gene or messenger RNA of Wnt2b), protein configured to bind Wnt2b (e.g., an anti-BMP synthase antibody (e.g., a monoclonal, polyclonal, murine, chimeric, humanized, or human antibody targeting an Wnt2b epitope, thus interfering with Wnt2b activity)), a small molecule inhibitor of Wnt2b, or combinations thereof. In some embodiments, the methods further comprise administration of a Wnt2b antibody or fragment thereof or an interfering
RNA to Wnt2b. In some embodiments, the Wnt2b inhibitor comprises an antisense oligonucleotide, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a guide RNA (gRNA), and combinations thereof.
[00131] Further disclosed herein are methods of treating a disease or disorder or inducing an immune response in a subject comprising administering an effective amount of an agent which downregulates or inhibits Wnt2b, to a subject in need thereof.
[00132] In some embodiments, the disease or disorder comprises cancer. In some embodiments, the subject is a human. In some embodiments, the subject has cancer, has had cancer, is predisposed to cancer, is suspected of having cancer, or has a family history of cancer.
[00133] The methods disclosed herein are suitable for any cancer type or subtype. In some embodiments, the cancer may be a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. The cancer may be a cancer of the bladder, blood, bone, brain, breast, cervix, colon/rectum, endometrium, head and neck, kidney, liver, lung, muscle tissue, ovary, pancreas, prostate, skin, spleen, stomach, testicle, thyroid, or uterus. In certain embodiments, the cancer is of the colon, breast, bladder, prostate, or kidney. In some embodiments, the cancer comprises breast cancer, lung cancer, skin cancer, and/or liver cancer.
[00134] In some embodiments, the cancer comprises a solid tumor. Examples of cancers that comprise solid tumors include, but are not limited to, pancreatic, bladder, non-small cell lung cancer (NSCLC), breast and ovarian cancers. In some embodiments, the cancer is metastatic cancer. In some embodiments, the methods result in decreased tumor growth. In some embodiments, the methods result in tumor regression. In some embodiments, the methods result in decreased numbers of tumor. In some embodiments, the methods result in decreased tumor growth. In some embodiments, the methods prevent tumor recurrence. In some embodiments, the methods result in increases in overall subject survival. In some embodiments, the methods result in long-term anti-tumor efficacy.
[00135] In some embodiments, the methods comprise an initial administration and at least one subsequent administration of the Wnt2b blocking agent. In any of the uses or methods described herein, administration may be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and/or intraperitoneal administration to a subject in need thereof, some embodiments, administration includes parenteral administration (including, but not limited to, subcutaneous, intramuscular, intravenous, intraperitoneal, intracardiac and intraarticular injections). In some embodiments, the administration is intratumoral. In some embodiments, the administration is systemic.
[00136] A wide range of second therapies may be used in conjunction with the Wnt2b blocking agent. The second therapy may be administration of an additional active agent or may be a second therapy not connected to administration of another agent. Such second therapies include, but are not limited to, surgery, immunotherapy, radiotherapy.
[00137] The second therapy may be administered at the same time as the initial therapy, either in the same composition or in a separate composition administered at substantially the same time as the first composition. In some embodiments, the second therapy may precede or follow the treatment of the first therapy by time intervals ranging from hours to months.
[00138] In some embodiments, the additional active agent comprises an immune modulator, a chemotherapeutic agent, a nucleic acid (e.g., mRNA, aptamers, antisense oligonucleotides, ribozyme nucleic acids, interfering RNAs, antigene nucleic acids), a steroid, an analgesic, and an antimicrobial agent, or a combination thereof.
[00139] Exemplary immune modulators include: STING agonists, signal transducer and activator of transcription 3 (Stat3) inhibitors and analogs thereof, toll-like receptor (TLR) agonists and analogs thereof, SM-360320, TMX-101, TMX-202, TMX-302, TMX-306, GSK2245035, CL097, 852A, AZD- 8848, DSP-3025, GS-9620, R07020531, RO6871765, ANA773, DSP-0509, NJH395, BNT411, TQ- A3334, JNJ-4964, LHC165, CV8102, VTX-1463, VTX-2337, IMO-8400, IMO-3100, IRS-954, and analogs thereof; and statins or other lipid-lowering medications and analogs thereof.
[00140] In some embodiments, the additional active agent comprises at least one chemotherapeutic agent. As used herein, the term “chemotherapeutic” or “anti-cancer drug” includes any small molecule or other drug used in cancer treatment or prevention. Chemotherapeutic s include, but are not limited to, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazine, cisplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib, afinitor (Everolimus), alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, toremifene, fulvestrant, letrozole, gemcitabine, goserelin, ixabepilone, emtansine, lapatinib, olaparib, megestrol, neratinib, palbociclib, ribociclib, talazoparib, thiotepa, toremifene, methotrexate, and tucatinib. In select embodiments, the chemotherapeutic agent comprises paclitaxel.
[00141] In some embodiments, the second therapy includes immunotherapy. Immunotherapies include chimeric antigen receptor (CAR) T-cell or T-cell transfer therapies, cytokine therapy, immunomodulators, immune checkpoint inhibitors, cancer vaccines, or administration of antibodies (e.g., monoclonal antibodies).
[00142] In some embodiments, the immunotherapy comprises administration of antibodies. The antibodies may target antigens either specifically expressed by tumor cells or antigens shared with normal cells. In some embodiments, the immunotherapy may comprise an antibody targeting, for example, CD20, CD33, CD52, CD30, HER (also referred to as erbB or EGFR), VEGF, CTLA-4 (also referred to as CD 152), epithelial cell adhesion molecule (EpCAM, also referred to as CD326), and PD- 1/PD-L1. Suitable antibodies include, but are not limited to, rituximab, blinatumomab, trastuzumab, gemtuzumab, alemtuzumab, ibritumomab, tositumomab, bevacizumab, cetuximab, panitumumab, ofatumumab, ipilimumab, brentuximab, pertuzumab, and the like). In some embodiments, the additional therapeutic agent may comprise anti-PD-l/PD-Ll antibodies, including, but not limited to, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab. The antibodies may also be linked to a chemotherapeutic agent. Thus, in some embodiments, the antibody is an antibody-drug conjugate.
7. Systems or Kits
[00143] Also disclosed herein is a system or kit comprising the compositions, polypeptides, polynucleotides, vaccines, or medicaments disclosed herein.
[00144] The kits can also comprise other agents and/or products co-packaged, co-formulated, and/or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed peptide and/or product and another agent (e.g., a chemotherapeutic, a monoclonal antibody, a pain reliever, a steroid, an antiemetic) for delivery to a patient or a cell.
[00145] The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
[00146] It is understood that the disclosed kits can be employed in connection with the disclosed methods. The kit may further contain containers or devices for use with the methods or compositions disclosed herein. The kits optionally may provide additional components such as buffers and disposable single-use equipment (e.g., pipettes, cell culture plates, flasks).
[00147] In some embodiments, the kits comprise a delivery device or container. In some embodiments, the delivery device or container comprises a syringe or syringe vial. In some embodiments, the delivery device or container is pre-filled with the composition.
[00148] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Individual member components of the kits may be physically packaged together or separately.
8. Examples
Materials and Methods
[00149] Materials COATSOME SS-OP, l,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), and l,2-Dimyristoyl-rac-glycero-3 -methylpolyoxyethylene (DMG-PEG2000) were purchased from NOF Corporation. Cholesterol was purchased from Avanti Polar Lipids. 2’3’-cGAMP was purchased from Invivogen. Luciferase Assay System was purchased from Promega. IVISbrite D-Luciferin potassium salt bioluminescent substrate was purchased from PerkinElmer. EndFree Plasmid Maxi Kit was purchased from Qiagen. All other reagents and chemicals were obtained from Sigma- Aldrich, Life Technologies, and TriLink BioTechnologies unless otherwise stated.
[00150] Cell culture 4T1 wild-type, 4T1-Luc2, and HEK293T cell lines were obtained from ATCC. ExpiCHO cell line was obtained from ThemoFisher Scientific. DC2.4 cell line was obtained from Millipore Sigma. E0771 cell line was from Dr. Jenny P.-Y. Ting’s lab at UNC Chapel Hill. 4T1 wildtype cells were stably transfected with a vector carrying the GFP and the puromycin resistance gene. 4T1-Luc2 cells were cultured in RPMI1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 8 pg/ml blasticidin at 37 °C and 5% CO2 in a humidified atmosphere. 4TLGFP cells were cultured in RPMI1640 supplemented with 10% FBS and 1 pg/ml puromycin at 37 °C and 5% CO2 in a humidified atmosphere. 4T1 wild-type cells were cultured in RPMI1640 supplemented with 10% FBS at 37 °C and 5% CO2 in a humidified atmosphere. DC2.4 cells were cultured with RPMI1640 supplemented with 10% FBS, lx non-essential amino acids (Gibco), lx HEPES buffer (Gibco), and 0.0054x p -mercaptoethanol (Gibco). HEK293T cells were cultured in high glucose DMEM (Gibco) supplemented with 10% FBS at 37 °C and 5% CO2 in a humidified atmosphere. ExpiCHO cells were cultured in ExpiCHO™ Expression Medium at 37 °C and 8% CO2 in a humidified atmosphere on an orbital shaker platform (125 ± 5 rpm).
[00151] Primary BMDCs were collected from WT, Tmeml73 /_, Irf3 /_, and Ifnar /_ C57BL/6 mice. Briefly, femurs and tibias were collected and bone marrow was flushed out with ice-cold RPMI1640 using a sterile syringe and a 21G needle. After red blood cell lysis, bone marrow cells were
resuspended in RPMI1640 medium containing 10% heat-inactivated FBS, murine recombinant GM- CSF (20 ng/ml, BioLegend) and 1% antibiotic-antimycotic (Gibco) and distributed into 100 mm Petri- dishes at a density of 3xl06 cells per dish for 6 days. Every 2 days, cells were washed and resupplemented with fresh complete medium. On day 6, highly adherent cells were discarded and CD1 lc+F4/80“ cells were sorted.
[00152] Experimental mouse models All animals were maintained at the animal facilities at University of North Carolina at Chapel Hill under specific pathogen-free conditions. Animal experiments were carried out in accordance with protocols approved by the University of North Carolina at Chapel Hill’s Institutional Animal Care and Use Committee. Six- week-old female wildtype C57BL/6J and BALB/cJ mice were purchased from Jackson Laboratories. Tmeml73 /_mice, Irf3 /_ mice, and Ifnar /_ mice were provided by Dr. Jenny P.-Y. Ting’s laboratory (UNC Chapel Hill). All KO strains were maintained on a C57BL/6J background. For E0771 tumor models, 4xl05to lx 106 cells in 20 pl of PBS were injected into the mammary fat pads of C57BL/6J and tumor size was measured every 2-3 days using a digital caliper. Tumor volume was estimated using the formula: tumor volume = lengthxwidth2/2. For 4T1-Luc2 tumor models, 2xl05 to IxlO6 cells in 20 pl of PBS were injected into the mammary fat pads of BALB/cJ mice. The tumor progression was monitored by an IVIS® Kinetics Optical System (Perkin Elmer, CA) after intraperitoneal injection of 100 pL of d-luciferin (10 mg/ml) to mice. For LLC tumor models, 5xl05 cells in 25 pl of PBS were injected into the right flank of C57BL/6J mice. For B16F10 tumor models, IxlO5 cells in 25 pl of PBS were injected into the right flank of C57BL/6J mice. For the rechallenge studies, mice that could be cured were inoculated with IxlO5 B16F10 cells for tumor rechallenging. Tumor size was directly measured by using a digital caliper. For orthotopic HCC tumor models, 5xl06 Hepal-6 cells in 30 pl of PBS were inoculated at the subcapsular region of the left lobes of the liver in C57BL/6J. To establish the hemi-spleen 4T1 liver metastasis model, the spleen was exposed and tied in half using suture. 1X1064T1-LUC2 tumor cells in 50 pl of PBS were inoculated directly into spleen, followed by resecting the inoculated portion of spleen. The rest of the spleen was returned before suturing. For all the different mouse models, mice were randomly assigned to treatment groups. The investigators were blinded to the group allocation during the animal experiments. On reaching an average tumor size of -60 mm3, mice were treated by intratumoral injection with either PBS, free cGAMP, or indicated mRNA/LNP formulations or intraperitoneal injection with a-Wnt2b antibody (Wnt2b Rabbit Polyclonal Antibody, Affinity Bioscicnccs DF12538 and Wnt2b Recombinant Polyclonal Antibody (17HCLC), ThermoFisher
Scientific). For survival studies, mice were euthanized when tumors reached -1000 mm3 or mice lost more than 30% of their weight.
[00153] In vitro transfection studies For plasmid transfection, HEK293T cells were cultured until 80% confluence and transfected with the expression vector encoding gene of interest through lipofectamine 2000, according to the manufacturer’s instructions. HEK293T cells were collected 48 h post transfection for further analysis. For recombinant expression of uniSTING, ExpiCHO cells were transfected with pCDNA3.4 plasmid encoding uniSTING using ExpiFectamine™ CHO Transfection Kit (ThermoFisher Scientific), according to the manufacturer’s instructions. After 6 days, cells were collected for protein purification. For mRNA transfection, cells were transfected with mRNA either mixed with TransIT®-mRNA transfection agent (Minis Bio) or encapsulated into SS-OP LNPs in DMEM culture medium supplemented with 10% FBS, according to the manufacturer’s instructions or optimized conditions. After 24 h or 48 h, cells or supernatant were collected for further analysis. For siRNA transfection, cells were transfected with 10 nM of ON-TARGETplus mouse Wnt2b siRNA pool or non-targeting Control siRNAs (Dharmacon, Horizon Discovery) using the INTERFERin transfection reagent (Polyplus-transfection) in a DMEM culture medium supplemented with 10% FBS, according to the manufacturer’s instructions. After 24 h or 48 h, cells were collected and followed by qPCR analysis. For miRNA transfection, DCs were transfected with 15 nM miRIDIAN mouse miRNA pool (miR-130b-3p mimic, miR-130a-3p mimic, and miR-19a-3p mimic) (Dharmacon, Horizon Discovery) or miRIDIAN miRNA mimic negative control using the DharmaFECT 1 transfection reagent (Dharmacon, Horizon Discovery) in DMEM culture medium, according to the manufacturer’ s instructions. After 4h, extracellular vesicles (EVs) isolated from tumor cells with different treatments were added into the culture medium, and the cells were incubated at 37 °C and 5% CO2 in a humidified atmosphere for 24 h followed by qPCR analysis.
[00154] uniSTING protein generation and characterization The constitutively active STING mimic protein was designed by genetically fusing a thermostable tetramerization motif (GIINETADDIVYRLTVIIDDRYESLKNLITLRADRLEMIINDNVSTILASIG - SEQ ID NO: 1) with the C-terminal cytoplasmic domain of STING (residues 138-378 of murine STING). A Flag tag was engineered at the N-terminus to facilitate protein purification and detection. The expression vectors encoding the fusion protein were generated by inserting synthetic DNAs (codon optimized for expression in mammalian cells) into pCDNA3.4 vector. For protein expression, ExpiCHO cells were transfected with pCDNA3.4 plasmid encoding uniSTING. The cell lysates from IxlO7 cells were collected 6 days after transfection by using 1 ml of lysis buffer (50 mM Tris HC1, pH 7.4, 150 mM
NaCl, 1 mM EDTA, and 1% TRITON X-100) with Halt™ protease inhibitor cocktail (ThermoFisher Scientific). The uniSTING protein was purified via anti-Flag M2 affinity gel (Sigma-Aldrich) according to the manufacturer’s protocol. The purified proteins were analyzed on 4-12% SDS-PAGE gel (Invitrogen) with Coomassie G-250 (Bio-Rad) stain. Molecular weight analysis was performed by FPLC (GE AKTA™) by using a Superdex 200 10/300 GL gel filtration column (MilliporeSigma). 200 pg of each standard (FIG. 9B) and 400 pg of uniSTING protein was dissolved in 200 pl of TBS (50 mM Tris HC1, 150 mM NaCl, pH 7.4) buffer with 0.02% Tween 80 and filtered through 0.45 pm filter, respectively. The protein samples were then injected to the column and separated at a flow rate of 0.3 ml/min and detected at 280 nm— . Molecular weight of uniSTING was finally determined from the standard curve. The melting temperature (Tm) of uniSTING was measured by using Protein Thermal Shift™ (PTS) kit (ThermoFisher Scientific) according to the manufacturer’s protocol. Briefly, uniSTING was mixed with different buffers (FIG. 9C) and PTS dye, and a mclt-curvc experiment was run on a real-time PCR instrument (Analytik Jena, qTOWER3 G). The Tm was calculated from the melt curve based on Protein Thermal Shift™ Software (ThermoFisher Scientific).
[00155] mRNA synthesis and LNPs preparation mRNA for studies of mCherry expression in vivo was obtained from TriLink BioTechnologies. The expression vectors encoding uniSTING and mSTING were generated by inserting the corresponding ORF into a modified pUC57 vector optimized for in vitro transcription. All plasmids were sequenced to confirm the sequence accuracy. The cDNA used for in vitro transcription was PCR-amplified using a T7 promoter primer and a 3’ primer containing 150 Ts. mRNAs were transcribed in vitro by using MEGAscript kits in the presence of Cleancap AG (TriLink) as a 5 ’-capping agent according to the manufacturer’s instructions. The resulting mRNAs were purified by using MEGAclear™ Transcription Clean-Up Kit (ThermoFisher Scientific) and characterized prior to being encapsulated into LNPs. All mRNAs were stored at -80 °C and were allowed to thaw on ice before use. The SS-OP LNPs were prepared according to previous reports (Tanaka H, et al., Molecular pharmaceutics 2018, 15(5): 2060-2067 and Sabnis S, et al. Molecular Therapy 2018, 26(6): 1509-1519) and manufacturer’s instructions. In brief, an organic phase was prepared by solubilizing with ethanol a mixture of ionizable SS-OP lipid, DOPC, cholesterol, DMG-PEG2000 at a 52.5/7.5/40/1.5 molar ratio. The mRNA solution was prepared in 20 mM malic acid buffer with 30 mM NaCl (pH 3.0) at a concentration of 66.7 p.g/ml. To prepare mRNA/LNPs, mRNA solution was rapidly mixed with the lipid ethanol solution at room temperature under vortexing. MES buffer (5.25 ml, pH 5.5, 20 mM) was then added to the mixture. Subsequent buffer exchange was conducted by using Amicon Ultra-15-100 K centrifugal units (Merck Millipore).
The size, polydispersity index and zeta potentials of LNPs were measured by using Malvern Nano ZS (Westborough, MA). Diameters were reported as the volume mean peak average. LNPs were also characterized by using cryogenic electron microscopy. To define the cell types responsible for mRNA uptake and its translation in vivo, 4T1-GFP tumor-bearing mice were intratumorally administered with mCherry-mRNA/LNPs, followed by flow cytometric and confocal microscope analyses for mCherry+ cells in tumor tissues.
[00156] Western blot analysis Cells were lysed in radio-immunoprecipitation buffer (RIPA buffer, Invitrogen). Protein concentration of samples was determined using a BCA Protein Assay Kit (Pierce). 10-30 pg of protein were mixed with NuPage LDS sample buffer (Invitrogen) and NuPage sample reducing agent (Invitrogen) and heated at 95 °C for 5 min. The denatured samples were loaded and separated on a 4-12% Bis-Tris NuPage gel (Invitrogen). The proteins were then blotted on a PVDF membrane (Bio-Rad) for 1 h at 20 V. The membrane was blocked in 5% bovine scrum albumin in TBST for 1 h at room temperature and then incubated with primary antibodies overnight at 4 °C while shaking. The membranes were washed and further incubated with a secondary antibody for 40 min at room temperature and then detected using the Clarity Western ECL Substrate (Bio-Rad). GAPDH was used as the control. ChemiDoc™ Imaging System was used to acquire images (Bio-Rad).
[00157] Colocalization and co-immunoprecipitation of uniSTING with TBK1 and IRF3 The interactions between uniSTING and TBK1 or IRF3 were analyzed through confocal microscope imaging and co-immunoprecipitation. For confocal microscope imaging, the expression vectors encoding Flag-tagged uniSTING, TBK1-GFP, and IRF3-HA were generated by inserting synthetic coding DNAs (codon optimized for expression in mammalian cells) into pCDNA3.4 vectors. HEK293T cells were cultured until 80% confluence and transfected with the expression vector encoding a gene of interest. HEK293T cells were rinsed in PBS 48 h post transfection and then placed in 4% paraformaldehyde (PF A) for 15 min at 4 °C. Following 4% PFA fixation, cells were processed through permeabilization and blocking in 5% goat serum at room temperature for 1 h. Primary antibodies were incubated overnight at 4 °C and rinsed with PBS for 3 times, followed by fluorescent secondary antibody staining (37 °C, 1 h). Finally, the cells were mounted with Prolong® Diamond Antifade Mountant with DAPI (ThermoFisher Scientific).
[00158] Immunofluorescence images were taken by using a laser scanning confocal microscope (Zeiss LSM 700) to analyze the colocalization of uniSTING, TBK1, and IRF3. For coimmunoprecipitation experiments, Flag-tagged uniSTING was first captured on anti-Flag M2 affinity resin in TBS buffer with protease inhibitor cocktails. Cell lysates from DC2.4 cells were incubated
with uniSTING bound to anti-Flag M2 resin for 4 h at 4 °C and washed three times with TBS buffer. The resin was then boiled in SDS-loading buffer and analyzed by immunoblotting. The bound Flag fusion protein was also eluted with 0.1 M glycine HC1, pH 3.5 into vials containing 15-25 pl of 1 M Tris, pH 8.0 and analyzed by immunoblotting.
[00159] Enzyme-linked immunosorbent assay (ELISA) The expression of Flag-tagged uniSTING protein in the tumor tissue, normal organs and serum was measured after intratumoral injection of uniSTING-mRNA/LNPs. Briefly, tissue and plasma samples were collected at different time points post injection. The tissue samples were lysed using RIPA lysis buffer containing protease inhibitor cocktail mix and 5 mM EDTA. The cell lysates were centrifuged, and the proteins in the supernatant were collected. BCA kit was used to measure protein concentration according to the manufacturer’s protocols. For ELISA, flat-bottomed 96-well plates (ThermoFisher Scientific) were precoated with anti-Flag-tag monoclonal antibody (ThermoFisher Scientific) at a concentration of 0.5 pg/ml per well in 100 mM carbonate buffer (pH 9.6) at 4 °C overnight. 5% BSA in PBST was then used to block the non-specific binding for 1 h at room temperature. The samples were diluted 50 times in PBST buffer and added to the wells. After incubation for 2 h at room temperature, the plate wells were washed with PBST for 5 times, followed by incubation with the detective antibody (rabbit polyclonal anti-STING antibody, 1: 1000 dilutions, ThermoFisher Scientific) for 1 h at room temperature. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (CST) was used at a dilution of 1: 10000 in the PBST buffer with 5% BSA for labeling. After adding the HRP substrates, optical densities were determined at a wavelength of 450 nm in the plate reader (Bio-Rad). IFN-0 expression in DCs and tumor cells after treatment with either PBS, EGFP-mRNA (mock), uniSTING-mRNA, mSTING- mRNA, or free cGAMP was also measured by using mouse IFN-P ELISA Kit (R&D systems) according to the manufacturer’s instructions.
[00160] Flow cytometry assay Tumor tissues were harvested and digested in RPMI1640 medium containing 2% heat-inactivated FBS, collagenase type I (200 U/ml, Invitrogen), collagenase type IV (200 U/ml, Invitrogen), and DNAase I (100 pg/ml, Invitrogen) in 2% FBS at 37 °C for 1 h to generate single-cell suspensions. The tumor-infiltrating lymphocytes were isolated by ficoll-paque density gradient centrifugation and diluted to IxlO6 cells/ml for staining with LIVE/DEAD™ Fixable Near-IR dye, followed by surface staining with fluorescently conjugated antibodies. For determination of cytokine production and granzyme B levels, tumor-infiltrating lymphocytes were incubated with PMA/Ionomycin for at least 5 h. Brcfcldin A (Biolcgcnd) was added 30 minutes after beginning of the stimulation. After stimulation, cell surfaces were stained, and cells were subsequently fixed and
permeabilized with Cytoperm™ Fixation/Permeabilization Solution Kit (BD) for intracellular staining, according to the manufacturer’s protocol. Samples were analyzed by BD FACSARIA II flow cytometer. Data was analyzed via FlowJo software (TreeStar, Ashland, OR) and the gating strategy is shown in FIGS. 23 and 24.
[00161] For sorting BMDCs, cells were stained with BV510 anti-mouse CD45, APC anti-mouse CD11c, and PE anti-mouse F4/80 and sorted according to the gating strategy displayed in FIGS. 20 and 18. Dead cells were excluded via LIVE/DEAD fixable dye staining.
[00162] Quantitative reverse transcription polymerase chain reaction (qPCR) assay Total RNAs were extracted from cell or tumor tissue samples by RNeasy Plus Mini Kit (Qiagen) according to manufacturer’s instructions. cDNA was reverse transcribed using iScript Reverse Transcription Supermix (Bio-Rad) and amplified with the TaqManTM Gene Expression Master Mix (Applied Biosystems) or iTaq Universal SYBR Green Supcrmix (Bio-Rad). qPCR was performed using 7500 Real-Time PCR System and data were analyzed with the 7500 v2.3. Expression was normalized to internal control GAPDH.
[00163] EV isolation Tumor cells were cultured in medium supplemented with 10% EV-depleted FBS. After treatment with mock- or uniSTING-mRNA, EVs were collected by serial centrifugation as reported (Torralba D, et al., Nature Communications 2018, 9(1): 2658). Briefly, the supernatants were sequentially centrifuged at 500 g for 10 min and 1,000 g for 20 min at 4 °C to remove debris and dead cells. The collected supernatant was then ultracentrifuged at 12,000 x g for 20 min at 4 °C (Beckman Coulter Optima L-100 XP, Beckman Coulter) to remove apoptotic bodies and shedding vesicles. EVs were pelleted by ultracentrifugation at 100,000 x g for 70 min at 4 °C. The collected EVs were washed twice with PBS. The biomarkers of EVs were characterized by western blot using CD9 and CD81 antibodies. EVs were also characterized by TEM.
[00164] miRNA array analysis miRNAs were extracted from EVs using miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The isolated RNAs were converted into cDNA by using miScript II reverse transcription reaction with HiSpec Buffer (Qiagen). cDNA was used as the template in qPCR with the miScript miRNA PCR Array Mouse Inflammatory Response & Autoimmunity and miScript SYBR Green PCR Kit (Qiagen). The qPCR was performed on the ABI9700 real-time PCR system using the following thermocycling parameters: 94 °C for 15 min, 40 cycles at 94 °C for 10 s, 55 °C for 30 s and 70 °C for 30 s, followed by a melting curve analysis with QuantStudio software (Applied Biosystems). The data were further analyzed by using the online miRNA PCR array data analysis tool (qiagen.com/us/shop/pcr/primer-sets/miscript-mima-pcr-
arrays/#resources) according to the manufacturer’s instructions. miRNAs with a threshold cycle value greater than 35 were considered as undetermined in order to minimize the potential noise introduced by measurements below the detection threshold.
[00165] RNA deep-sequencing Total RNA was extracted from DC2.4 cells after different treatments by using RNeasy Plus Mini Kit (Qiagen) according to manufacturer’s instructions. Contaminating genomic DNA was removed by using the specific column from the kit. Library preparation and RNA deep-sequencing were performed by Novogene. Briefly, library for transcriptome sequencing was generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB) following manufacturer’ s recommendations and the index codes were added to attribute sequences to each sample. The library quality was determined in the Agilent Bioanalyzer 2100 system. After removing reads with adapter or of low quality, clean reads were aligned to the mouse GRCm39 reference genome using hisat2 software, and only the uniquely mapped reads were retained for downstream analysis. The gene expression level was quantified using FeatureCounts function from Subread package. Data were then processed with a pipeline that used the EdgeR Bioconductor package for normalization and differential expression analysis in a paired strategy. The differential expression genes were analyzed by DESeq2 package with default settings using total read counts as input. Fold change and log2(ratio) values were calculated to represent gene expression differences between conditions. Heatmaps of gene expression were generated based on z-score values of normalized expression matrix from DESeq2 analysis (broadinstitute.org/GENE-E/).
[00166] GSEA GSEA was performed by using the Java application available from The Broad Institute (broadinstitute.org/gsea/). Gene set database Hallmarks (h.all.v6.1.symbols.gmt) from the Molecular Signatures Database (MSigDB) were used in the analysis. One thousand gene set permutations were performed. An FDR cutoff of <0.05 was used for enriched terms, as is recommended when performing permutations by gene set. R version 3.5.0 was used for analysis. [00167] Statistical analysis Data were expressed as the mean ± standard deviation (s.d.). Statistical significance was determined by unpaired two-tailed Student’s t-test when only two value sets were compared or by ANOVA comparison between multiple groups. Log-rank Mantel-Cox test was used for survival curves. Exact P values are documented in the figures. All statistical analyses were done by using GraphPad Prism 7.0. No exclusion criteria were incorporated in the design of the experiments.
Example 1
Construction and characterization of universal STING activator
[00168] To overcome the lack of functional STING in TME and undesired toxicity after systemic administration, the universal STING activator was developed by genetically fusing an extremely thermostable tetramerization motif (52 residues) with the C-terminal cytoplasmic domain of STING (residues 138-378 of murine STING for the mouse version). Four copies of the resulting fusion protein self-assembled into a subunit with tetrameric structure independent of membrane association, mimicking the side-by-side tetrameric STING on the membrane. Further oligomerization based on this tetrameric core could result in polymeric STING with higher-order architecture for TBK1 recruitment and downstream signaling activation (FIG. 2A). This recombinant uniSTING fusion protein was expressed in ExpiCHO cells (37.5 kDa, monomer in SDS-PAGE) and purified based on the engineered N-tcrminal Flag tag. FPEC analyses confirmed that under non-denaturing conditions uniSTING existed predominantly as an octamer with an estimated molecular weight around 300 kDa (FIGS. 9 A and 9B) and good stability (Tm -52.7 °C, FIG. 9C). By co-expressing TBK1 and IRF3 with uniSTING gene in HEK293T cells, it was demonstrated that TBK1 and IRF3 colocalized with uniSTING in the cytosol. Notably, IRF3 was additionally observed in the nucleus that usually reflected its activated state (FIG. 2B). Likewise, phosphorylated (p) TBK1 and pIRF3 were co -immunoprecipitated with uniSTING in DC2.4 cells (FIG. 2C), confirming that uniSTING was able to associate with TBK1 and IRF3 in agreement with the activation of STING signaling pathway. Gene set enrichment analysis (GSEA) showed IFN-a and IFN-y response genes were markedly enriched in uniSTING-mRNA treated DC2.4 cells compared to EGFP-mRNA (mock) group, according to RNA sequencing (RNA-seq) results (FIGS. 2D and 10A).
[00169] STING’s high mobility allows its trafficking through subcellular organelle membranes which initiates a chain reaction of cellular immune pathways. It is reported proinflammatory NF-KB signal occurs when STING is in the ER or ERGIC which is not necessary for IRF3 transcriptional activity. uniSTING was designed to form a tetrameric structure independent of membrane association. NF-KB phosphorylation was observed in the group treated with 2’3’-cGAMP, whereas there was no substantial activation of NF-KB signaling after treatment with uniSTING (FIG. 2F). RNA-seq was performed on 4T1 tumor cells via an unbiased analysis. Applying differentially expressed genes (DEGs) in uniSTING-versus mock-treated cells allowed us to examine the transcriptional factor binding gene clusters. Surprisingly, transcriptomic study revealed uniSTING’ s functions were mediated by IRF3/IFN-I pathway without impacting NF-KB signal (FIGS. 2E-2F). Specifically, 235
DEGs were elevated in 4T1 tumor cells after uniSTING treatment, with 80% of them connected to IRF3 binding and only 5% related to p65 binding, emphasizing the highly selective activation of STING downstream pathways by uniSTING, which favors IRF3/IFN-I activity over the pro- inflammatory yet also pro-tumorigenic NF-KB signaling (FIG. 2E). Pathway enrichment analysis was performed and STING signals that reported to be IRF3 -dependent and NF-KB -dependent were displayed (FIG. 2F). IRF3 -dependent signalings were extensively upregulated by uniSTING treatment (p<0.01) while NF-KB target signaling was unaffected by uniSTING. Furthermore, both uniSTING and cGAMP treatments elevated IFN- and CXCE10 transcripts (FIG. 2G). However, cGAMP treatment increased the production of IE6 and TNF-a, whereas uniSTING treatment showed no discernible change in these downstream molecules of NF-KB signaling (FIG. 2G). Additionally, a dendritic cell line (DC2.4) and two murine TNBC cell lines (4T1 and E0771) were driven to increase STING downstream pTBKl, pIRF3, and IFN-P secretion after treatment with uniSTING-mRNA but not with a monomeric form of STING (mSTING, which lacked the tetramerization motif (FIGS. 2H and 10B). Human polymeric STING (huSTING) activated STING signals in a STING-deficient human ovarian cancer cell line ES2, as well as the human monocyte THP-1 cell line, confirming the universal function of this polymeric STING protein across the human population (FIGS. 2H, 21, and 10C). Moreover, the NF-KB phosphorylation was significantly increased by cGAMP group but not uniSTING in these different types of cell lines (FIG. 2H), consistent with the observation by RNA-seq. These data indicated uniSTING functioned as a universal activator in mice and human cells. Thus, the specificity assessment of downstream pathways upon uniSTING treatment indicate a strong preference for IRF3/IFN-I over NF-KB signaling.
[00170] To delineate whether uniSTING relies on cGAS/STING pathway, cGAS inhibitor and cGAS knockout cells were used to assess the biological activities of uniSTING. As shown in FIG. 11, the activation of uniSTING downstream signaling was resistant to a cGAS inhibitor, Ru.521. Furthermore, BMDCs were collected from WT,
and Ifnar 1' mice and treated with STING agonists. Elevated mRNA expression of IFN-P and CXCL10 by uniSTING or 2’3’cGAMP in WT BMDCs was abolished in lrf3~!~ and Ifnarl'1' BMDCs, consistent with their action via the type I IFN pathways (FIG. 2K). However, uniSTING but not 2’3’cGAMP retained its ability to activate type I IFN and CXCL10 in STING deletion (Tmeml73~l~') BMDCs (FIG. 2J). As expected, phosphorylation of STING and IRF3 were induced by uniSTING in both WT and 'I'meml73^^ BMDCs, as assessed by western blotting (FIG. 12). Thus, uniSTING initiated IRF3/IFN-I-mediated signaling without requiring endogenous STING expression. Through uniSTING treatment, the remarkable STING-mediated IRF3
activity and the absence of STING-mediated NF-KB signaling established its characterization arose from a unique design distinctive from existing STING agonists. More importantly, the selectivity of uniSTING could reduce the side effects from the multifunctional NF-KB signaling which has been documented to promote tumor progression while strengthening the IRF3 -dependent type I IFN release for antitumor immunity.
Example 2
Cytosolic delivery of uniSTING based on mRNA/LNPs eliminates established tumors [00171] Clinically, low expression level of STING protein in human breast cancer correlates with poor prognosis (FIG. 13A). Compared to nontumor sites, endogenous STING expression is significantly downregulated in human breast tumor tissues (FIG. 13B). Among all breast cancer subtypes, triple-negative breast cancer (TNBC), as defined by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 expression, has the poorest outcomes. The therapeutic potential of the uniSTING mRNA against murine TNBC models was explored. LNP-mediated delivery of mRNA therapeutics is a powerful strategy to ensure transient and local expression of therapeutic proteins. To deliver uniSTING into the cytosol of target cells, mRNA encoding uniSTING was encapsulated into LNPs (uniSTING mRNA/LNPs) based on ionizable lipid- COATSOME SS-OP for efficient intracellular delivery. SS-OP contains two sensing motifs: a tertiary amine that is responsive for endosomal membrane destabilization and a disulfide bond that can be cleaved by GSH in a reductive environment (cytoplasm) for spontaneous particle collapse. The resulting mRNA/LNPs represent surface-neutral particles with a median diameter of -100 nm (FIGS. 3A and 14A). When luciferase mRNA was delivered via such SS-OP LNPs with redox-sensitivity, higher luciferase activity was observed in tumor cells compared to DC2.4 cells (FIG. 14B), presumably due to the enhanced GSH expression and thus more reductive conditions in the cytoplasm of tumor cells. To identify the cell types responsible for mRNA/LNP uptake and its translation in TME of 4T1-GFP tumors, mCherry-mRNA/LNPs were intratumorally administered into established tumors of mice followed by flow cytometric and confocal microscope analyses for mCherry+ cells. Both tumor cells and CDllc+ cells showed pronounced uptake and translation of the LNP-delivered mRNA, while cancer cells displayed the highest mCherry expression in accordance with the in vitro assay (FIGS. 3B- 3C, and 14C). Additionally, in vivo bioluminescent imaging showed that SS-OP LNPs had approximately 85% of luciferase activity in the tumor site 24 h after dosing, compared to -10% when the clinically approved MC3 LNPs were used, indicating SS-OP-bascd LNP functioned as an efficient delivery system (FIG. 3D). The in vivo translation efficacy of uniSTING mRNA was evalutated.
uniSTING was enriched in tumors compared to that in nontumor tissues or plasma. Intratumoral injection of 10 pg of LNP-formulated mRNA resulted in peak production of uniSTING protein after 4 hrs with decreased yet detectable measurements that lasted up to day 7 (FIG. 3E). uniSTING protein was detected in the liver from 2 hr to 72 hr post injection (FIG. 9D). This solved the poor stability problem of the current STING agonist by demonstrating continuous translation of the mRNA inside the transfected cells prior to its degradation.
[00172] The antitumor effect of uniSTING-mRNA/LNPs was investigated in vivo. Administration of uniSTING-mRNA/LNP by intratumoral injection efficiently inhibited 4T1-Luc2 breast tumor growth and prolonged survival benefit, compared with mock-mRNA/LNPs, mSTING-mRNA/LNPs, or soluble 2’3’-cGAMP (FIGS. 3F-3H, 15, and 16). Longer term monitoring showed that uniSTING promoted survival for the entire monitoring period of 50 days (FIG. 15D). Such antitumor efficacy was not limited to the 4T1 or breast tumors. Similar results were obtained in the Lewis lung carcinoma (LLC), B 16F10 melanoma tumor, and breast cancer E0771, demonstrating the potent and prevalent therapeutic efficacy of uniSTING-mRNA/LNPs to suppress multiple malignancies (FIGS. 3L3M and 17). In particular, uniSTING significantly eliminated 50% of B16F10 tumors and effectively prevented tumor rechallenge (FIGS. 3L-3N). It should be noted that the antitumor efficacy of uniSTING displayed an independence on STING expression based on the observed antitumor activity in Tmeml S'1' mice. Consistent with the IFNaR-dependent signaling of STING, uniSTING failed to inhibit tumor growth or prolong the survival time in tumor-bearing Ifna '~ mice, confirming its action mechanism via type I IFNs (FIGS. 3O-3P and 18). Together, uniSTING mRNA delivered by SS-OP LNPs boosted robust antitumor response in multiple tumor models within a manner independent of endogenous STING.
[00173] Although localized intratumoral administration of STING agonists is emerging as a clinically viable treatment for specific cancer types, it is still not clinically feasible for most solid tumors, particularly in the setting of advanced metastatic tumors. The therapeutic efficacy of uniSTING-mRNA/LNP through systemic treatment was examined in the 4T1-Luc2 metastatic tumor model and orthotopic liver cancer model, which are more challenging to cure than subcutaneous solid tumor models. Systemic administration of uniSTING significantly inhibited 4T1 liver metastatic tumor growth and eliminated established metastatic tumors in 40% of mice compared to cGAMP treated group that showed negligible response (FIGS. 4A-4D). An orthotopic HCC liver cancer model was also constructed by inoculating three million Hcpal-6 tumor cells into the subcapsular region of the left lobes of the liver. Intravenous administration with uniSTING-mRNA/LNPs significantly reduced
HCC tumor burden and prolonged survival in comparison with PBS, mock, or cGAMP groups (FIGS. 4E-4G). Thus, uniSTING-mRNA/LNPs effectively restrained advanced orthotopic/metastatic tumors by systemic administration.
[00174] The effect of uniSTING treatment on immune cell profile in tumors was investigated. The overall number of CD45+ leukocytes, as well as that of total DCs were not obviously changed among different groups (FIG. 5A). However, a substantial increase in the expression of the co stimulatory, CD86, was noticed on the DCs, suggesting improved DC co-stimulation by uniSTING treatment (FIG. 5A). A similar shift of immune cell composition was also validated in E0771 tumor models (FIG. 19). A significant expansion of tumor-infiltrating cytotoxic CD8+ T and NK1.1+ cells was observed in uniSTING treated group in comparison to that in the mock or PBS groups, while the change of the number of CD4+ T cells was minor (FIG. 5A), demonstrating STING signaling drives immune cell activation. The frequency of intratumoral functional IFN-v+ was increased by two fold while granzymc B + CD8+ T cells were dramatically increased by 7 fold after uniSTING treatment, indicating enhanced cytotoxicity of TILs in uniSTING-treated tumors (FIG. 5B). Increased transcripts of Thl cytokines, including IFN-P, IFN-y, and IL12A , were observed in uniSTING-treated group relative to control groups (FIG. 5C). To determine whether uniSTING treatment affects memory T cell populations, mice were vaccinated with or without uniSTING-mRNA/LNPs combined with ovalbumin (OVA) antigens. Specifically, four groups of C57BL/6 mice were immunized on day 0 and boosted at 3 weeks later with OVA alone or OVA mixed with uniSTING-mRNA/LNPs or mock LNPs via tail base injection, as illustrated in FIG. 5D. ADU-S100, a STING agonist that has been used in clinical trials, was used as a control. Consistent with tumor studies (FIG. 5A), uniSTING-mRNA/LNPs increased CD8+ T cells in PBMC compared to the ADU-S100 treated group (FIG. 5E). Additionally, uniSTING treatment decreased the level of CD44 CD62L+ naive CD8 T cell without obvious change for CD44+CD62L+central memory subpopulation (FIG. 5F). Importantly, the frequency of CD44+CD62L“ effect memory CD8 T cell were dramaticly increased in uniSTING groups compared to other groups, indicating that the uniSTING treatment promoted CD8 memory T cell differentiation (FIGS. 5F and 5G). It was reported that immunotherapy induces stem-like CD44+TCF1+CD27+CD8+ T cells, which were capable of proliferating, regenerating, and differentiating into effector T cells. Similar to this report, a surge in intratumoural CD44+TCF1+CD27+CD8+ T cells was observed with a higher frequency in the former group after injection of uniSTING-mRNA/LNPs and ADUS100 (FIG. 5H). These findings illustrated that uniSTING-mRNA/LNPs promoted DC maturation and strong CD8+ T cell responses.
Example 3 uniSTING triggers crosstalk between tumor cells and DCs in TME through silencing of Wnt2b by tumor-derived exosomal miRNAs
[00175] To explore the underlying mechanism of uniSTING that sensitizes antitumor immunity in TME, the dendritic cell line, DC2.4, was incubated with conditioned medium (CM) from tumor cells treated with either uniSTING-mRNA (CMuniSTiNG) or mock-mRNA (CMmock) control. Significant upregulation of IFN-P and CXCL10 mRNA was observed in the CMuniSTiNG treated DC2.4 cells compared to those incubated with CMmock (FIG. 6B), indicating CM from uniSTING-treated tumor cells may contribute to DC activation. Extracellular vesicles (EVs) are released by cells as way of communication with other nearby or distant cells. To decipher the molecule mechanism that governs the cell-cell communication, EVs were isolated from mock- or uniSTING-treated tumor cell CM (EXOmock or EXOUIUSTING) through differential ultracentrifugation. Average diameters of EV s obtained in the 100,000 x g fraction was around 130 nm (FIG. 6A), in agreement with the reported size of EVs. The presence of the canonical exosomal proteins CD9 and CD81 in the purified 100,000 x g fraction suggested an endosomal origin (FIG. 6A). The resulting EVs or the supernatants were then separately added into DC2.4 cells. Surprisingly, a drastic increase of IFN- and CXCL10 mRNA was observed in DC2.4 cells exposed to EXOuniSTiNG compared to EXOmock, indicating enhanced Type I IFN signal cascade in EXOuniSTiNG treated group (FIG. 6C-6D). The antitumor and immune modulative activities of EXOuniSTiNG were further evaluated in 4Tl-Luc2-tumor bearing mice. Compared to EXOmock-treated group, intratumoral administration of EXOuniSTiNG significantly retarded tumor growth, in parallel with increased release of type I IFN and reduced expression of immunosuppressive cytokines in the TME (FIG. 6E). These results highlighted the predominant role of EXOuniSTiNG in mediating the crosstalk between tumor cells and DCs as well as in modulating immune response. As the major exosome substances, miRNAs often act as intercellular communicators to reorganize the microenvironment during tumor evolution. miRNA profiling analysis was used to determine whether specific exosomal miRNAs derived from EXOuniSTiNG could participate in the crosstalk between tumor cells and DCs. Eleven miRNAs were considerably altered between EXOmock and EXOuniSTiNG, according to scatter plots with the boundaries set at plus or minus threefold changes. Five top-ranked miRNAs (miR-130b- 3p, miR-130a-3p, miR-19a-3p, miR-16-5p, and miR-15b-5p) were upregulated significantly in EXOuniSTiNG compared with those in EXOmock (FIGS. 6F-6G). The role of exosomal miRNA in triggering the activation of IFN signal in recipient DCs was first evaluated by treating EVs with RNase. As shown in FIG. 6H, RNase treatment did not change the level of the target miRNAs in the
EXOuniSTiNG, suggesting that the miRNA packaged into EVs, but not outer surface of EVs, is important for induction of antitumor immunity in recipient cells. To further examine whether these miRNAs are indeed involved in the induction of immune response, the corresponding synthetic miRNAs, including miR-130b-3p, miR-130a-3p and miR-19a-3p mimics that have been implicated in the immune response modulation, were obtained and mixed as a pool in treating DCs. Compared with the miRNA negative control group, treatment with the synthetic miRNA pool elicited upregulation of IFN-P and CXCL10 in addition to the downregulation of immunosuppressive molecules including TGF- , IL10 and MRC1 in DC2.4 cells (FIG. 61 and 20A), suggesting that exosomal miRNAs derived from EXOuniSTiNG modulated immune response through the regulation of their target genes and subsequent activation of relative signaling pathways. It should be noted that three out of the top-five upregulated miRNAs in EXOuniSTiNG, including miR-130a, miR-19a, and miR-16, showed a favorable correlation with the prognosis of human breast cancer (FIG. 2B). Together, these data support the hypothesis that uniSTING-treated tumor cells modulate DCs’ immunological responses via secreting exosomal miRNAs.
[00176] To further delineate genes in DCs that are regulated by the miRNAs of EVs derived from uniSTING-treated tumor cells and accountable for the improved immune response, RNA-seq of DC2.4 cells was conducted post exposure to EXOuniSTiNG or EXOmock. More than 2000 significantly altered genes were identified. Indeed, EXOuniSTiNG regulated the expression of a major set of genes involved in IFN signaling responses, including Ifit2, Ifit3, Isgl5, and Uspl8 (FIG. 7A). GSEA results showed IFN- a response genes were markedly enriched in recipient DC2.4 cells exposed to EXOunisi iNG (FIG. 7A). These findings confirmed that EVs from uniSTING-treated tumor cells promoted immune activation in DCs, resulting in a gene signature that improved antitumor responses. Interestingly, DC2.4 cells drastically downregulated Wnt signaling-associated genes, particularly Wnt2b and Snail, after exposure to EXOuniSTiNG (FIG. 7B). It has been demonstrated that Wnt signaling activation in TME can drive DCs into tolerogenic regulatory state, increase infiltration of regulatory T cells, and impair differentiation of CD8+ effector T cells. Wnt2b transcripts were examined in DC2.4 cells after indicated treatments to validate the RNA-seq result. A slight increase in Wnt2b (about twofold) was unexpectedly observed in uniSTING-treated DC2.4 cells, implying the activation of STING in DCs might stimulate putative negative feedback in Wnt2b signaling pathway. Nevertheless, EXOuniSTiNG treatment was able to mitigate this negative impact in DCs as shown by substantially reduced expression of Wnt2b (about sixfold) (FIG. 7C). Similar outcomes were also validated in EVs derived from E0771 tumor cells and BMDCs (FIGS. 21A-12C). Blockade of Wnt2b function in DC2.4 cells by
either silencing Wnl2b using siRNA or by neutralizing with a-Wnt2b antibody upregulated IFN-P, CXCL9, and CXCL10, while downregulated immunosuppressive TGF- and IL10 (FIGS. 7D and 21D-21E). These results suggest that Wnt2b serves as a regulatory signal for type I IFN response, in line with the earlier finding that Wnt2b negatively regulated the IFN-P signaling. Moreover, Wnt2b levels were correlated with poor prognosis in human breast cancer (FIG. 21F). Since uniSTING-treated tumor cells triggered Wnt2b downregulation and inflammatory response in recipient DCs through the transfer of exosomal components, whether the downregulation of Wnt2b in DCs was indeed mediated by miRNAs secreted from EXOunisnNG was assessed. The gene targets for the elevated miRNAs from EXOuniSTiNG were determined by using a sophisticated database for miRNA target prediction. Interestingly, among the fourteen top-ranked Wnt2b associated miRNAs, four of them (miR-130b-3p, miR-130a-3p, miR-16-5p, and miR-15b-5p) were detected in EXOuniSTiNG, statistically indicating the consequence of such miRNA-targct interactions (FIG. 7E). Treatment with the corresponding synthetic miRNA pool indeed decreased Wnl2b expression in DC2.4 cells (FIG. 7F). Additionally, EXOuniSTiNG co-treatment with the synthetic miRNA pool showed more pronounced Wnt2b inhibition relative to other treatment groups. To further determine exosomal miRNAs’ dominancy in regulating Wnt2b level in DCs, the knockout of miR-130a-3p and miR-130b-3p in tumor cells was performed. A more than 95% downregulation of miR-130a-3p and miR-130b-3p was observed in 4T1 cells after their relative hairpin inhibitor (miRi Pool) transfection compared to that of the miRNA hairpin inhibitor negative control (miRi Ctr) treatment (FIG. 7G). uniSTING co-treatment with miRi Pool failed to recover the miR-130a-3p and miR-130b-3p levels. Intriguingly, EVs derived from 4T1 cells treated with miRi Pool + uniSTING failed to downregulate Wnt2b relative to those derived from cells treated with miRiCtr + uniSTING (FIGS. 7H and 71). More importantly, miRi Pool + uniSTING treatment was not able to prime the immune response as indicated by the mRNA expression of IFN-P, CXCL10, CD80, and CD86, while miRi Ctr + uniSTING showed remarkable increase of those gene signatures (FIG. 71). Thus, exosomal transfer during the contact between DC cells and uniSTING-treated tumor cells could be mainly attributed to miRNAs, which targeted the critical regulatory Wnt2b signal and improved the antitumor response in DCs.
Example 4
Wnt2b blockade enhances in vivo antitumor activity of uniSTING
[00177] Wnt signaling is one of the well-known oncogenic drivers in many cancer types, notably suppressing the maturation and differentiation of DCs and T cells. Whether Wnt2b blockade further enhanced antitumor activity of uniSTING was investigated. The therapeutic efficacy of uniSTING-
mRNA/LNP or/and a-Wnt2b antibody was evaluated by treating mice bearing 4T1-Luc2 tumors (FIGS. 8A-8C). Dual treatment of uniSTING-mRNA/LNPs with a-Wnt2b greatly augmented antitumor efficacy as assessed by reduced tumor weight, prolonged survival in comparison with monotherapies and the soluble 2’3’-cGAMP plus a-Wnt2b group, indicating the advantage of synergistic effect of uniSTING-mRNA/LNPs and a-Wnt2b in improving antitumor immunity. Enhanced therapeutic efficacy of uniSTING-mRNA/LNPs and a-Wnt2b antibody combination was further proved in murine E0771 tumor model and Lewis lung carcinoma LLC models (FIGS. 8D-8E and 22). Similarly, a-Wnt2b with uniSTING-mRNA/LNPs but not with ADU-S100 exhibited most potent tumor control (FIGS. 8D-8E). In addition to the functions of promoting memory differentiation by vaccination with uniSTING-mRNA/LNPs (FIG. 5G) or strengthening cytotoxic CD8+ T cell responses by uniSTING-mRNA/LNPs treatment for tumors (FIGS. 5A, 5B and 5D), Wnt2b blockade together with uniSTING-mRNA/LNPs further drastically increased CD8+ T cells frequency in CD45+ cells as well as the TCF1+CD27+ memory subpopulation in lung tumor bed (FIGS. 8F-8G). When paired with uniSTING-mRNA/LNPs but not with ADU-S100, the cytotoxic Gzmb+CD8+ T cell infiltrated more in the group treated with the Wnt2b inhibition (FIG. 8H). These findings suggested that the combination approach of uniSTING-mRNA/LNPs plus a-Wnt2b elicited synergistic antitumor immunity in the severely unfavorable TME, leading to robust therapeutic efficacy through this uniSTING activation strategy.
Sequences
Tetramerization domains derived from the tetrabrachion protein in Staphylothermus marinus and related organisms
UNETADDIVYRLTVIIDDRYESLKNLITLRADRLEMIINDNVSTILAS - SEQ ID NO: 2 VIYDVRDDVLTNLTITLDDIKTDLTTLINTRVDKLEMMINDNVTTILAT - SEQ ID NO: 3 IIGSVERAVDDIIYTITLEINWLRENLTGLINARIDELKLFINDNITTVLT - SEQ ID NO: 4
Tetramerization domains derived from human potassium voltage-gated channel subfamily A members (the orthologs of these sequences in other organisms, including in mouse and rat can also be used)
ERVVINISGLRFETQLKTLAQFPNTLLGNPKKRMRYFDPLRNEYFFDRNRPSFDAILYYYQSGG RLRRPVNVPLDMFSEEIKFYELG - SEQ ID NO: 5
ERVVINISGLRFETQLKTLAQFPETLLGDPKKRMRYFDPLRNEYFFDRNRPSFDAILYYYQSGG
RLRRPVNVPLDIFSEEIRFYELG - SEQ ID NO: 6
ERVVINISGLRFETQLKTLCQFPETLLGDPKRRMRYFDPLRNEYFFDRNRPSFDAILYYYQSGG
RIRRPVNVPIDIFSEEIRFYQLG - SEQ ID NO: 7
ERVVINVSGLRFETQMKTLAQFPETLLGDPEKRTQYFDPLRNEYFFDRNRPSFDAILYYYQSG
GRLKRPVNVPFDIFTEEVKFYQLG - SEQ ID NO: 8
QRVHINISGLRFETQLGTLAQFPNTLLGDPAKRLRYFDPLRNEYFFDRNRPSFDGILYYYQSGG
RLRRPVNVSLDVFADEIRFYQLG - SEQ ID NO: 9
ERLVINISGLRFETQLRTLSLFPDTLLGDPGRRVRFFDPLRNEYFFDRNRPSFDAILYYYQSGGR
LRRPVNVPLDIFLEEIRFYQLGDEALAAF - SEQ ID NO: 10
Tetramerization domains derived from human potassium voltage-gated channel subfamily KCNQ1
SNTIGARLNRVEDKVTQLDQRLALITDMLHQLLSLHG - SEQ ID NO: 11
Tetramerization domain derived from human potassium voltage-gated channel subfamily KQT member
GAVDEISMMGRVVKVEKQVQSIEHKLDLLLGFY - SEQ ID NO: 12
Tetramerization domain derived from human P53
EYFTLQIRGRERFEMFRELNEALELKDAQAG - SEQ ID NO: 13
Tetramerization domain derived from human vasodilator-stimulated phosphoprotein
SSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKR - SEQ ID NO: 14
Tetramerization domain derived from human acetylcholinesterase
DTLDEAERQWKAEFHRWSSYMVHWKNQFDHYSKQDR - SEQ ID NO: 15
Tetramerization domain derived from human butyrylcholinesterase
MTGNIDEAEWEWKAGFHRWNNYMMDWKNQFNDYTSKKESCVGL - SEQ ID NO: 16
Tetramerization domain derived from measles virus phosphoprotein
YDDELFSDVQDIKTALAKIHEDNQKIISKLESLLLLKGEVESIKKQINRQNISI - SEQ ID NO: 17
Tetramerization domain derived from Nipah virus phosphoprotein
SNDSLDDKYIMPSDDFSNTFFPHDTDRLNYHADHLGDYDLETLCEESVLMGVINSIKLINLDM
RLNHIEEQVKEIPKIINKLESIDRVLAKTNTALSTIEGHLVSMMIMI - SEQ ID NO: 18
Tetramerization domain derived from human metapneumovirus phosphoprotein
LSIEARLESIEEKLSMILGLLRTLN - SEQ ID NO: 19
Tetramerization domain derived from rotavirus NSP4
IEKQMDRVVKEMRRQLEMIDKLTTREIEQVELLKRIYDKLTVQ - SEQ ID NO: 20
Tetramerization domain derived from Sendai virus nucleocapsid phosphoprotein
ENTSSMKEMATLLTSLGVIQSAQEFESSRDASYVFARRALKSANYAEMTFNVCGLILSAEKSS
ARKVDENKQLLKQIQESVESFRDIYKRFSEYQKEQNSLLMSNLSTLHIITD - SEQ ID NO: 21
Tetramerization domain of Bacillus cereus TubY
NPDFIEALTEKITEEVTAKVTEELTKQNMEFFAAVAKQSQDNFDRINKRLEERDEKLMSTIRLI
QE - SEQ ID NO: 22
Tetramerization domain derived from Cauliflower mosaic virus VAP
ANLNQIQKEVSEILSDQKSMKADIKAILELLGSQNPIKESLETVAAKIVNDLTKLINDCPCNKEI
LEALGTQP- SEQ ID NO: 23
Tetramerization domain derived from Newcastle disease virus hemagglutinin-neuraminidase
NQDVVDRIYKQVALESPLALLNTESIIMNAITSLSYQIN - SEQ ID NO: 24
Tetramerization domain derived from Enterococcus phage N-acetylmuramoyl-L-alanine amidase
YCLYERPINSKTGVLEWNGDAWTVMFCNGVNCRRVSHPDEMKVIEDIYRKNNGKDIPFYSQK
EWNKNAPWYNRLETVCPVVGITKK - SEQ ID NO: 25
Tetramerization domain derived from Pseudomonas phage 201phi2-lp060
RKNLEKFKFSKGDGIKFSNTTFHIYEATRNYVTIHILKKYATAELMEFMHTRHDAVYIGPILEW
TDGVHLTFRRKS - SEQ ID NO: 26
Tetramerization domain derived from Drosophila anastral spindle 2
SDLAALVSLVESVRHEQQQLRNLCEMILEQQQRAKEFGENLYFQ - SEQ ID NO: 27
Tetramerization domain derived from Gibberella zeae TRP channel analog
VRKLRAEMEELKSMLSQLGKT - SEQ ID NO: 28
Engineered tetramerization domains:
GEIQKQLKEIQKQLKEIQWQLKEIQKQLKG - SEQ ID NO: 29
GEIKQQLAEIKQQLAEIKWQLAEIKQQLAG - SEQ ID NO: 30
AEIEQAKKEIAYLIKKAKEEILEEIKKAKQEIA - SEQ ID NO: 31
Tetramerization domains derived from yeast GCN4
MKQLEDKVEELLSKNYHLENEVARLKKLVNSD - SEQ ID NO: 32
MKQIEDKLEEILSKLYHIENELARIKKLLGER - SEQ ID NO: 33
MKVKQLVDKVEELLSKNYHLVNEVARLVKLVGER - SEQ ID NO: 34
MKQIEDKLEEILSKLYHISNELARIKKLLGER - SEQ ID NO: 35
MKQIEDKLEEILSKGYHICNELARIKKLLGER - SEQ ID NO: 36
MKQIEDKGEEILSKLYHIENELARIKKLLGER - SEQ ID NO: 37
C-terminal cytoplasmic domain of mouse STING:
LTPAEVSAVCEEKKLNVAHALAWSYYIGYLRLILPGLQARIRMFNQLHNNMLSGAGSRRLYIL
FPLDCGVPDNLSVVDPNIRFRDMLPQQNIDRAGIKNRVYSNSVYEILENGQPAGVCILEYATPL
QTLFAMSQDAKAGFSREDRLEQAKLFCRTLEEILEDVPESRNNCRLIVYQEPTDGNSFSLSQEV
LRHIRQEEKEEVTMNAPMTSVAPPPSVLSQEPRLLISGMDQPLPLRTDLI - SEQ ID NO: 38
C-terminal cytoplasmic domain of human STING:
LAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYIL
LPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATP
LQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQE
VLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS - SEQ ID NO: 39 uniSTING for mouse studies described in the proof-of-concept experiments:
MDYKDDDDKGGGTGGIINETADDIVYRLTVIIDDRYESLKNLITLRADRLEMIINDNVSTILASI
GGGGSGGGGSTGLTPAEVSAVCEEKKLNVAHGLAWSYYIGYLRLILPGLQARIRMFNQLH
NNMLSGAGSRRLYILFPLDCGVPDNLSVVDPNIRFRDMLPQQNIDRAGIKNRVYSNSVYEI LENGQPAGVCILEYATPLQTLFAMSQDAKAGFSREDRLEQAKLFCRTLEEILEDVPESRN NCRLIVYQEPTDGNSFSLSQEVLRHIRQEEKEEVTMNAPMTSVAPPPSVLSQEPRLLISGM DQPLPLRTDLI - SEQ ID NO: 40
Monomeric STING control without the tetramerization domain:
MDYKDDDDKGGGTGGGGGSTGLTPAEVSAVCEEKKLNVAHALAWSYYIGYLRLILPGLQ
ARIRMFNQLHNNMLSGAGSRRLYILFPLDCGVPDNLSVVDPNIRFRDMLPQQNIDRAGIK NRVYSNSVYEILENGQPAGVCILEYATPLQTLFAMSQDAKAGFSREDRLEQAKLFCRTLE EILEDVPESRNNCRLIVYQEPTDGNSFSLSQEVLRHIRQEEKEEVTMNAPMTSVAPPPSVL SQEPRLLISGMDQPLPLRTDLI - SEQ ID NO: 41 uniSTING for human applications:
MDYKDDDDKGGGTGGIINETADDIVYRLTVIIDDRYESLKNLITLRADRLEMIINDNVSTILASI
GGGG5GGGG5TGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHY
NNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYE LLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPES QNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLIS
GMEKPLPLRTDFS SEQ ID NO: 42
The underlined sequence is the tetramerization domain/motif that can be replaced with other tetramerization domain/motif disclosed herein.
The italic sequence is the flexible linker.
The bolded sequence is the non-membrane bound region of mouse or human STING.
[00178] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.
[00179] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope thereof.
Claims
1. A polypeptide comprising: a tetramerization motif; and
C-terminal cytoplasmic domain of a stimulator of interferon genes (STING) protein.
2. The polypeptide of claim 1, further comprising a linker between the tetramerization motif and the C-terminal cytoplasmic domain of a STING protein.
3. The polypeptide of claim 2, wherein the linker is a flexible linker.
4. The polypeptide of any of claims 1-3, wherein the tetramerization motif is N-terminal to the C- terminal cytoplasmic domain of a STING protein.
5. The polypeptide of any of claims 1-4, wherein the tetramerization motif comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-37.
6. The polypeptide of any of claims 1-5, wherein the tetramerization motif comprises an amino acid sequence of SEQ ID NO: 1.
7. The polypeptide of any of claims 1-6, wherein the C-terminal cytoplasmic domain of a STING protein comprises an amino acid sequence of at least 70% similarity to SEQ ID NOs: 38 or 39.
8. The polypeptide of any of claims 1-7, wherein the C-terminal cytoplasmic domain of a STING protein comprises an amino acid sequence of SEQ ID NO: 38 or 39.
9. A polynucleotide encoding a polypeptide of any of claims 1-8.
10. The polynucleotide of claim 9, wherein the polynucleotide is RNA or DNA.
11. The polynucleotide of claim 9 or 10, wherein the polynucleotide is mRNA.
12. The polynucleotide of any of claim 9-11, wherein the polynucleotide further comprises or encodes: a 5’ untranslated region (UTR), a 5’ cap, a 3’ UTR, a 3’ tailing sequence, or any combination thereof.
13. The polynucleotide of claim 12, wherein the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.
14. The polynucleotide of any of claim 9-13, wherein the polynucleotide comprises at least one chemically modified nucleotide.
15. The polynucleotide of claim 14, wherein the at least one chemically modified nucleotide comprises a modified uracil, a 5 -methylcytosine or a combination thereof.
16. A composition comprising a polypeptide of any of claims 1-8 or a polynucleotide of any of claims 9-15 and a pharmaceutical acceptable excipient or carrier.
17. A vaccine or medicament comprising: a polypeptide of any of claims 1-8, a polynucleotide of any of claims 9-15, or a composition of claim 16; and an adjuvant, a delivery vehicle, or a combination thereof.
18. The vaccine or medicament of claim 17, wherein the delivery vehicle comprises a lipid nanoparticle encapsulating the polynucleotide or polypeptide.
19. The vaccine or medicament of claim 18, wherein the lipid nanoparticle comprises a cationic lipid, a neutral and/or non-cationic lipid, a sterol, or any combination thereof.
20. The vaccine or medicament of claim 19, wherein the non-cationic lipid comprises a phospholipid.
21. The vaccine or medicament of claim 19 or 20, wherein the sterol comprises cholesterol or a modification or ester thereof.
22. The vaccine or medicament of any of claims 19-21, wherein the lipid nanoparticle comprises a polyethylene glycol (PEG)-lipid conjugate.
23. A method of treating a disease or disorder comprising administering a polypeptide of any of claims 1-8, a polynucleotide of any of claims 9-15, a composition of claim 16, or a vaccine or medicament of any of claims 17-22 to a subject in need thereof.
24. The method of claim 23, wherein the disease or disorder is cancer.
25. The method of claim 24, wherein the cancer comprises breast cancer, lung cancer, skin cancer, liver cancer, or a combination thereof.
26. A method of inducing an immune response in a subject comprising administering a polypeptide of any of claims 1-8, a polynucleotide of any of claims 9-15, a composition of claim 16, or a vaccine of any of claims 17-22 to a subject in need thereof.
27. The method of any of claims 23-26, further comprising administering a Wnt2b blocking agent based on an antibody or a non-immunoglobulin scaffold, or an interfering RNA to Wnt2b.
28. The method of any of claims 23-27, wherein the administering comprises an initial immunization and at least one subsequent immunization.
29. The method of any of claims 23-28, wherein the administering comprises intratumoral administration and/or systemic administration.
30. The method of any of claims 23-29, wherein the subject has cancer, has had cancer, is predisposed to cancer, is suspected of having cancer, or has a family history of cancer.
31. A method of treating a disease or disorder or inducing an immune response in a subject comprising administering a Wnt2b blocking agent to a subject in need thereof.
32. The method of claim 31, wherein the disease or disorder is cancer.
33. The method of claim 32, wherein the cancer comprises breast cancer, lung cancer, skin cancer, liver cancer, or a combination thereof.
34. The method of any of 31-33, therein the Wnt2b blocking agent is an antibody or an interfering RNA to Wnt2b.
35. The method of any of claims 31-34, wherein the administering comprises an initial administration and at least one subsequent administration of the Wnt2b blocking agent.
36. The method of any of claims 31-35, wherein the administering comprises intratumoral administration and/or systemic administration.
37. The method of any of claims 31-36, wherein the subject has cancer, has had cancer, is predisposed to cancer, is suspected of having cancer, or has a family history of cancer.
38. A vaccine or medicament comprising a polypeptide of any of claims 1-7, a polypeptide of any of claims 1-8, a polynucleotide of any of claims 9-15, or a composition of claim 16, for use in treating a disease or disorder or inducing an immune response in a subject.
39. The vaccine or medicament of claim 38, further comprising a Wnt2b blocking agent based on an antibody or a non-immunoglobulin scaffold, or an interfering RNA to Wnt2b.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202263350714P | 2022-06-09 | 2022-06-09 | |
US63/350,714 | 2022-06-09 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2023240225A1 true WO2023240225A1 (en) | 2023-12-14 |
Family
ID=89119055
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2023/068182 WO2023240225A1 (en) | 2022-06-09 | 2023-06-09 | Constitutively active polymeric sting mimics for antitumor immunity |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2023240225A1 (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20160068596A1 (en) * | 2014-07-11 | 2016-03-10 | Genentech, Inc. | Notch pathway inhibition |
US20180085432A1 (en) * | 2008-08-04 | 2018-03-29 | University Of Miami | STING (Stimulator of Interferon Genes), A Regulator of Innate Immune Responses |
US20180303931A1 (en) * | 2012-07-26 | 2018-10-25 | The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. | Multimeric fusion protein vaccine and immunotherapeutic |
-
2023
- 2023-06-09 WO PCT/US2023/068182 patent/WO2023240225A1/en unknown
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20180085432A1 (en) * | 2008-08-04 | 2018-03-29 | University Of Miami | STING (Stimulator of Interferon Genes), A Regulator of Innate Immune Responses |
US20180303931A1 (en) * | 2012-07-26 | 2018-10-25 | The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. | Multimeric fusion protein vaccine and immunotherapeutic |
US20160068596A1 (en) * | 2014-07-11 | 2016-03-10 | Genentech, Inc. | Notch pathway inhibition |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11344504B1 (en) | Combinations of mRNAs encoding immune modulating polypeptides and uses thereof | |
US11660341B2 (en) | mRNA combination therapy for the treatment of cancer | |
JP6990176B2 (en) | Methods for therapeutic administration of messenger ribonucleic acid drugs | |
JP2022519557A (en) | Method for preparing lipid nanoparticles | |
WO2023240225A1 (en) | Constitutively active polymeric sting mimics for antitumor immunity | |
TW202245808A (en) | Therapeutic rna for treating cancer |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23820674 Country of ref document: EP Kind code of ref document: A1 |